Antibodies against dll3 and uses thereof

Abstract

The application discloses an antibody against DLL3 and application thereof, and belongs to the field of antibodies. The antibody against DLL3 comprises CDRs with amino acid sequences as shown in SEQ ID NO:1-3 and 5-7, has advantages of high affinity, high specificity and high stability, and a conjugate and a conjugate prepared based on the antibody or an antigen binding fragment thereof have excellent in-vivo tumor targeting enrichment capacity and biological distribution characteristics, can specifically target DLL3 positive tumor cells, and significantly reduce off-target toxicity, so that the prepared conjugate or conjugate has good safety and therapeutic activity. The application makes up for the deficiency of existing DLL3 targeted diagnosis and treatment technology, provides a novel scheme for precise diagnosis and targeted treatment of DLL3 positive malignant tumors, and has important clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, to an anti-DLL3 antibody and its applications. Background Technology

[0002] Lung cancer is the leading cause of cancer-related death worldwide, consistently ranking first and posing a serious threat to human health. Small cell lung cancer (SCLC), a highly aggressive subtype, accounts for approximately 13% to 15% of all lung cancers. It is characterized by rapid proliferation, early metastasis, and an extremely poor prognosis. Among these, the median survival for patients with extensive-stage SCLC is only about 12 months, the 5-year survival rate for limited-stage SCLC is less than 30%, and for metastatic advanced-stage patients, it is less than 5%, making it the deadliest type of lung cancer.

[0003] Currently, the standard first-line treatment for SCLC is a platinum-based chemotherapy regimen combined with etoposide, plus an immune checkpoint inhibitor. While this regimen has some short-term efficacy in treatment-naïve patients, it is highly prone to drug resistance, and most patients experience disease progression within a short period. Second-line and subsequent-line treatment options are extremely limited; traditional drugs such as topotecan have low efficacy and significant side effects, resulting in poor overall clinical benefit. Although new strategies such as immunotherapy and anti-angiogenic therapy are constantly emerging, they still cannot effectively solve the core dilemmas of rapid relapse, high drug resistance rates, and limited effectiveness of subsequent-line treatments. There is an urgent clinical need to develop novel targeted drugs that are highly effective, low in toxicity, and long-acting.

[0004] Delta-like ligand 3 (DLL3), a key ligand in the Notch signaling pathway, is a type I transmembrane glycoprotein. Its extracellular DSL domain specifically binds to Notch receptors, regulating tumor proliferation, differentiation, and metastasis. Studies have shown that DLL3 is almost not expressed in normal adult tissues, but is abnormally highly expressed on the surface of over 80% of SCLC tumor cells, and its expression level is significantly correlated with poor patient prognosis. This distribution characteristic of "tumor-specific high expression and normal tissue low expression" makes DLL3 an ideal target and research hotspot for precision diagnosis and treatment of SCLC. In addition, reports show that DLL3 is also expressed to varying degrees in large cell neuroendocrine carcinoma (LCNEC), neuroendocrine carcinoma (NEC), neuroendocrine prostate cancer (NEPC), bladder neuroendocrine tumors, Merkel cell carcinoma, medullary thyroid carcinoma, glioma, medulloblastoma, melanoma, pituitary neuroendocrine tumors, and gastrointestinal and pancreatic neuroendocrine carcinomas, making DLL3 an ideal target for precision diagnosis and treatment.

[0005] Targeted therapies for DLL3 mainly include three major directions: antibody-drug conjugates (ADCs), bispecific T-cell conjugates (BiTEs), and CAR-T cell therapy. However, all existing technologies have significant limitations. Rovalpituzumab tesirine (Rova-T), the world's first DLL3-targeted ADC to enter Phase III trials, failed to meet its survival endpoint in the pivotal Phase III trial due to insufficient antibody affinity, poor linker stability, and off-target toxicity, leading to the termination of its development. While the BiTE drug Tarlatamab (AMG 757) has recently been approved for second-line treatment of SCLC and has shown some efficacy in relapsed patients, its short half-life, frequent dosing requirements, and tendency to induce cytokine release syndrome limit its long-term efficacy due to T-cell exhaustion, resulting in a still high relapse rate. In recent years, targeted radionuclide therapy (RDC) has emerged in DLL3-positive tumors, based on... 89 Zr、²² 5 Ac、¹ 77 Antibodies labeled with radionuclides such as Lu have shown potential in both imaging and therapeutic applications, but they also face key bottlenecks such as antibody affinity, specificity, in vivo distribution, and toxicity to normal organs.

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

[0007] The purpose of this invention is to provide an anti-DLL3 antibody and its application.

[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 and LCDR1, LCDR2 and LCDR3 in the light chain variable region; the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:4 and 8, respectively.

[0009] Secondly, embodiments of the present invention provide a biomaterial selected from any of the following: (1) An isolated nucleic acid molecule that encodes the antibody or its antigen-binding fragment as described in the foregoing embodiments; (2) A recombinant vector comprising the isolated nucleic acid molecule; (3) Recombinant cells, including the recombinant vector.

[0010] Thirdly, embodiments of the present invention provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in the foregoing embodiments and its conjugated portion; the conjugated portion comprising at least one of a marker, a purification tag, and a solid-phase carrier.

[0011] Fourthly, embodiments of the present invention provide a composition comprising: the antibody or its antigen-binding fragment described in the foregoing embodiments, or the antibody conjugate described in the foregoing embodiments.

[0012] Fifthly, embodiments of the present invention provide a reagent or kit comprising: the antibody or its antigen-binding fragment described in the foregoing embodiments or the antibody conjugate described in the foregoing embodiments.

[0013] In a sixth aspect, embodiments of the present invention provide the use of the antibody or antigen-binding fragment thereof as described in the foregoing embodiments or the antibody-drug conjugate as described in the foregoing embodiments in the preparation of any of the following: (I) a product for detecting DLL3; (II) a medicament for the prevention or treatment of DLL3-positive tumors.

[0014] In a seventh aspect, embodiments of the present invention provide a method for preparing the antibody or its antigen-binding fragment as described in the foregoing embodiments, comprising: culturing the recombinant cells described in the foregoing embodiments.

[0015] The present invention has the following beneficial effects: (1) The anti-DLL3 antibody provided by the present invention has excellent antigen binding affinity and high specificity, can effectively recognize and bind to DLL3 positive tumor cell surface antigens, and has low cross-reactivity with normal tissues and good safety.

[0016] (2) The anti-DLL3 antibody or its antigen-binding fragment and its derivative products (conjugates / conjugates / derivatives, etc.) provided by the present invention can be used to prepare drugs for the prevention, diagnosis or treatment of various DLL3 positive tumors, especially in refractory tumors such as small cell lung cancer that have failed traditional treatments, which have important clinical application value.

[0017] (3) This invention provides new candidate drugs for DLL3 targeted diagnosis and treatment, which helps to overcome the technical bottleneck of poor efficacy of existing DLL3 targeted therapy and has good industrialization and application prospects. Attached Figure Description

[0018] 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.

[0019] Figure 1 In this example, SDS-PAGE was used to detect the expression and purification of the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1; 1: reduced; 2: non-reduced.

[0020] Figure 2 In this example, the binding specificity of the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 was analyzed using indirect ELISA. Figure 3 The example uses flow cytometry to detect the binding epitopes of the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1; Figure 4 In this example, flow cytometry was used to detect the species cross-reactivity of the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1; Figure 5 In this example, SDS-PAGE was used to detect and purify the expression of the anti-DLL3 22C3-scFv-hFc recombinant protein; 1: reduced; 2: non-reduced; Figure 6 In this example, indirect ELISA was used to analyze the affinity activity of the anti-DLL3 antibody; Figure 7 In this example, flow cytometry was used to analyze the binding activity of anti-DLL3 human-mouse chimeric antibodies 22C3-hIgG1 and 22C3-scFv-hFc to DLL3-positive cells; A: SHP-77 cells; B: NCI-H82 cells; C: Binding of anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 to DLL3-H1975 cells.

[0021] Figure 8 The following figures illustrate the in vivo distribution of the anti-DLL3 antibody-Cy7 conjugate in a DLL3-positive tumor mouse xenograft model: A: DLL3-H1975-ff.Luc cell model; B: Visceral distribution of the anti-DLL3 antibody-Cy7 conjugate in the DLL3-H1975-ff.Luc cell model 336 h after administration; C: SHP-77 cell model; LP: Lateral position; SP: Supine position. Detailed Implementation

[0022] 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.

[0023] The term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance containing the antibody's CDR (Cellular Receptor Diagram), lacking some amino acids present in the full-length chain, but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest unit of antibody recognition. The antigen-binding fragments of the aforementioned antibodies can bind to the same antigen as the parent antibody.

[0024] Antigen-binding fragments of antibodies typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments. Antigen-binding fragments can also be obtained using recombinant genetic techniques known to those skilled in the art or synthesized using, for example, automated peptide synthesizers, such as those sold by Applied BioSystems.

[0025] The terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody. The heavy chain complementarity-determining region is denoted by HCDR, and the three CDRs contained in the heavy chain variable region include HCDR1, HCDR2, and HCDR3; the light chain complementarity-determining region is denoted by LCDR, and the three CDRs contained in the light chain variable region include LCDR1, LCDR2, and LCDR3.

[0026] The term “treatment” 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 stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0027] 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.

[0028] The term "suitable conditions" refers to conditions suitable for the expression of the antibody or its antigen-binding fragment as described in any embodiment of the present invention. It will be readily understood by those skilled in the art that suitable conditions for the expression of the antibody or its antigen-binding fragment include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody or its antigen-binding fragment according to the specific environment of their laboratory.

[0029] 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 and LCDR1, LCDR2 and LCDR3 in the light chain variable region; the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:4 and 8, respectively.

[0030] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

[0031] In some embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:1, 2, 3, 5, 6 and 7 respectively; In some embodiments, the heavy chain variable region and the light chain variable region further include a skeleton region.

[0032] In this article, the "backbone region" or "FR region" refers to the region in the variable region of the antibody heavy or light chain, excluding the CDRs. The backbone region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4). The heavy chain backbone region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 backbone regions. The heavy chain variable region is obtained by arranging the following CDRs with FRs (from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. The light chain backbone region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 backbone regions. The light chain variable region is obtained by arranging the following CDRs with FRs (from the amino terminus to the carboxyl terminus): LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0033] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, with amino acid sequences as shown in SEQ ID NO:4 and 8, respectively.

[0034] In some embodiments, the antibody or its antigen-binding fragment further includes a constant region.

[0035] 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.

[0036] In some embodiments, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0037] In some embodiments, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and / or the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0038] In some embodiments, the antibody is a single-chain scFv antibody, a humanized antibody, a chimeric antibody, or a fully human antibody, and the antigen-binding fragment is selected from Fab, Fab', F(ab')2, scFv, or Fv.

[0039] In some embodiments, the amino acid sequence of the scFv is shown in SEQ ID NO:9.

[0040] On the other hand, embodiments of the present invention provide a biomaterial selected from any of the following: (1) An isolated nucleic acid molecule that encodes the antibody or its antigen-binding fragment as described in any of the foregoing embodiments; (2) A recombinant vector comprising the isolated nucleic acid molecule; (3) Recombinant cells, including the recombinant vector.

[0041] In some embodiments, 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 a target DNA fragment into a host cell 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 a target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package a target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). These vectors, after entering the host cell, can express the target gene.

[0042] In some embodiments, the recombinant cells include at least one of prokaryotic cells, eukaryotic cells, and bacteriophages. The prokaryotic cells may be Escherichia coli, Streptomyces, or Bacillus subtilis, etc. The eukaryotic cells include mammalian cells (such as 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, or PerC6 cells), yeast cells (such as Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces hansenii, or Candida albicans), or insect cells. Among these, 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 provide an antibody conjugate comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments and its conjugated portion; The coupling component includes at least one of a marker, a purification tag, and a solid-phase carrier.

[0044] In some embodiments, a marker refers to a substance that has properties that can be directly observed by the naked eye or detected or probed by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0045] In some embodiments, the markers are selected from, but are not limited to, metal ions, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, electron-dense markers, adamantane, and nanoparticle markers.

[0046] In some embodiments, the fluorescent dye is selected from, but not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy...). 5. Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0047] In some embodiments, the enzyme is selected from, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0048] In some embodiments, the chemiluminescent reagent is selected from, but not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0049] In some embodiments, the nanoparticle-type markers are selected from, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0050] In some embodiments, the purification tag includes His tag, GST tag, FLAG tag, Fc tag, etc., to facilitate antibody separation and purification.

[0051] In some embodiments, the solid support includes microspheres, latex particles, microfluidic chips, magnetic beads, microplates, or nitrocellulose membranes, etc., for antibody immobilization applications.

[0052] On the other hand, embodiments of the present invention provide a composition comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or the antibody conjugate as described in any of the foregoing embodiments.

[0053] In some embodiments, the composition further includes a pharmaceutically acceptable carrier.

[0054] In some embodiments, the composition is a pharmaceutical composition or an immunoconjugate.

[0055] 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.

[0056] In some embodiments, the pharmaceutical composition represents a combination of at least one drug combined together to achieve a specific purpose, and optionally a pharmaceutically acceptable carrier or excipient. 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.

[0057] In some embodiments, the pharmaceutical composition further includes at least one of a pharmaceutically acceptable carrier, an excipient, a diluent, or an excipient. The pharmaceutically acceptable carrier includes buffers, stabilizers, preservatives, isotonic agents, or solubilizers.

[0058] On the other hand, embodiments of the present invention provide a reagent or kit comprising: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments, or the antibody-drug conjugate as described in any of the foregoing embodiments.

[0059] On the other hand, embodiments of the present invention provide the use of 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, in the preparation of any of the following: (I) Products that test DLL3; (II) Drugs for the prevention or treatment of DLL3-positive tumors.

[0060] 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-resistant prostate cancer, large cell neuroendocrine carcinoma, small cell bladder cancer, pulmonary neuroendocrine tumor, and glioblastoma multiforme.

[0061] In some embodiments, the drug may be administered via intravenous injection, subcutaneous injection, intradermal injection, intratumoral injection, or infusion. The dosage and administration regimen of the drug are determined based on factors such as the patient's age, weight, disease state, and any concurrent medication use.

[0062] Furthermore, embodiments of the present invention provide a method for preparing the antibody or antigen-binding fragment thereof described in any of the foregoing embodiments, comprising: culturing the recombinant cells described in any of the foregoing embodiments; and, under suitable conditions, enabling the cells to effectively express the antibody or antigen-binding fragment thereof described in any of the foregoing embodiments.

[0063] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0064] Example 1: Preparation of recombinant DLL3 protein and construction of stable cell lines expressing human DLL3 antigen 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.

[0065] 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.

[0066] 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 NCI-H1975 human lung adenocarcinoma cells. Stable NCI-H1975 cells expressing DLL3 were obtained by flow cytometry sorting and named DLL3-H1975 cells.

[0067] 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 initial 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 two 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 once the antibody titer met the requirements.

[0068] 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.

[0069] ELISA screening: DLL3 recombinant protein was coated onto 96-well microplates at 50 ng / well and incubated overnight at 4°C. The next day, the plates were blocked with 5% skim milk powder. 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 color development, the reaction was terminated with 2M H2SO4. The OD450 nm value was read, and positive clones were screened.

[0070] Immunofluorescence: DLL3-H1975 cells and wild-type NCI-H1975 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, washed three times with PBS, and Alexa Fluor 594-labeled Goat Anti-Mouse IgG (1:500) was used as the secondary antibody. The plates were incubated at room temperature for 1 hour, washed three times with PBS, and images were collected and analyzed using a fluorescence microscope to identify positive clones.

[0071] Flow cytometry: DLL3-H1975 cells and wild-type NCI-H1975 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.

[0072] 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.

[0073] 2.3 Amplification of antibody VH and VL genes First, total RNA was extracted from hybridoma cells according to the instructions. cDNA was then obtained by reverse transcription using the RNA as a template. Using the cDNA as a template, the VH and VL genes were amplified using mouse antibody VH and VL amplification primers. These 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. The VH (amino acid sequence shown in SEQ ID NO:4) and VL (amino acid sequence shown in SEQ ID NO:8) sequences were obtained by alignment with the IgBlast database.

[0074] Example 3: Preparation of anti-DLL3 recombinant chimeric antibody Using the plasmid containing the anti-DLL3 antibody gene from step 2.3 of Example 2 as a template, the VH and VL genes were amplified and constructed into the eukaryotic expression vectors pcDNA3.1-hCH1-hCH2-hCH3 and pcDNA3.1-hCK, respectively, via homologous recombination. After sequencing confirmed, the plasmids were extracted and transfected into HEK293T cells at a mass ratio of 1:1. After 5 days of expression, the supernatant was collected and purified using Protein G packing material via affinity chromatography to obtain the human-mouse chimeric antibody 22C3-hIgG1 (e.g., ...). Figure 1 (As shown).

[0075] Example 4: Specificity of indirect ELISA detection of anti-DLL3 antibody The antigen-specific binding activity of the anti-DLL3 antibody in Example 3 was detected by indirect ELISA: DLL3-His recombinant protein and a series of unrelated antigens (B7H3-His, MSLN-His, CD16a-His, CDH3-His and CD117-His) were coated on a 96-well microplate at a concentration of 0.5 μg / mL and incubated overnight at 4°C. After blocking with 5% skim milk powder, the antibodies to be tested (including the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 prepared in Example 3 of this invention, and the positive control antibody, Tarlatamab (AMG757), which binds to human DLL3 and is named AMG757 scFv-hFc in this invention, with an antibody concentration of 0.1 μg / mL) were added to the above-mentioned ELISA plate and incubated at 37°C for 1 hour. The plate was washed three times with PBST buffer, and 100 μL of HRP-labeled goat anti-human secondary antibody (1:5000) was added to each well. The plate was incubated at 37°C for 1 hour. After washing three times with PBST buffer, TMB chromogenic solution was added and reacted for 5 minutes. The reaction was then terminated by adding 2 M H2SO4 solution, and the absorbance was measured at 450 nm using an ELISA reader. The results are as follows: Figure 2 As shown, the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 binds to the DLL3 protein and does not react with other unrelated antigens, indicating that antibody 22C3-hIgG1 has good specific binding activity to the DLL3 protein.

[0076] Example 5: Analysis of anti-DLL3 antibody binding epitopes Based on the classification of extracellular domains of human DLL3 in the Uniprot database, the full-length DLL3 sequence (27-618 AA) and truncated sequences containing different domains (DSL: 176-618 AA; EGF1: 216-618 AA; EGF2: 274-618 AA; EGF3: 312-618 AA; EGF4: 353-618 AA; EGF5: 391-618 AA; EGF6: 429-618 AA) were cloned into the eukaryotic expression vector pcDNA3.1 containing the EGFP tag, and transfected into HEK293T cells. Cells were collected 48 hours after transfection.

[0077] The transfected cells were incubated with anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 and an irrelevant control antibody (Nb26 targeting CLEC12A, application number CN2023116121645) at 37°C for 1 hour. After washing three times with PBS, APC-labeled anti-human secondary antibody (1:500) was added, and the cells were incubated at 37°C for 1 hour. After washing with PBS, the cells were analyzed by flow cytometry. The results are as follows: Figure 3As shown, the binding region of antibody 22C3 is located in the N-terminal domain of the DLL3 protein, specifically amino acids 27 to 176.

[0078] Example 6: Species Cross-Reactivity Analysis of Anti-DLL3 Antibody To verify the species cross-reactivity of the anti-DLL3 antibody 22C3 of this invention, eukaryotic expression vectors for human, mouse, and cynomolgus monkey DLL3 proteins were constructed: the three DLL3 genes were cloned into eukaryotic expression vectors containing EGFP marker proteins, respectively, to obtain recombinant plasmids pCMV-hDLL3-EGFP, pCMV-mDLL3-EGFP, and pCMV-cyDLL3-EGFP. These recombinant plasmids were transfected into HEK293T cells, and cells were collected 48 hours after transfection for subsequent analysis. HEK293T cells transfected with the test antibody 22C3-hIgG1 (5 μg / mL) were co-incubated. An irrelevant control antibody group (Nb26-hFc) and a positive antibody group (AMG757-scFv-hFc) were also included. Incubation was performed at 37°C for 1 hour. After washing three times with PBS, APC-labeled goat anti-human secondary antibody (1:500) was added, and incubation was performed at 37°C in the dark for 1 hour. After washing three times with PBS, the APC fluorescence signal intensity was detected by flow cytometry. Experimental results are as follows: Figure 4 As shown, the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 of the present invention can cross-react with mouse and cynomolgus monkey DLL3 proteins, indicating that the antibody has cross-species recognition capabilities.

[0079] Example 7: Preparation of anti-DLL3 protein scFv antibody 7.1 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 (amino acid sequence as shown in SEQ ID NO:9) by overlap PCR, and then ligated it into the pcDNA3.1-hFc vector 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.

[0080] 7.2 Expression and purification of scFv-hFc antibody The pcDNA3.1-22C3-scFv-hFc recombinant plasmid prepared in step 7.1 was transfected into HEK293T cells using PEI. After 5 days of expression, the supernatant was collected, and the recombinant antibody was obtained by affinity chromatography using Protein G packing material. Figure 5 (As shown).

[0081] Example 8: Indirect ELISA detection of anti-DLL3 antibody binding activity DLL3-His recombinant protein was coated into 96-well microplates at a rate of 100 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 the recombinant antibodies prepared in Examples 3 and 7, as well as the positive control antibody AMG757-scFv-hFc, were added to each well. The plates 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 applied for 5 min, and the reaction was terminated with 2M H2SO4. The absorbance at OD450 nm was then read. The results are as follows: Figure 6 As shown, the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 (EC50=15.51 ng / mL), scFv antibody 22C3-scFv-hFc (EC50=16.29 ng / mL), and positive control antibody AMG757-scFv-hFc (EC50=20.92 ng / mL) prepared in this invention all have good binding activity with DLL3 protein.

[0082] Example 9: Biacore detection of anti-DLL3 antibody binding activity The anti-DLL3 antibodies 22C3-hIgG1, 22C3-scFv-hFc recombinant antibodies, and positive control antibody (AMG757-scFv-hFc) prepared in this invention were used to bind to the DLL3-His antigen coated on a CM5 chip using a Biacore 8k instrument. The results are shown in Table 1. The affinity of the anti-DLL3 recombinant antibodies 22C3-hIgG1 and 22C3-scFv-hFc to the DLL3 protein was 10. -11 M is significantly superior to the positive control antibody AMG757-scFv-hFc.

[0083] Table 1. Affinity and kinetics of anti-DLL3 antibody binding to DLL3 protein.

[0084] Example 10: Flow cytometry detection of the binding of anti-DLL3 antibody to tumor cells Small cell lung cancer cell lines SHP-77 and NCI-H82, which are positive for DLL3 expression, and NCI-H1975 cells (stably expressing DLL3) prepared in Example 1 (DLL3-H1975) were collected in flow cytometry tubes. After blocking with 2% BSA, the cells were resuspended with anti-DLL3 antibodies prepared in Examples 3 and 7 and an irrelevant control antibody (2 μg / mL), incubated at room temperature for 1 hour, washed three times with PBS, and incubated with APC-labeled Goat Anti-human IgG (1:500) as a secondary antibody at room temperature for 1 hour, washed three times with PBS, and the binding activity was analyzed by flow cytometry. The results are as follows: Figure 7 As shown, the anti-DLL3 antibody prepared in this invention has good binding activity with DLL3-positive tumor cells.

[0085] Example 11: Preparation of anti-DLL3 antibody-Cy7 conjugate The anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1, scFv antibody 22C3-scFv-hFc, positive control antibody AMG757-scFv-hFc, and irrelevant control antibody Nb26-hFc prepared in Examples 3 and 7 were conjugated with Cy7 fluorescent dye according to the instructions. After conjugation, desalting and recovery were performed using a PD-10 desalting column.

[0086] Example 12: In vivo distribution analysis of anti-DLL3 antibody The NCI-H1975 cells DLL3-H1975-ff.Luc, which stably express DLL3 as prepared in Example 1, and the small cell lung cancer cell line SHP-77 (3E6 / mouse) were subcutaneously inoculated into nude mice. After tumor formation, the anti-DLL3 antibody-Cy7 conjugates (22C3-hIgG1-Cy7, 22C3-scFv-hFc-Cy7, AMG757-scFv-hFc-Cy7) prepared in Example 11, as well as the irrelevant control antibody-Cy7 conjugate (isotype), were injected into the mice via the tail vein at a rate of 100 μg per mouse. In vivo imaging was performed at different time points after administration to detect the in vivo biodistribution of the anti-DLL3 antibody-Cy7 conjugates and to analyze the targeting and tumor enrichment of the anti-DLL3 antibody. The results showed that the anti-DLL3 human-mouse chimeric antibody 22C3-hIgG1 and scFv antibody 22C3-scFv-hFc prepared in this invention were effective in DLL3-H1975-ff.Luc cells ( Figure 8 (A) and SHP-77 cells ( Figure 8In the C) mouse xenograft model, both antibodies showed excellent tumor targeting and in vivo distribution, significantly superior to the positive control antibody AMG757-scFv-hFc. Furthermore, in the DLL3-H1975-ff.Luc cell model, mice were sacrificed 336 hours after tail vein injection of the anti-DLL3 antibody-Cy7 conjugate (22C3-hIgG1-Cy7). Visceral and subcutaneous tumors were removed and observed in vivo using an in vivo imaging system. The results showed that the anti-DLL3 antibody-Cy7 conjugate (22C3-hIgG1-Cy7) was mainly enriched in tumor tissue, while the irrelevant control antibody was mainly distributed in the liver. Figure 8 (B in the middle).

[0087] In summary, the anti-DLL3 antibody prepared by this invention has high affinity, high specificity, excellent biodistribution in vivo, and high tumor enrichment efficiency. It can be used to prepare drugs for the prevention, diagnosis, or treatment of various DLL3-positive tumors, and has good prospects for industrialization and application.

[0088] The sequence information involved in this application is as follows:

[0089] 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 HCDR1, HCDR2 and HCDR3 in the heavy chain variable region and LCDR1, LCDR2 and LCDR3 in the light chain variable region; the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:4 and 8 respectively.

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 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, 2, 3, 5, 6 and 7 respectively; Optionally, the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, with amino acid sequences as shown in SEQ ID NO:4 and 8, respectively.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment 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 constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and / or the light chain constant region is selected from the κ-type or λ-type light chain constant region.

4. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody is a single-chain scFv antibody, a humanized antibody, a chimeric antibody, or a fully human antibody, and the antigen-binding fragment is selected from Fab, Fab', F(ab')2, scFv, or Fv; Optionally, the amino acid sequence of the scFv is shown in SEQ ID NO:

9.

5. A biomaterial, characterized in that, It is selected from any of the following: (1) An isolated nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4; (2) A recombinant vector comprising the isolated nucleic acid molecule; (3) Recombinant cells, including the recombinant vector.

6. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1 to 4, characterized in that, It includes: Cultivate the recombinant cells as described in claim 5.

7. An antibody conjugate, characterized in that, It includes: The antibody or its antigen-binding fragment and its conjugated portion as described in any one of claims 1 to 4; The coupling component includes at least one of a marker, a purification tag, and a solid-phase carrier.

8. A composition, characterized in that, It includes: The antibody or its antigen-binding fragment according to any one of claims 1 to 4, or the antibody conjugate according to claim 7.

9. A reagent or kit, characterized in that, It includes: The antibody or its antigen-binding fragment according to any one of claims 1 to 4, or the antibody conjugate according to claim 7.

10. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, or the antibody conjugate as described in claim 7, in the preparation of any one of the following; (I) Products that test DLL3; (II) Drugs for the prevention or treatment of DLL3-positive tumors; Optionally, 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-resistant prostate cancer, large cell neuroendocrine carcinoma, small cell bladder cancer, pulmonary neuroendocrine tumor, and glioblastoma multiforme.

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