Antibodies targeting dll3 and uses thereof

Abstract

The application discloses an antibody targeting DLL3 and application thereof, and relates to the field of biological medicines. Specific anti-DLL3 nanobodies are screened in the embodiment of the application, and a bispecific antibody constructed from the anti-human DLL3 nanobody or humanized nanobody and an anti-human CD3 antibody is prepared, the obtained bispecific antibody has good antitumor activity, can be applied to preparation of at least one drug for preventing or treating a DLL3 expression abnormality related tumor or cancer disease including small cell lung cancer, and has wide application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to antibodies targeting DLL3 and their applications. Background Technology

[0002] DLL3 (Delta-like canonical Notch ligand 3) is a ligand for the Notch signaling pathway, belonging to the DSL (Delta / Serrate / Lag-2) family. DLL3 is a single-pass transmembrane protein composed of an extracellular, transmembrane, and intracellular region. The extracellular region contains the DSL domain and six epidermal growth factor (EGF) repeat sequences, while the intracellular region is shorter and primarily involved in intracellular signal transduction. The DSL gene sequence is highly conserved within the ligand family and is essential for Notch receptor binding. DLL3 is highly expressed in SCLC (small cell lung cancer), especially in subtypes with neuroendocrine characteristics (such as SCLC-A and SCLC-N). Studies have shown that DLL3 expression in SCLC cells is significantly higher than in non-small cell lung cancer (NSCLC) and other normal tissues. The high expression of DLL3 in SCLC cells makes it an ideal therapeutic target. Treatment strategies targeting DLL3 include antibody-drug conjugates (ADCs), bispecific T-cell conjugates (BiTEs), and chimeric antigen receptor T-cell (CAR-T) therapy. High DLL3 expression is associated with poor prognosis in SCLC patients and may serve as a prognostic biomarker to help predict patient survival and treatment response. DLL3-targeted therapies have shown some efficacy in clinical trials. For example, the DLL3-targeting bispecific T-cell conjugate tarlatamab demonstrated an objective response rate of approximately 40% in relapsed SCLC patients, with some patients experiencing a response duration exceeding 6 months. With further research into DLL3 function and regulatory mechanisms, and the continuous development of novel DLL3-targeting drugs, DLL3 holds promise as an important target for SCLC treatment.

[0003] Currently, the market still lacks the development of high-performance anti-DLL3 antibodies.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide antibodies targeting DLL3 and their applications.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide an anti-DLL3 nanobody comprising: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4 or 8.

[0008] Secondly, embodiments of the present invention provide an antibody or an antigen-binding fragment thereof, comprising: the nanobody described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide an isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes a nanobody as described in the foregoing embodiments or an antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide a host cell comprising the recombinant vector described in the foregoing embodiments.

[0011] Fifthly, embodiments of the present invention provide a method for preparing an antibody, which includes: culturing the host cells described in the foregoing embodiments.

[0012] In a sixth aspect, embodiments of the present invention provide a conjugate comprising: the nanobody described in the foregoing embodiments or the antibody or antigen-binding fragment thereof described in the foregoing embodiments.

[0013] In a seventh aspect, embodiments of the present invention provide an immunoconjugate or pharmaceutical composition comprising: the nanobody described in the foregoing embodiments or the antibody or antigen-binding fragment described in the foregoing embodiments.

[0014] Eighthly, embodiments of the present invention provide the application of nanobodies as described in the foregoing embodiments, or antibodies or antigen-binding fragments thereof as described in the foregoing embodiments, or isolated nucleic acids or recombinant vectors containing said isolated nucleic acids as described in the foregoing embodiments, or host cells or conjugates as described in the foregoing embodiments, in the detection of DLL3 protein for purposes other than disease diagnosis or treatment.

[0015] Ninthly, embodiments of the present invention provide the use of nanobodies as described in the foregoing embodiments, or antibodies or antigen-binding fragments thereof as described in the foregoing embodiments, isolated nucleic acids as described in the foregoing embodiments, or recombinant vectors containing said isolated nucleic acids, or host cells as described in the foregoing embodiments, or conjugates as described in the foregoing embodiments, in the preparation of products for the prevention, diagnosis, treatment, or adjunctive treatment of diseases targeting DLL3.

[0016] The present invention has the following beneficial effects:

[0017] In the embodiments of the present invention, specific anti-DLL3 nanobodies were screened, and bispecific antibodies constructed from anti-human DLL3 nanobodies or humanized nanobodies and anti-human CD3 antibodies were prepared. The obtained bispecific antibodies all have good anti-tumor activity and can be used to prepare at least one drug for the prevention or treatment of tumors or cancers related to abnormal DLL3 expression, including small cell lung cancer, and have a wide range of applications. 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 embodiment of the invention, SDS-PAGE was used to detect the expression of DLL3-hFc-His recombinant protein. 1: reduced; 2: non-reduced.

[0020] Figure 2 In this embodiment of the invention, indirect ELISA was used to analyze the binding activity of anti-DLL3-Nb to DLL3 antigen;

[0021] Figure 3 Schematic diagram of the expression pattern of full-length human DLL3 and different domains fused with EGFP;

[0022] Figure 4 In this embodiment of the invention, flow cytometry is used to detect the binding domain of anti-DLL3-Nb to human DLL3.

[0023] Figure 5 Results of amino acid sequence similarity comparison of the extracellular region of DLL3 in humans, mice, and cynomolgus monkeys;

[0024] Figure 6 This embodiment of the invention utilizes flow cytometry to detect the binding of anti-DLL3-Nb to DLL3 in humans, mice, and cynomolgus monkeys;

[0025] Figure 7 In this embodiment of the invention, flow cytometry was used to detect the binding of anti-DLL3-Nb to SHP-77, NCI-H82 and DLL3-HeLa cells;

[0026] Figure 8 This invention provides an example of detecting the in vitro killing activity of anti-DLL3 / CD3 BiTE against DLL3-positive endogenous SCLC cell lines.

[0027] Figure 9In this embodiment of the invention, the in vivo antitumor effect of anti-DLL3 / CD3 BiTE was evaluated using an NCG mouse xenograft model. Detailed Implementation

[0028] 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.

[0029] On one hand, embodiments of the present invention provide an anti-DLL3 nanobody, which includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:4 or 8.

[0030] In some embodiments, HCDR1, HCDR2, and HCDR3 are defined by any one or a combination of systems selected from Kabat, Chothia, IMGT, AbM, or Contact.

[0031] In some embodiments, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:1~3 or 5~7, respectively.

[0032] In some embodiments, the heavy chain variable region further includes a backbone region. Specifically, the structure of the heavy chain variable region of the nanobody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.

[0033] In some embodiments, the amino acid sequence of the heavy chain variable region of the nanobody is as shown in SEQ ID NO:4, 8, 9 or 10.

[0034] On the other hand, embodiments of the present invention provide an antibody or an antigen-binding fragment thereof, which includes the nanobody described in any of the foregoing embodiments.

[0035] Nanobodies have a large number of hydrophilic residues on their surface, maintaining a strict monomeric structure, and can bind to their antigens with high specificity and high affinity solely in this monomeric form. Due to their small molecular weight and single-gene encoding, nanobodies are easily genetically engineered, and multiple nanobodies can be polymerized through short linker sequences to form multivalent or multispecific antibody structures.

[0036] In some embodiments, the antibody is selected from any one of: bivalent antibody, bispecific antibody, multivalent antibody, multispecific antibody, fusion antibody, and chimeric antibody.

[0037] In this article, "bivalent antibody" or "multivalent antibody" refers to a polymer of a monovalent antibody that recognizes the same epitope and has a higher antigen affinity than the corresponding monovalent antibody.

[0038] The "bispecific antibody" or "multispecific antibody" mentioned in this article is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, and has a stronger antigen recognition ability than the corresponding monovalent antibody. Optionally, multispecific antibodies can be trispecific antibodies, tetraspecific antibodies, etc.

[0039] The “chimeric antibody” mentioned in this article is usually an antibody formed by fusing the variable region of a non-human antibody with the constant region or backbone region of a human antibody, which can reduce the immune response induced by non-human antibodies.

[0040] Fusion antibodies include fusion nanobodies, which include, but are not limited to, new fusion molecules formed by combining with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering techniques, such as enzymes, antimicrobial peptides, or imaging substances that can prolong their half-life.

[0041] In some embodiments, the fusion antibody is formed by fusing a constant region of an antibody with a nanobody as described in any of the foregoing embodiments.

[0042] In some implementations, the constant region of the antibody can be the heavy chain constant region of the antibody, specifically selected from the heavy chain constant region of human antibodies, mouse heavy chain constant region, rabbit heavy chain constant region, sheep heavy chain constant region, or monkey heavy chain constant region.

[0043] In some embodiments, the heavy chain constant region of the human antibody is selected from the heavy chain constant regions of any one of hIgG1, hIgG2, hIgG3, and hIgG4, or a mutation thereof. Using the heavy chain constant region of IgG1 as the constant region of the anti-DLL3 nanobody results in an extremely high inhibition rate against target cells and maintains good binding activity with the antigen.

[0044] In some embodiments, the bispecific antibody includes: the nanobody and the anti-CD3 antibody described in any of the foregoing embodiments.

[0045] In some embodiments, the bispecific antibody is constructed by linking the nanobody described in any of the foregoing embodiments with the anti-CD3 antibody via a flexible linker G4S.

[0046] In some embodiments, the amino acid sequence of the bispecific antibody is any one of SEQ ID NO:12~15.

[0047] In some embodiments, the antigen-binding fragment includes any one selected from the antibody's F(ab')2, Fab', Fab, Fv, and scFv, provided that they exhibit the desired antigen-binding activity.

[0048] The aforementioned antigen-binding fragments, also known as functional 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 herein, that these functional fragments of 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 functional fragments.

[0049] The aforementioned antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0050] On the other hand, embodiments of the present invention also provide an isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes a nanobody as described in any of the foregoing embodiments or an antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0051] The isolated nucleic acid encodes a nanobody as described in any of the foregoing embodiments or an antibody or antigen-binding fragment as described in any of the foregoing embodiments. Considering the degeneracy of codons, the gene sequence encoding the above-mentioned antibody or antigen-binding fragment can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody or antigen-binding fragment; alternatively, the gene can be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the antibody expression efficiency.

[0052] In some embodiments, the isolated nucleic acid has 80% identity with any one of SEQ ID NO:16-19.

[0053] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged into a virus) into host cells for target gene expression through transformation, transfection, or transduction. One type of vector is a plasmid, i.e., 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 the viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0054] On the other hand, embodiments of the present invention also provide a host cell comprising the recombinant vector described in any of the foregoing embodiments.

[0055] 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*, *Saccharomyces hansenii*, *Candida*, some insect cells, and plant 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.

[0056] Based on the disclosure of the amino acid sequence of the antibody or its functional fragment in this invention, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment. For example, the antibody or its functional fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0057] On the other hand, embodiments of the present invention also provide a method for preparing an antibody, which includes: culturing the host cells described in any of the foregoing embodiments.

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

[0059] In some embodiments, the conjugate further includes a conjugation portion conjugated to the nanobody or the antibody or its antigen-binding fragment.

[0060] In some embodiments, the coupling portion includes any one or more of the following: a protein tag for purification, a marker for detection or tracing, and a solid-phase support.

[0061] In some embodiments, the marker is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0062] On the other hand, embodiments of the present invention also provide an immunoconjugate or pharmaceutical composition, the active ingredient of which includes: the nanobody described in any of the foregoing embodiments or the antibody or its antigen-binding fragment described in any of the foregoing embodiments.

[0063] 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.

[0064] The term "pharmaceutical composition" as used in this invention refers to a combination of at least one drug substance and optionally a pharmaceutically acceptable carrier or excipient, combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes combinations that are separate in time and / or space, provided that they can work together to achieve the objectives of this invention.

[0065] In some embodiments, the pharmaceutical composition further includes at least one of a pharmaceutical excipient, a carrier, and a diluent.

[0066] The aforementioned carriers are pharmaceutically acceptable carriers, including but not limited to fillers, lubricants, disintegrants, binders, and flow aids.

[0067] The pharmaceutically acceptable carriers include, but are not limited to, one or a combination of polyvinylpyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methylcellulose and its derivatives, ethylcellulose and its derivatives, hydroxypropylcellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, dicalcium phosphate, calcium stearate, sodium stearoyl fumarate, silicon dioxide, titanium dioxide, talc, and indigo.

[0068] On the other hand, embodiments of the present invention also provide the application of nanobodies as described in any of the foregoing embodiments, or antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, or isolated nucleic acids or recombinant vectors containing said isolated nucleic acids as described in any of the foregoing embodiments, or host cells or conjugates as described in any of the foregoing embodiments, in the detection of DLL3 protein for non-disease diagnosis or treatment purposes.

[0069] There are many situations where testing is conducted for purposes other than disease diagnosis or treatment. For example, when the sample to be tested is selected from artificially prepared samples, negative samples, and environmental samples, the testing is conducted for purposes other than disease diagnosis or treatment.

[0070] Furthermore, embodiments of the present invention also provide the use of nanobodies as described in any of the foregoing embodiments, or antibodies or antigen-binding fragments thereof as described in any of the foregoing embodiments, or isolated nucleic acids or recombinant vectors containing said isolated nucleic acids as described in any of the foregoing embodiments, or host cells as described in any of the foregoing embodiments, or conjugates as described in claim 7, in the preparation of products for the prevention, diagnosis, treatment, or adjunctive treatment of diseases targeting DLL3.

[0071] In some embodiments, the related diseases include any one or more of the following: small cell lung cancer, melanoma, glioma, bladder cancer, prostate cancer, gastric cancer, liver cancer, and vertebral rib dysplasia.

[0072] 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.

[0073] 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.

[0074] In some embodiments, the product includes at least one of immune cells, reagents, kits, drugs, and drug compositions.

[0075] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0076] Example 1: Construction, expression, and purification of the eukaryotic expression vector for DLL3 protein

[0077] 1.1 Carrier Construction

[0078] 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 ECD gene was then ligated into the pcDNA3.1-hFc-His vector, which had been digested with restriction endonucleases BamHI and EcoRI, 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. Plasmids were extracted from successfully constructed clones through amplification culture.

[0079] 1.2 Expression and purification of recombinant DLL3 protein

[0080] The recombinant plasmid pcDNA3.1-DLL3-hFc-His, containing the extracellular region of the DLL3 gene, was successfully transfected into HEK293T cells. After transient transfection for 8 h, the medium was replaced with 293 freestyle medium. After 5 days of culture, the cell culture supernatant was collected, and high-purity DLL3-hFc-His recombinant protein was obtained by affinity chromatography using Protein G Beads. Figure 1 ).

[0081] Example 2: Screening of anti-DLL3 protein nanobodies

[0082] 1 mg of purified DLL3 recombinant protein was emulsified with an equal volume of adjuvant and used to immunize Bactrian camels. After three consecutive immunizations, the antibody titer against DLL3-His recombinant protein (ACRO: DL3-H52H4) in the camel peripheral blood was 1:256000, indicating a good immunization effect and suitable for subsequent antibody library construction. 100 mL of peripheral blood was collected to isolate PBMCs, and total mRNA was extracted and reverse transcribed into cDNA. The VHH gene was obtained by nested PCR amplification. The primers used in the first round of PCR were CALL001 (nucleotide sequence as shown in SEQ ID NO: 20) and CALL002 (nucleotide sequence as shown in SEQ ID NO: 21), and approximately 700 bp of band was recovered after amplification. Then, the recovered 700 bp product was used as a template for a second round of PCR amplification. The primers used in the second round of PCR were VHH-FOR (nucleotide sequence as shown in SEQ ID NO: 22) and VHH-REV (nucleotide sequence as shown in SEQ ID NO: 23), and a 400 bp band was recovered.

[0083] PCR products and the phage display vector pMECS were digested with Pst I and Not I and then recovered, followed by ligation using T4 DNA ligase. The ligation product was added to *E. coli* TG1 competent cells and electroporated to induce entry into TG1 cells. After electroporation, the cells were activated at 37°C and 200 rpm for 1 h, then plated on LB / Amp-Glu plates and incubated overnight at 37°C to obtain the phage library. Using the prepared phage library as the antibody source, three rounds of screening were performed using phage display technology. First, DLL3-His recombinant protein (2 μg / mL) was coated onto 96-well microplates. The next day, the plates were blocked with 3% skim milk powder at 37°C for 1 h, and 1×10⁻⁶ oz. of phage was added to each well. 10 Recombinant phages containing nanobodies were incubated at 37°C for 1 hour, washed five times with PBST, and then eluted with 0.1 M glycine (pH 1.5) to remove phages bound to DLL3-His. The elution was neutralized with 1 M Tris-HCl (pH 8.0). The elution solution was then used to infect the host bacterium TG1 and cultured. After three rounds of screening, two specific anti-DLL3 nanobodies, NbA2 and NbB1, were obtained.

[0084] Example 3: Preparation of anti-DLL3 protein-specific nanobodies

[0085] The genes of anti-DLL3 nanobodies and humanized nanobodies were cloned into the eukaryotic expression vector pcDNA3.1-MCS-hFc. After sequencing confirmed, the plasmids were extracted and transfected into HEK293T cells. After 5 days of expression, the supernatant was collected and purified by affinity chromatography using Protein G Beads to obtain recombinant nanobodies (NbA2, NbB1, huA2, and huB1).

[0086] Example 4: Indirect ELISA detection of the binding of nanobodies to DLL3 protein

[0087] DLL3-His recombinant protein was coated into 96-well microplates at a rate of 100 ng per well. The plates were blocked with 3% skim milk at 37°C for 1 h. After washing three times with PBST, 100 μL of recombinant nanobodies prepared in Example 3 and positive control antibody AMG757 (patent number: CN108271376A) at different concentrations were added to each well and 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 developed for 5 min, and the reaction was terminated with 2M H2SO4. The OD450 nm absorbance was read, and binding curves were plotted. The results are as follows: Figure 2 As shown.

[0088] The two obtained nanobodies and the humanized nanobodies all exhibited good binding activity to the DLL3 protein, and the irrelevant control antibody isotype did not bind to the DLL3-His protein. The EC50 values ​​of the recombinant antibodies NbA2-hFc, huA2-hFc, NbB1-hFc, huB1-hFc, and AMG757-hFc were 6.811 ng / mL, 4.953 ng / mL, 5.458 ng / mL, 8.007 ng / mL, and 20.52 ng / mL, respectively.

[0089] Example 5: Analysis of the binding epitope between anti-DLL3 nanobody and DLL3

[0090] Based on the Uniprot database's classification of the extracellular domains of human DLL3, the full-length DLL3 gene (27-618 AA) and genes containing different domains extending into the intracellular domain: DSL (176-618 AA), EGF1 (216-618 AA), EGF2 (274-618 AA), EGF3 (312-618 AA), EGF4 (353-618 AA), EGF5 (391-618 AA), and EGF6 (429-618 AA) were cloned into eukaryotic expression vectors containing EGFP, as shown in the schematic diagram below. Figure 3As shown. The above eukaryotic expression vector was then transfected into HEK293T cells. After transient transfection for 48 h, cells were collected for analysis. These cells were co-incubated at 37°C for 1 h with recombinant anti-DLL3 nanobodies (NbA2-hFc, NbB1-hFc), positive control antibody AMG757-hFc (patent number: CN108271376A), and an irrelevant control antibody isotype. After washing three times with PBS, the cells were co-incubated at 37°C for 1 h with APC-labeled anti-human secondary antibody. After washing three times with PBS, the cells were analyzed by flow cytometry. The results are shown below. Figure 4 As shown, both NbA2 and the positive control antibody AMG757 bind to the EGF3 domain, while NbB1 binds to the EGF1 domain.

[0091] Example 6: Species Cross-Reactivity Analysis of Anti-DLL3 Nanobodies

[0092] To further investigate the cross-reactivity of the anti-DLL3 nanobody with mouse and cynomolgus monkey DLL3, the similarity of the extracellular amino acid sequences of human DLL3 (NM_016941.4), mouse DLL3 (NM_007866.2), and cynomolgus monkey DLL3 (XM_045378439.2) was first analyzed. The results are as follows: Figure 5 As shown, the extracellular similarity of the DLL3 gene between humans and mice was 85.6%, and the similarity between humans and cynomolgus monkeys was 96.4%. Then, the DLL3 genes from humans, mice, and cynomolgus monkeys were cloned into eukaryotic expression vectors containing EGFP. These recombinant vectors were transfected into HEK293T cells, and cells were collected for analysis after 48 h of transient transfection. The cells were co-incubated at 37°C for 1 h with recombinant anti-DLL3 nanobodies (NbA2-hFc, NbB1-hFc, 5 μg / mL) and an irrelevant control antibody isotype, respectively. After washing three times with PBS, the cells were co-incubated at 37°C for 1 h with APC-labeled anti-human secondary antibody, washed three times with PBS, and analyzed by flow cytometry. The results are shown below. Figure 6 As shown, both the anti-DLL3 nanobodies NbA2 and NbB1 can cross-react with DLL3 in mice and cynomolgus monkeys.

[0093] Example 7: Biacore detection of the affinity between nanobodies and DLL3 protein

[0094] In this invention, the binding affinity of the prepared anti-DLL3 recombinant nanobody to the DLL3-His antigen coated on a CM5 chip was detected using a Biacore 8k instrument. The results are shown in the table below. The affinity between the nanobody and the DLL3 protein is within 10... -10 ~10 -9 M is a high-affinity antibody.

[0095] Table 1. In vitro binding affinity and kinetics analysis of anti-DLL3 nanobodies to DLL3 protein.

[0096]

[0097] Example 8: FACS detection of the binding of nanobodies to DLL3 at the cellular level

[0098] The recombinant nanobodies prepared in Example 3 were incubated with SHP-77, NCI-H82, and HeLa cells overexpressing DLL3 (DLL3-HeLa) at 37°C for 40 min. After washing three times with PBS, they were incubated with APC-labeled Goat anti-human secondary antibody (1:600) at 37°C for 40 min, washed three times with PBS, and detected by flow cytometry. The results are as follows: Figure 7 As shown, the above-mentioned nanobody exhibits good binding activity with SHP-77, NCI-H82, and DLL3-HeLa cells, indicating that the anti-DLL3 nanobody in this invention has good specific binding activity with DLL3 at the cellular level.

[0099] Example 9: Preparation of Bispecific Antibody and Detection of its In Vitro Antitumor Activity

[0100] Using the anti-DLL3 nanobody and humanized nanobody plasmid of this invention as templates, the antibody gene targeting DLL3 was amplified and cloned into a vector containing anti-human CD3 antibody (OKT3). After sequencing confirmed, the plasmid was extracted and transfected into HEK293T cells. After 5 days of expression, the supernatant was collected and purified by affinity chromatography using an NTA-Ni column to obtain bispecific antibodies (NbA2 / CD3, huA2 / CD3, NbB1 / CD3, huB1 / CD3), the sequences of which are shown in Table 3. The anti-DLL3 / CD3 bispecific antibodies (NbA2 / CD3, huA2 / CD3, NbB1 / CD3, huB1 / CD3), positive control bispecific antibody (AMG757 / CD3), and irrelevant bispecific antibody (isotype / CD3) of this invention were co-cultured with DLL3-expressing SCLC endogenous cells NCI-H69-Luciferase, SHP-77-Luciferase, NCI-H82-Luciferase, and human peripheral blood-derived T cells at an effector-to-target ratio of 1:1. A control containing only SCLC endogenous cells was also included. After 24 hours of co-culture, fluorescein potassium substrate was added and incubated for 5 minutes. The Luminescence fluorescence value (RLU) was read using a microplate reader. The killing efficiency of the bispecific antibodies was calculated using the formula [1 - RLU(experimental group) / RLU(control group)] × 100. The killing efficiency curves of different concentrations of bispecific antibodies are shown below. Figure 8As shown in Table 2, its EC50 values ​​are as follows.

[0101] Table 2 Bispecific antibody EC50

[0102]

[0103] The results showed that the bispecific antibody huA2 / CD3 in this invention exhibited the strongest killing activity against NCI-H69-Luc, SHP-77-Luc, and NCI-H82-Luc cell lines, with EC50 values ​​of 2.827 pM, 0.367 pM, and 0.289 pM, respectively, which were significantly superior to the positive control AMG757 / CD3 (29.47 pM, 0.846 pM, and 5.366 pM).

[0104] Example 10: Evaluation of the antitumor activity of DLL3 / CD3 bispecific antibody in a SCLC xenograft model

[0105] 5×10 6 SHP-77 cells in the logarithmic growth phase were subcutaneously in the right posterior back of 6-8 week old NCG mice, and inoculated until the tumor volume reached 150 mm. 3 Mice with uniform tumor volume were randomly divided into groups of 5 mice each. A blank control group (PBS), experimental groups (huA2 / CD3, huB1 / CD3, AMG757 / CD3), and an irrelevant control group (isotype / CD3) were established. Bispecific antibodies were administered via tail vein every 2 days for 5 consecutive doses at a dose of 5 mg / kg. The blank control group received the same volume of PBS. During the first administration, each mouse in the experimental and irrelevant control groups was injected via tail vein with 1 × 10⁻⁶ PBS. 7 Each mouse in the control group was injected with the same volume of PBS via the tail vein after in vitro expansion and activation of CD3+ T cells. The average tumor volume was calculated using the formula V = 1 / 2(L × W × W), where L represents the length of the tumor and W represents the width of the tumor. When the tumor volume in the mouse reached 1500 mm², the tumor was considered to be enlarged. 3 If obvious ulceration occurs on the surface of the tumor, the mouse is euthanized, and the animal experiment is terminated. All data are mean ± SD.

[0106] Depend on Figure 9 The results showed that, compared with the control group without drug administration, the anti-DLL3 / CD3 bispecific antibodies (huA2 / CD3, huB1 / CD3, AMG757 / CD3) all had significant inhibitory effects on the growth of tumor cells, indicating that the DLL3 / CD3 bispecific antibodies prepared in this invention have good anti-tumor activity in the SCLC mouse xenograft model.

[0107] The experimental results above show that the anti-DLL3 nanobodies obtained in this invention all possess excellent antigen-binding activity and specificity. The DLL3 / CD3 bispecific antibody, composed of the anti-DLL3 nanobodies and the anti-CD3 antibody (OKT3) of this invention, exhibits good killing activity against tumor cells both in vitro and in vivo, and can be used to prepare drugs for the prevention or treatment of tumors.

[0108] The sequence information involved in this application is shown in the table below.

[0109] Table 3 Sequence Information

[0110]

[0111] 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. A nanobody against DLL3, 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 or 8; The HCDR1, HCDR2, and HCDR3 are defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact.

2. The nanobody according to claim 1, characterized in that, The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1~3 or 5~7, respectively.

3. The nanobody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the nanobody is shown in SEQ ID NO:4, 8, 9 or 10.

4. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment is a fusion antibody, which is composed of the following: the nanobody according to any one of claims 1 to 3, and an Fc fused to the nanobody.

5. An antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment is selected from any one of bispecific antibodies and chimeric antibodies; The chimeric antibody includes: the nanobody according to any one of claims 1 to 3; The bispecific antibody comprises the following: the nanobody as described in any one of claims 1 to 3 and the anti-CD3 antibody.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The amino acid sequence of the bispecific antibody is any one of SEQ ID NO:12~15.

7. An isolated nucleic acid, an expression cassette containing the isolated nucleic acid, or a recombinant vector containing the isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the nanobody as described in any one of claims 1 to 3 or encodes the antibody or antigen-binding fragment thereof as described in any one of claims 4 to 6.

8. The isolated nucleic acid according to claim 7, characterized in that, The isolated nucleic acid has 80% identity with any one of SEQ ID NO:16~19.

9. A host cell, characterized in that, It comprises the recombinant vector as described in claim 7.

10. A method for preparing an antibody, characterized in that, It includes: Cultivate the host cells as described in claim 9.

11. A coupling, characterized in that, It comprises the following parts: the nanobody according to any one of claims 1 to 3 or the antibody or antigen-binding fragment thereof according to any one of claims 4 to 6; and a coupling part coupled to the nanobody or the antibody or antigen-binding fragment thereof; The coupling portion is selected from any one or more of the following: protein tags for purification, markers for detection or tracing, and solid-phase supports.

12. A pharmaceutical composition, characterized in that, Its active ingredient is: the nanobody according to any one of claims 1 to 3 or the antibody or its antigen-binding fragment according to any one of claims 4 to 6.

13. The use of the nanobody as described in any one of claims 1 to 3, or the antibody or its antigen-binding fragment as described in any one of claims 4 to 6, or the isolated nucleic acid or recombinant vector containing the isolated nucleic acid as described in claim 7 or 8, or the host cell as described in claim 9, or the conjugate as described in claim 11, in the detection of DLL3 protein for non-disease diagnosis or treatment purposes.

14. The use of the nanobody as described in any one of claims 1 to 3, or the antibody or its antigen-binding fragment as described in any one of claims 4 to 6, or the isolated nucleic acid as described in claim 7 or 8, or the recombinant vector containing said isolated nucleic acid, or the host cell as described in claim 9, or the conjugate as described in claim 11, in the preparation of a product for the treatment or adjuvant treatment of small cell lung cancer.

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