Anti-DLL3 antibody and related product and application thereof

Abstract

The invention discloses an anti-DLL3 antibody as well as a related product and application thereof, and relates to the field of antibodies. According to the invention, the anti-DLL3 antibody with a remarkable effect is screened, and the antibody not only can be subjected to high-specificity binding with a DLL3 target spot, but also has excellent binding affinity. The anti-DLL3 antibody is used as an antigen binding domain, CAR-T cells are further prepared, and experiments prove that the CAR-T cells show strong and stable anti-tumor activity, can specifically recognize and kill DLL3 positive tumor cells, and effectively inhibit tumor growth and proliferation. The anti-DLL3 antibody and the corresponding CAR-T cell can be widely applied to preparation of drugs for preventing or treating DLL3 positive tumors, provide a new targeted therapy choice for patients with the DLL3 positive tumors, and have important clinical application value and wide industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, to antibodies against DLL3 and related products and applications. Background Technology

[0002] Lung cancer is a leading cause of cancer-related deaths worldwide, with small cell lung cancer (SCLC) accounting for approximately 15%–20%. SCLC is a high-grade neuroendocrine tumor closely associated with smoking, and common symptoms include cough, dyspnea, and hemoptysis. Most patients have distant metastases at diagnosis, resulting in a very poor prognosis. While current chemotherapy and radiotherapy are initially effective, their effects are short-lived, with a median survival of only about one year for metastatic patients and less than two years for limited-stage patients. Therefore, in-depth exploration of SCLC from epidemiological and pathogenesis perspectives to novel treatment methods is of significant clinical importance.

[0003] Delta-like ligand 3 (DLL3) belongs to the Notch ligand family and is a type I single-pass transmembrane protein. Its structure includes an extracellular DSL domain, six EGF-like repeat sequences, a transmembrane region, and an intracellular domain, with the DSL domain responsible for binding to the Notch receptor. DLL3 is almost unexpressed in normal tissues, but it is significantly highly expressed on the surface of tumor cells in over 80% of SCLC patients, particularly in the neuroendocrine subtype, making it a highly promising therapeutic target. Targeting strategies against DLL3, such as antibody-drug conjugates, bispecific T-cell conjugates, and CAR-T therapy, are under investigation. For example, the bispecific antibody talatumab, targeting DLL3, has shown an objective response rate of approximately 40% in relapsed SCLC, with some patients experiencing a response duration exceeding 6 months. High DLL3 expression is also associated with poor prognosis and may serve as a biomarker for predicting treatment response. In summary, the specific high expression of DLL3 in SCLC provides important evidence for its targeted therapy. Further research into its biological functions and regulatory mechanisms holds promise for providing new treatment directions for SCLC patients.

[0004] Chimeric antigen receptor T-cell (CAR-T) therapy is a groundbreaking technology in the field of tumor immunotherapy. Through genetic engineering, T cells express chimeric receptors on their surface that specifically recognize tumor antigens, thereby precisely targeting and eliminating tumor cells. When CAR-T cells bind to tumor antigens, they are activated and proliferate, killing tumors by releasing toxic substances such as perforin and granzymes, as well as cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). This technology has achieved remarkable efficacy in the treatment of various hematologic malignancies, not only revolutionizing the treatment of hematologic tumors but also driving the development of other immunotherapies.

[0005] Currently, investigational drug formulations targeting DLL3 for the treatment of SCLC include monoclonal antibodies, ADCs, bispecific antibodies, multiclonal antibodies, CAR-T, and CAR-NK, but their efficacy still needs improvement.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide antibodies against DLL3 and related products and applications.

[0008] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide an antibody against DLL3 or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:7.

[0009] Secondly, embodiments of the present invention provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes an anti-DLL3 antibody or its antigen-binding fragment as described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide a CAR-T cell comprising the chimeric antigen receptor as described in the foregoing embodiments.

[0012] Fifthly, embodiments of the present invention provide a reagent or kit comprising: an anti-DLL3 antibody or its antigen-binding fragment as described in the foregoing embodiments, or an antibody-drug conjugate as described in the foregoing embodiments, or a chimeric antigen receptor as described in the foregoing embodiments, or CAR-T cells as described in the foregoing embodiments.

[0013] In a sixth aspect, embodiments of the present invention provide an immune conjugate or pharmaceutical composition comprising: an anti-DLL3 antibody or its antigen-binding fragment as described in the foregoing embodiments, or an antibody-drug conjugate as described in the foregoing embodiments, or a chimeric antigen receptor as described in the foregoing embodiments, or CAR-T cells as described in the foregoing embodiments.

[0014] In a seventh aspect, embodiments of the present invention provide the use of anti-DLL3 antibodies or antigen-binding fragments thereof as described in the foregoing embodiments, or antibody conjugates as described in the foregoing embodiments, or chimeric antigen receptors as described in the foregoing embodiments, or CAR-T cells as described in the foregoing embodiments, in the preparation of medicaments for the prevention or treatment of DLL3-positive tumors.

[0015] The present invention has the following beneficial effects: This invention has identified a highly effective anti-DLL3 antibody that not only binds specifically to the DLL3 target but also possesses excellent binding affinity. Using this anti-DLL3 antibody as the antigen-binding domain, CAR-T cells were further prepared. Experimental results demonstrated that these CAR-T cells exhibit potent and stable anti-tumor activity, specifically recognizing and killing DLL3-positive tumor cells, effectively inhibiting tumor growth and proliferation. This anti-DLL3 antibody and its corresponding CAR-T cells can be widely used in the preparation of drugs for the prevention or treatment of DLL3-positive tumors, providing a new targeted therapy option for patients with DLL3-positive tumors, and possessing significant clinical application value and broad industrialization prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 In this embodiment of the invention, SDS-PAGE was used to analyze the expression of anti-DLL3 scFv antibody; 1: reduced; 2: non-reduced; Figure 2 In this embodiment of the invention, the specificity of the anti-DLL3 scFv antibody is analyzed using indirect ELISA. Figure 3 In this embodiment of the invention, indirect ELISA is used to analyze the affinity activity of anti-DLL3 scFv antibody; Figure 4 In this embodiment of the invention, flow cytometry was used to analyze the binding activity of anti-DLL3 scFv antibody to DLL3-positive cells; Figure 5 This is a schematic diagram of the chimeric antigen receptor structure against DLL3 in an embodiment of the present invention; Figure 6 This invention relates to the detection of CAR expression efficiency on the surface of T cells in an embodiment of the invention. Figure 7 This invention relates to the in vitro killing efficiency of CAR-T cells against DLL3-positive tumor cells in this embodiment. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] On one hand, embodiments of the present invention provide an antibody against DLL3 or an antigen-binding fragment thereof, comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4, and LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:7.

[0020] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

[0021] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:1~3, SEQ ID NO:5, KVS, and SEQ ID NO:6, respectively.

[0022] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region.

[0023] In some embodiments, the heavy chain variable region and the light chain variable region further include a skeleton region.

[0024] In this article, the "backbone region" or "FR region" refers to the region in the antibody heavy chain variable region other than the CDRs. The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4), which include the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0025] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0026] In some embodiments, the antibody further includes a constant region.

[0027] In some embodiments, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0028] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and / or; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0029] "Antigen-binding fragment" is a portion of the intact antibody that specifically binds to the antigen to which the intact antibody is bound. Those skilled in the art will readily understand from the description of this invention that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds, or synthesized using recombinant genetics techniques or automated peptide synthesizers (such as AppliedBioSystems' automated peptide synthesizers).

[0030] In some embodiments, the antigen-binding fragment includes any one of Fab, Fab', F(ab')2, scFv, and Fv.

[0031] In some embodiments, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is as shown in SEQ ID NO:8.

[0032] On the other hand, embodiments of the present invention provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0033] In some embodiments, the antibody-drug conjugate further includes a label, purification tag, and / or solid-phase carrier conjugated to the anti-DLL3 antibody or its antigen-binding fragment.

[0034] On the other hand, embodiments of the present invention provide a chimeric antigen receptor, wherein the antigen-binding domain of the chimeric antigen receptor includes an anti-DLL3 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0035] In some embodiments, the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0036] In some embodiments, the signal transduction structural domain includes CD3ζ.

[0037] In some embodiments, the signal transduction domain further includes a 4-1BB intracellular region.

[0038] On the other hand, embodiments of the present invention provide an isolated nucleic acid molecule encoding the antibody or chimeric antigen receptor described in any of the above embodiments.

[0039] On the other hand, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in any of the foregoing embodiments.

[0040] The recombinant vector is an expression vector or a cloning vector, optionally an expression vector. It can refer to any recombinant polynucleotide construct that can directly or indirectly (e.g., packaged as a virus) introduce the target DNA fragment into host cells for target gene expression via transformation, transfection, or transduction. One type of vector is a plasmid, a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After entering the host cell, these vectors can express the target gene.

[0041] On the other hand, embodiments of the present invention provide a host cell containing the recombinant vector described in any of the foregoing embodiments.

[0042] Specifically, the host cell includes at least one of prokaryotic host cells, eukaryotic host cells, and bacteriophages. The prokaryotic host cell can be *Escherichia coli*, *Streptomyces*, or *Bacillus subtilis*, etc. The eukaryotic host cell can be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Hansenula polymorpha*, *Candida*, and some insect cells. The 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

[0043] On the other hand, embodiments of the present invention also provide a method for preparing an antibody or its antigen-binding fragment, comprising: culturing host cells as described in the foregoing embodiments to obtain an antibody. Specifically, the present invention does not specifically limit the culture conditions of the host cells; culture conditions capable of enabling the host cells to express and produce the antibody can be obtained based on conventional technical knowledge.

[0044] On the other hand, embodiments of the present invention provide a CAR-T cell that includes the chimeric antigen receptor as described in any of the foregoing embodiments.

[0045] In some embodiments, the CAR-T cells include at least one of universal CAR-T cells and autologous CAR-T cells.

[0046] On the other hand, embodiments of the present invention provide a reagent or kit comprising: an anti-DLL3 antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or an antibody-drug conjugate as described in any of the foregoing embodiments, or a chimeric antigen receptor as described in any of the foregoing embodiments, or CAR-T cells as described in any of the foregoing embodiments.

[0047] On the other hand, embodiments of the present invention provide an immune conjugate or pharmaceutical composition comprising: an anti-DLL3 antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or an antibody-drug conjugate as described in any of the foregoing embodiments, or a chimeric antigen receptor as described in any of the foregoing embodiments, or a CAR-T cell as described in any of the foregoing embodiments.

[0048] In some embodiments, the immune conjugate further includes a therapeutic agent. The therapeutic agent includes at least one of: immune checkpoint-related agents, antibody-drug conjugates, bispecific (multispecific) antibodies, radionuclides, toxins, factors, kinase inhibitors, and cytotoxic agents.

[0049] In some embodiments, the term "pharmaceutical composition" refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient, grouped together to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes combinations that are separate in time and / or space, provided they can work together to achieve the objectives of the invention. Some pharmaceutical compositions enhance the biological efficacy of the invention or reduce drug side effects by combining several pharmaceutically acceptable ingredients or compounds (e.g., they can be used in combination with other antitumor drugs to enhance antitumor effects). Other pharmaceutical compositions aim to promote administration to the organism, facilitate the absorption of the active ingredient, enhance stability or targeting, prolong half-life, and thus better exert the biological efficacy of the invention.

[0050] Furthermore, the pharmaceutical composition further includes at least one of a pharmaceutical excipient, a carrier, and a diluent.

[0051] On the other hand, embodiments of the present invention provide the use of anti-DLL3 antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, or antibody conjugates as described in any of the foregoing embodiments, or chimeric antigen receptors as described in any of the foregoing embodiments, or CAR-T cells as described in any of the foregoing embodiments in the preparation of medicaments for the prevention or treatment of DLL3-positive tumors.

[0052] In some embodiments, the DLL3-positive tumor includes at least one of the following: small cell lung cancer, neuroendocrine prostate cancer, prostate cancer, melanoma, gastrointestinal pancreatic neuroendocrine tumor, metastatic castration prostate cancer, large cell neuroendocrine carcinoma, small cell bladder cancer, pulmonary neuroendocrine tumor, or glioma multiforme.

[0053] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0054] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Example 1: Expression, purification, and construction of a stable cell line expressing human DLL3 antigen for recombinant DLL3 protein. 1.1 Construction of DLL3 recombinant protein expression vector Using a plasmid containing the full-length DLL3 gene (gene number NM_016941.4) as a template, primers were designed to amplify the extracellular domain (ECD) of DLL3. The gene was then amplified by PCR and ligated into the pcDNA3.1-His vector via homologous recombination. The vector was transformed into DH5α competent cells, plated on ampicillin-resistant plates, and incubated overnight at 37°C. Single clones were picked and sequenced for identification. Recombinant plasmids were extracted from successfully constructed clones through further culture.

[0057] 1.2 Expression and purification of recombinant DLL3 protein HEK293T cells were transiently transfected with the recombinant plasmid pcDNA3.1-DLL3-His, which contained the extracellular region of the DLL3 gene. The cells were cultured in FreeStyle™ serum-free medium for 5-7 days. The culture supernatant was collected and purified by nickel column affinity chromatography to obtain the recombinant protein DLL3-His.

[0058] 1.3 Construction of stable cell lines expressing human DLL3 antigen The full-length gene encoding human DLL3 was constructed into the lentiviral vector pLenti-EGFP and co-transfected with the lentiviral packaging plasmids psPAX2 and pMD2.G into HEK293T cells. After 48 hours of transfection, the supernatant was collected, filtered through a 0.45 μm filter, and then used to infect HeLa cells. He1a cells that stably express DLL3, i.e., DLL3-HeLa cells, were obtained by flow cytometry sorting.

[0059] Example 2: Screening of anti-DLL3 monoclonal antibodies 2.1 Mouse Immunization Female BALB / c mice aged 6-8 weeks were immunized at a dose of 100 μg per mouse. For the first immunization, 100 μL of Freund's complete adjuvant (Sigma) was mixed with an equal volume of DLL3 recombinant protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Every 2 weeks, an equal volume of Freund's incomplete adjuvant was mixed with the recombinant protein, thoroughly emulsified, and then injected subcutaneously at multiple sites. Hybridoma fusion was performed when the antibody titer met the requirements.

[0060] 2.2 Cell fusion and screening of positive hybridoma cells 2.2.1 Hybridoma cell fusion After sterile dissection of mice, the spleen was removed, ground, and prepared into a cell suspension. The spleen cell suspension was then fused with mouse myeloma cells SP2 / 0 using PEG4000. The fused cells were cultured in HAT medium, and on day 10 post-fusion, the medium was partially replaced with fresh HT medium. Positive clones were screened using ELISA, immunofluorescence, and flow cytometry on days 11-15 post-fusion.

[0061] ELISA screening: DLL3 recombinant protein was coated onto 96-well microplates at 100 ng / well and incubated overnight at 4°C. The next day, the plates were blocked with 5% skim milk. 50 μL of hybridoma culture supernatant was used as the primary antibody, and HRP-labeled anti-mouse IgG antibody (1:5000) was used as the secondary antibody. After TMB staining, the reaction was terminated with 2M H2SO4, and the OD values ​​were read. 450 nm value was used to screen for positive clones.

[0062] Immunofluorescence: DLL3-HeLa cells and wild-type HeLa cells were seeded in 96-well plates. The next day, the plates were blocked with 2% BSA and 50 μL of hybridoma supernatant was added as the primary antibody. The plates were incubated at room temperature for 1 hour and washed three times with PBS. Alexa Fluor 594-labeled Goat Anti-Mouse IgG (1:500) was used as the secondary antibody and the plates were incubated at room temperature for 1 hour and washed three times with PBS. Images of positive clones were collected and analyzed using a fluorescence microscope.

[0063] Flow cytometry: DLL3-HeLa cells and wild-type HeLa cells were collected, blocked with 2% BSA, resuspended in 50 μL of hybridoma supernatant, incubated at room temperature for 1 hour, washed 3 times with PBS, and APC-labeled Goat Anti-Mouse IgG (1:500) (Cat#405308, BioLegend) was used as a secondary antibody. The cells were incubated at room temperature for 1 hour, washed 3 times with PBS, and positive clones were analyzed by flow cytometry.

[0064] 2.2.2 Subcloning to screen positive hybridoma cells Based on the results of the ELISA, immunofluorescence, and flow cytometry experiments, positive candidate clones were subcloned 2-3 times to obtain monoclonal hybridoma cells, which were then cultured and total RNA was extracted.

[0065] 2.3 Amplification of antibody VH and VL genes First, total RNA was extracted from hybridoma cells according to the instructions (Cat# 74134, QIAGEN). Then, cDNA was obtained by reverse transcription using RNA as a template using a reverse transcription kit (Cat# 18080051, Invitrogen). Using cDNA as a template, the VH and VL genes were amplified using mouse antibody VH and VL amplification primers. The genes were then cloned into the pMD-19T vector, transformed into DH5α bacterial competent cells, and cultured overnight at 37°C. The next day, single clones were picked and sequenced, and the VH (amino acid sequence as shown in SEQ ID NO:4) and VL (amino acid sequence as shown in SEQ ID NO:7) sequences were obtained by alignment with the IgBlast database.

[0066] Example 3: Preparation of anti-DLL3 protein scFv antibody 3.1 Construction of scFv vector The first round of PCR used the plasmid containing the anti-DLL3 antibody gene from step 2.3 of Example 2 as a template to amplify the VH and VL genes, respectively. The PCR products were identified and separated by agarose gel electrophoresis, and the DNA was recovered by gel extraction. The second round of PCR used the recovered product from the first round as a template to amplify the complete scFv fragment (741 bp, amino acid sequence as shown in SEQ ID NO:8) by overlap PCR. The fragment was then ligated into the pcDNA3.1-hFc vector, which had been digested with restriction endonucleases BamH Ⅰ and EcoR Ⅰ, by homologous recombination. The vector was transformed into DH5α competent cells, plated on ampicillin-resistant plates, and incubated overnight at 37°C. Single clones were picked and sequenced for identification. The plasmids were extracted from the successfully constructed clones through amplification culture.

[0067] 3.2 Expression and purification of scFv-hFc antibody The plasmid obtained in step 3.1 was transfected into HEK293T cells using PEI. After 5 days of expression, the supernatant was collected and purified by affinity chromatography using Protein G packing material to obtain the recombinant antibody (e.g., ...). Figure 1 (As shown).

[0068] Example 4: Specificity of indirect ELISA detection of anti-DLL3 antibody The anti-DLL3 scFv antibody (1G1-scFv) prepared in Example 3 and the positive control antibody AMG757 (patent number: CN108271376A) were co-incubated with DLL3-His recombinant protein and other irrelevant antigens (CD5-His, CD7-His, CD47-His and B7H3-His) coated on an ELISA plate (200 ng / well) at 37°C for 1 h. After washing three times with PBST, 100 μL of HRP-labeled Goat anti-human antibody (1:5000) was added to each well and incubated at 37°C for 1 h. After washing three times with PBST, the reaction was developed with TMB for 5 min and then terminated with 2M H2SO4. The OD was then read. 450 absorbance value in nm.

[0069] The results are as follows Figure 2 As shown, the anti-DLL3 antibody 1G1-scFv can bind to both DLL3 protein and does not react with other unrelated antigens, indicating that the above antibody has good specific binding activity to DLL3 protein.

[0070] Example 5: Indirect ELISA detection of anti-DLL3 antibody binding activity DLL3-His recombinant protein was coated into 96-well microplates at a rate of 200 ng per well. The plates were blocked with 5% skim milk at 37°C for 1 h. After washing three times with PBST, 100 μL of different concentrations of (10...) were added to each well. -2 ~10 2 The recombinant scFv antibody and positive control antibody AMG757-hFc prepared in Example 3 (nM) were incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of HRP-labeled Goat anti-human antibody (1:5000) was added to each well and incubated at 37°C for 1 h. After washing three times with PBST, TMB was used for color development for 5 min, and the reaction was terminated with 2M H2SO4. The OD was then read. 450 absorbance value in nm.

[0071] The results are as follows Figure 3 As shown, the anti-DLL3 scFv antibody 1G1 scFv-hFc and the positive control antibody AMG757-hFc prepared in this invention both have good binding activity with DLL3 protein.

[0072] Example 6: Flow cytometry detection of the binding of anti-DLL3 antibody to tumor cells Small cell lung cancer cell lines NCI-H69 and NCI-H82, which are positive for DLL3 expression, were collected in flow cytometry tubes. After blocking with 2% BSA, the cells were resuspended with the anti-DLL3 antibody prepared in Example 3 and an irrelevant control antibody (2 μg / mL), incubated at room temperature for 1 hour, washed three times with PBS, and incubated at room temperature for 1 hour with APC-labeled Goat Anti-Mouse IgG (1:500) (Cat#405308, BioLegend) as a secondary antibody. After washing three times with PBS, the binding activity was analyzed by flow cytometry.

[0073] The results are as follows Figure 4 As shown, the anti-DLL3 antibody prepared in this invention exhibits excellent binding activity with DLL3-positive tumor cells.

[0074] Example 7: Construction of chimeric antigen receptor (CAR) lentiviral vector Using the anti-DLL3 scFv antibody (amino acid sequence as shown in SEQ ID NO:8), the positive control antibody AMG757-scFv plasmid, and the anti-BCMA antibody 388 plasmid (patent number: ZL2022111680520) as templates, the antibody gene targeting DLL3 was amplified and cloned into the lentiviral vector pSLCAR-BBz via homologous recombination to construct a second-generation CAR. This CAR mainly contains the following elements: CD8α signal peptide, antigen-binding domain (αDLL3-scFv), CD8α hinge region, CD28 transmembrane domain, 4-1BB intracellular signal transduction domain, and CD3ζ signal transduction domain. Figure 5 ).

[0075] Example 8: Detection of in vitro antitumor activity of CAR-T cells 8.1 CAR-T cell preparation Peripheral blood was collected from healthy donors and lymphocytes were isolated. The cells were stimulated with CD3 / CD28 magnetic beads for 48 h to activate and expand high-purity T cells. HEK293T cells were co-transfected with the CAR lentiviral plasmid constructed in Example 7 of this invention and the packaging plasmids psPAX2 and pMD2.G for 48 h. The culture supernatant was collected, filtered through a 0.45 μm filter, and concentrated by ultracentrifugation to obtain CAR lentiviral. The obtained CAR lentiviral was then used to infect and stimulate activated T cells to prepare CAR-T cells.

[0076] The results are as follows Figure 6 As shown, the expression efficiencies of 388-CAR, 1G1-CAR, and AMG757-CAR in T cells were 77.49%, 79.68%, and 78.25%, respectively.

[0077] 8.2 Detection of CAR-T cell antitumor activity The anti-tumor effect of CAR-T cells was detected by in vitro co-culture assay. DLL3-positive tumor cells carrying Luciferase in logarithmic growth phase (2 × 10⁻⁶ cells) were cultured together. 4 CAR-T cells were seeded in 96-well cell culture plates at an effector-to-target ratio of 5:1 and co-cultured for 24 h. After co-culturing, 3 μL of fluorescein potassium salt (15 mg / mL) was added to each well, mixed and incubated for 5 min. The absorbance at OD562 nm was then measured using a multi-functional microplate reader. The killing efficiency of CAR-T cells was calculated based on the absorbance values ​​of different treatment groups. The killing efficiency was calculated as follows: Killing efficiency % = (1 - fluorescence value of experimental group / fluorescence value of negative control group) × 100%. The irrelevant control group was supplemented with 388-CAR-T cells targeting BCMA, and the positive control group was supplemented with AMG757-CAR-T cells targeting DLL3.

[0078] The results are as follows Figure 7 As shown, the killing efficiencies of DLL3-targeting 1G1-CART, AMG757-CART, and the unrelated control BCMA-targeting 388-CART against DLL3-positive cells were 65.59%–75.15%, 87.10%–88.49%, and 2.58%–3.91%, respectively, indicating that the DLL3-targeting CAR-T cells prepared in this invention have good specific killing activity against DLL3-positive tumor cells. P <0.001).

[0079] The sequence information involved in this application is as follows.

[0080]

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody against DLL3 or its antigen-binding fragment, characterized in that, It includes: The amino acid sequences are HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4, and LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:

7.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact. Optionally, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:1~3, SEQ ID NO:5, KVS, and SEQ ID NO:6, respectively.

3. The antibody or its antigen-binding fragment according to claim 1, 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 shown in SEQ ID NO:4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7; Optionally, the antibody further includes a constant region; Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; and / or; the light chain constant region is selected from the κ-type or λ-type light chain constant region; Optionally, the antigen-binding fragment includes any one of: Fab, Fab', F(ab')2, scFv, and Fv; Optionally, when the antigen-binding fragment is scFv, the amino acid sequence of scFv is as shown in SEQ ID NO:

8.

4. An antibody conjugate, characterized in that, It includes: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3; Optionally, the antibody conjugate may further include a label, purification tag, and / or solid-phase carrier conjugated to the anti-DLL3 antibody or its antigen-binding fragment.

5. A chimeric antigen receptor, characterized in that, The antigen-binding domain of the chimeric antigen receptor includes the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

6. A CAR-T cell, characterized in that, It includes the chimeric antigen receptor as described in claim 5.

7. A reagent or kit, characterized in that, It includes: the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the antibody conjugate as described in claim 4, the chimeric antigen receptor as described in claim 5, or the CAR-T cell as described in claim 6.

8. An immunoconjugate or pharmaceutical composition, characterized in that, It includes: The antibody against DLL3 as described in any one of claims 1 to 3 or its antigen-binding fragment, or the antibody conjugate as described in claim 4, or the chimeric antigen receptor as described in claim 5, or the CAR-T cell as described in claim 6.

9. The use of the anti-DLL3 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the antibody conjugate as described in claim 4, or the chimeric antigen receptor as described in claim 5, or the CAR-T cell as described in claim 6, 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 at least one of the following: small cell lung cancer, neuroendocrine prostate cancer, prostate cancer, melanoma, gastrointestinal pancreatic neuroendocrine tumors, metastatic castration prostate cancer, large cell neuroendocrine carcinoma, small cell bladder cancer, pulmonary neuroendocrine tumor, or glioma multiforme.

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