Anti-human dll3 recombinant monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of anti-human DLL3 recombinant monoclonal antibody and its preparation method and application, belong to the technical field of monoclonal antibody.The anti-human DLL3 recombinant monoclonal antibody provided by the present application includes the amino acid sequence as shown in SEQ ID NO.1~3 VHCDR1, VHCDR2, VHCDR3, and the amino acid sequence as shown in SEQ ID NO.4~6 VLCDR1, VLCDR2, VLCDR3.It also provides the nucleotide sequence of the variable region of heavy chain and light chain variable region of the monoclonal antibody, and the monoclonal antibody can be prepared using genetic engineering technology.The monoclonal antibody prepared by the present application has better specificity and sensitivity compared with imported DLL3 antibody, and can be applied to clinical immunohistochemical detection.

Description

Technical Field

[0001] This invention relates to a recombinant monoclonal antibody against human DLL3, its preparation method, and its application, belonging to the field of monoclonal antibody technology. Background Technology

[0002] DLL3 is an inhibitory ligand of the Notch signaling pathway and is closely related to enhanced tumor cell proliferation, migration, and invasion. In gastric cancer, upregulation of DLL3 activates Notch signaling and accelerates cell cycle transition, thereby promoting cancer cell proliferation. DLL3 is highly expressed on the cell surface of various neuroendocrine tumors (highly expressed in approximately 80% of small cell lung cancer patients), while it is rarely expressed or not expressed at all in normal tissues. This significant difference makes it an ideal target for precise targeting of tumor cells. In extensive-stage small cell lung cancer, invasive breast cancer, and colorectal cancer, studies have found that high expression of DLL3 is significantly associated with shortened overall survival and poor prognosis.

[0003] Current research on DLL3 antibodies focuses on cancer treatment. An international PCT patent document (published as WO2025067294A1) discloses a DLL3 antibody for use in cancer immunotherapy or diagnosis; a Chinese patent document (published as CN121909211A) also discloses a DLL3 antibody for cancer treatment. In recent years, DLL3 has become an effective therapeutic target for small cell lung cancer (SCLC) and other neuroendocrine malignancies. The recent FDA approval of tarlatamab for recurrent small cell lung cancer highlights the promising application of DLL3 targeting in pulmonary and extrapulmonary neuroendocrine tumors. Therefore, clarifying DLL3 expression as a screening biomarker for patients with these malignancies is crucial for treatment selection and clinical trial design.

[0004] Currently, commonly used DLL3 antibodies in clinical research and companion diagnostics include clones such as SP347, E3J5R, and S207-179A. Among them, the DLL3 antibody with clone number E3J5R from Cell Signaling Technology has a significant competitive advantage due to its superior biological characteristics and extensive validation data. However, this product is imported and expensive. Furthermore, the lack of standardized DLL3 detection kits and varying cutoff values ​​across studies lead to biases in clinical trial results, a critical issue that urgently needs to be addressed. In addition, research in the field of DLL3 immunodiagnostics in my country is severely lagging, and domestically produced antibodies cannot meet the needs of SCLC patients. Therefore, developing a DLL3 antibody with higher sensitivity and specificity could provide a more reliable detection method for many patients and provide a basis for precision medicine, which has profound and positive significance. Summary of the Invention

[0005] The first objective of this invention is to provide a recombinant monoclonal antibody against human DLL3, which is stable, has high experimental reproducibility, and exhibits good specificity and sensitivity.

[0006] A second objective of this invention is to provide the application of the above-mentioned anti-human DLL3 recombinant monoclonal antibody in the preparation of DLL3 in vitro detection reagents or kits.

[0007] A third objective of this invention is to provide a detection reagent or kit comprising the above-described anti-human DLL3 recombinant monoclonal antibody.

[0008] A fourth objective of this invention is to provide a nucleic acid molecule encoding the aforementioned anti-human DLL3 recombinant monoclonal antibody.

[0009] The fifth objective of this invention is to provide a method for preparing a recombinant monoclonal antibody against human DLL3.

[0010] To achieve the above objectives, the technical solution adopted by the anti-human DLL3 recombinant monoclonal antibody in this invention is as follows: A recombinant monoclonal antibody against human DLL3, the recombinant monoclonal antibody against human DLL3 comprising VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO.1~3, and VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO.4~6.

[0011] The beneficial effects of the above technical solution are as follows: This invention provides a recombinant monoclonal antibody against human DLL3, which has better specificity and sensitivity compared with imported antibodies, and can be applied to clinical immunohistochemical detection.

[0012] Specifically, the heavy chain variable region of the anti-human DLL3 recombinant monoclonal antibody has the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.8.

[0013] To achieve the above objectives, the technical solution adopted in this invention for the application of anti-human DLL3 recombinant monoclonal antibody in the preparation of DLL3 in vitro detection reagents or kits is as follows: Application of anti-human DLL3 recombinant monoclonal antibody in the preparation of DLL3 in vitro detection reagents or kits.

[0014] The beneficial effects of the above technical solution are as follows: Experimental verification shows that the anti-human DLL3 recombinant monoclonal antibody has good specificity and affinity, and can be used to prepare DLL3 detection reagents or kits to detect the expression of DLL3 in tissues and cells, and can further study the function of DLL3.

[0015] To achieve the above objectives, the technical solution adopted in the detection reagent or kit containing the above-mentioned anti-human DLL3 recombinant monoclonal antibody in this invention is as follows: A detection reagent or kit containing the above-mentioned recombinant monoclonal antibody against human DLL3.

[0016] The beneficial effects of the above technical solution are as follows: the recombinant monoclonal antibody against human DLL3 with good specificity and affinity of the present invention can be used to prepare detection reagents or kits for detecting DLL3 in tissues or cells, such as immunohistochemical staining, immunocellular staining, Western blot or ELISA, according to the needs of users, which helps to meet diverse market demands.

[0017] Preferably, the detection reagent or kit includes an immunohistochemical detection reagent or kit.

[0018] To achieve the above objectives, the technical solution adopted in this invention for a nucleic acid molecule encoding a recombinant monoclonal antibody against human DLL3 is as follows: A nucleic acid molecule encoding the aforementioned anti-human DLL3 recombinant monoclonal antibody.

[0019] The beneficial effects of the above technical solution are as follows: This invention provides a nucleic acid molecule encoding a recombinant monoclonal antibody against human DLL3, which preserves the recombinant monoclonal antibody against human DLL3 in the form of DNA, resulting in higher stability and facilitating subsequent exogenous expression and large-scale industrial production.

[0020] Specifically, the nucleotide sequence of the heavy chain variable region gene of the anti-human DLL3 recombinant monoclonal antibody is shown in SEQ ID NO.20, and the nucleotide sequence of the light chain variable region gene of the anti-human DLL3 recombinant monoclonal antibody is shown in SEQ ID NO.21.

[0021] To achieve the above objectives, the technical solution adopted in the preparation method of the anti-human DLL3 recombinant monoclonal antibody of the present invention is as follows: A method for preparing an anti-human DLL3 recombinant monoclonal antibody involves introducing the above-mentioned nucleic acid molecules into host cells, collecting the cell supernatant, and purifying and ultrafiltration the antibody.

[0022] The beneficial effects of the above technical solution are as follows: Compared with traditional monoclonal antibody preparation methods, the method for preparing anti-human DLL3 recombinant monoclonal antibodies provided by this invention has advantages such as known antibody sequences, long-term preservation of antibody genes, stable antibody properties, and good experimental reproducibility. It is a standardized antibody production process that avoids the risk factors that occur in the traditional monoclonal antibody production and preservation process. Utilizing recombinant technology to prepare anti-human DLL3 recombinant monoclonal antibodies ensures the controllability and traceability of the antibody sequence, solves the batch-to-batch differences and preservation problems that may occur in traditional antibody production, and provides a more stable and reliable antibody tool suitable for long-term and standardized clinical diagnostic reagent development and basic research applications.

[0023] Preferably, the host cell includes mammalian cells; the mammalian cells include HEK293 cells and CHO cells.

[0024] HEK293 and CHO cells have accurate post-translational modification capabilities, are easy to transfect, have high tolerance to shear stress and osmotic pressure, express recombinant antibodies in near-native state, and can produce large amounts of recombinant proteins. Attached Figure Description

[0025] Figure 1 The immunohistochemical detection results (10×) of the 3B8 antibody in small cell lung cancer tissue in Example 3 of the present invention. Figure 2 The results of immunohistochemical detection of E3J5R antibody in small cell lung cancer tissue in Example 3 of this invention (10×). Detailed Implementation

[0026] Small cell lung cancer (SCLC) is one of the most malignant types of lung cancer, with extremely high invasive and metastatic capabilities. After recurrence, it exhibits high resistance to conventional chemotherapy, resulting in a very poor overall prognosis. For decades, treatment options have been limited. Although first-line chemotherapy combined with immune checkpoint inhibitors (ICIs) has improved the median overall survival (OS) of extensive-stage (ES) SCLC by 2-3 months compared to chemotherapy alone, the benefit remains very limited. In this area urgently needing breakthroughs, novel precision immunotherapies targeting DLL3-specific T-cell connectors have emerged. These therapies can precisely activate the body's immune system, redirecting T cells to kill DLL3-positive tumor cells while reducing off-target effects. DLL3 expression is highest in SCLC type A and lowest in type P / I, making it a key biomarker for differentiating subtypes and screening for beneficiaries. In recent years, DLL3 has become an effective therapeutic target for small cell lung cancer and other neuroendocrine malignancies, with related ADCs, bispecific T-cell connectors, and CAR-T therapies all in clinical development.

[0027] Currently, the DLL3 antibody products sold on the market for immunohistochemical pathological diagnosis are mainly expensive imported products, such as the E3J5R antibody from Cell Signaling Technology and the SP347 antibody from Ventana. Research in the field of DLL3 immunodiagnostics in my country lags significantly, and domestically produced antibodies cannot meet the needs of SCLC patients. Therefore, developing a DLL3 antibody with higher sensitivity and specificity could provide a more reliable detection method for many patients and provide a basis for precision medicine, which has profound and positive significance.

[0028] Based on this, this application provides an anti-human DLL3 recombinant monoclonal antibody and its preparation method, comprising the following steps: (1) Antigen preparation: The C-terminal 465-489 amino acid region of DLL3 was selected as the antigen sequence. The antigen was expressed by linking it to the pET28a or pET32a vector in prokaryotic cells and then purified by chromatography.

[0029] (2) Rabbits were immunized simultaneously with DLL3 antigen and chaperone protein CSF2. Rabbit peripheral blood was collected, rabbit peripheral blood lymphocytes were isolated, and antigen-specific B lymphocytes were sorted out.

[0030] (3) The B lymphocytes obtained in step (2) are cultured and identified, and positive cell pores are screened out. Then, the heavy chain and light chain gene sequences of the antibody are obtained, and rabbit monoclonal antibodies are obtained through recombination and expression.

[0031] (4) The antibody obtained in step (3) was verified for its specificity and sensitivity by immunohistochemistry (IHC).

[0032] The present invention also provides the light and heavy chain variable region sequence information of the above-mentioned anti-human DLL3 recombinant monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and simultaneously includes VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO.1~3 in sequence; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8, and simultaneously includes VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO.4~6 in sequence.

[0033] The present invention also provides a nucleic acid molecule encoding the above-mentioned anti-human DLL3 recombinant monoclonal antibody, wherein the nucleotide sequence of the heavy chain variable region gene of the anti-human DLL3 recombinant monoclonal antibody is shown in SEQ ID NO.20, and the nucleotide sequence of the light chain variable region gene is shown in SEQ ID NO.21.

[0034] This invention provides the amino acid and nucleotide sequences of the heavy and light chain variable regions of the anti-human DLL3 recombinant monoclonal antibody. Based on these sequences, the monoclonal antibody of this invention can be obtained using conventional genetic engineering methods.

[0035] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art, and the equipment and raw materials used are commercially available or commonly used in the art.

[0036] Example 1: Variable region sequence of anti-human DLL3 recombinant monoclonal antibody This embodiment provides a method for obtaining the amino acid and nucleotide sequences of the light and heavy chain variable regions of the anti-human DLL3 recombinant monoclonal antibody. The C-terminal 465-489 amino acid fragment of DLL3 was selected as the design scheme to prepare the antigen. The corresponding nucleic acid sequence was artificially synthesized, and the target protein was obtained through genetic engineering technology. Rabbits were immunized, and B lymphocytes were screened. After obtaining monoclonal antibodies with high positive values, total RNA was extracted, reverse transcribed to obtain cDNA, and sequencing analysis was performed to obtain the sequence information of the anti-human DLL3 monoclonal antibody. The specific operation is as follows: 1. Antigen Acquisition According to Uniprot's published protein sequence number Q9NYJ7, the DLL3 protein is a single-transmembrane protein. The C-terminal 465-489 amino acid region was selected as the antigen sequence, as shown in SEQ ID NO.9. The nucleotide sequence was codon-optimized, and the optimized nucleic acid sequence was artificially synthesized and constructed into the pET-28a vector, which was then transformed into *E. coli* for protein expression. The target protein was purified by nickel affinity chromatography with a purity of up to 90%.

[0037] 2. Animal immunization To further improve the immunization effect in animals, CSF2 cytokine was added as an immune chaperone. CSF2 is a self-constructed and expressed protein. The immunogen and immune chaperone (CSF2 protein) were added to a water-soluble adjuvant to obtain an immunization reagent. The concentration of the immune chaperone in the immunization reagent was controlled at 50 μg / mL, and the immunization dose of the immunogen was 100 μg / rabbit. New Zealand white rabbits were then immunized via intramuscular injection in the leg, for a total of three immunizations, each 21 days apart, with the same immunization dose. Seven days after the third immunization, blood was collected from the marginal ear vein to assess the immunization effect. Once the expected results were achieved, the rabbits underwent a pulse immunization with DLL3 immunogen; 10 mL of blood was collected from the marginal ear vein on the fourth day.

[0038] 3. Rabbit peripheral blood collection and PBMC isolation, B lymphocyte sorting and culture PBMCs were isolated from the peripheral blood of rabbits after shock immunization using a commercial rabbit peripheral blood lymphocyte isolation kit (Solepro, P6280). Subsequently, they were screened again using one or more antibodies (Lsbio, LS-C58688) against T lymphocyte surface markers such as CD4, CD8, CD14, CD28, and CD80 to remove T cells, monocytes, etc.

[0039] Activated B lymphocytes were then selected using FITC-labeled goat anti-rabbit IgG (Thermo, A10931).

[0040] Immunogen DLL3 was labeled using a commercially available biotin labeling kit (Frdbio, ARL0020S) according to the manufacturer's instructions. This was then conjugated with commercially available avidin-conjugated fluorescein PE (Thermofisher, 12-4317-87) and APC-780 (Thermofisher, 47-4317-82), and incubated at 37°C for 1 hour (with agitation several times during incubation) to obtain the antigen-biotin-avidin screening reagent. When using, the reagent should be prepared according to the cell quantity (1×10⁻⁶). 6 Add cells (5 μL) to the B lymphocytes and perform positive screening again to obtain antigen-specific B lymphocytes.

[0041] The sorted B lymphocytes were seeded into 96-well plates with 1-2 cells per well, which were pre-filled with feeder cells. One or more cytokines from IL-2, IL-4, IL-6, IL-10, TNF-α, TGF-β, and SAC were added for co-culture.

[0042] 4. ELISA detection of B lymphocyte culture supernatant After culturing for 7-12 days, the cell supernatant was collected for ELISA detection. The cells were coated with immunogen DLL3 protein, and serum collected from rabbits before immunization was selected as negative control wells. A positive result was defined as an OD450 reading greater than 2.1 times that of the negative control wells, and the selected positive wells were selected.

[0043] 5. cDNA acquisition, antibody heavy and light chain gene amplification and sequencing Cells from the selected positive cell wells were lysed, and total RNA was extracted to obtain cDNA via RT-PCR. Antibody heavy and light chain gene bands from the cells in the wells were amplified using conventional PCR with one pair of primers for each of the heavy and light chains. The primer sequences are shown in SEQ ID NO. 10-13 (where SEQ ID NO. 10-11 are heavy chain primers, and SEQ ID NO. 12-13 are light chain primers). The obtained bands were recovered from the gel to obtain the antibody heavy and light chain gene products. A portion of the obtained products was sequenced using primer sequences shown in SEQ ID NO. 14-15.

[0044] 6. Determination of antibody heavy and light chain gene information After analyzing the correctly sequenced antibody heavy and light chain gene sequences, the heavy and light chain sequences were extracted. The nucleotide sequence of the variable region of the heavy chain of the anti-human DLL3 recombinant monoclonal antibody of this invention is shown in SEQ ID NO.20, the amino acid sequence is shown in SEQ ID NO.7, and the amino acid sequences of VHCDR1-3 are shown in SEQ ID NO.1-3 respectively. The nucleotide sequence of the variable region of the light chain of the anti-human DLL3 monoclonal antibody is shown in SEQ ID NO.21, the amino acid sequence is shown in SEQ ID NO.8, and the amino acid sequences of VLCDR1-3 are shown in SEQ ID NO.4-6 respectively.

[0045] Example 2: Preparation method of recombinant monoclonal antibody against human DLL3 This embodiment provides a method for preparing a recombinant monoclonal antibody against human DLL3. Based on the antibody light and heavy chain sequence information obtained in Example 1, the variable region genes of the heavy and light chains are amplified by PCR. These genes are then recombined with the self-modified vectors pRCH and pRCK, which contain the constant region sequences of the heavy and light chain antibody genes. The resulting recombinant monoclonal antibody is then transformed into host cells, expressed, and purified to obtain the recombinant monoclonal antibody against human DLL3. The specific steps are as follows: 1. Construction of recombinant plasmids DH5α strain containing pRCH and pRCK plasmids were cultured on a large scale, and plasmids were extracted and double-digested (EcoRI and HindIII). Plasmid extraction was performed according to the Axygen plasmid extraction kit instructions. The double-digestion reaction system was: 2 μg pRCH or pRCK plasmid, 2 μL EcoRI, 2 μL HindIII, 5 μL Buffer, and 40 μL ddH2O; the digestion conditions were 37°C water bath for 2 h. The digested system was subjected to agarose gel electrophoresis, and the double-digested plasmid fragments were recovered from the gel, following the Axygen gel recovery kit instructions.

[0046] The antibody heavy and light chain variable region gene fragments were obtained by PCR. The upstream and downstream primer sequences were designed as shown in SEQ ID NO. 16-19 (SEQ ID NO. 16-17 are primers for the heavy chain variable region, and SEQ ID NO. 18-19 are primers for the light chain variable region). The PCR amplification cycle settings were: I: 98℃ 3 min; II: 98℃ 10 s; III: 56℃ 10 s; IV: 72℃ 10 s; II-IV: 30 cycles; V: 72℃ 5 min. The target band was obtained by 1.5% agarose gel electrophoresis, and the target fragment was excised and recovered. This procedure was performed according to the instructions of the Axygen gel recovery kit.

[0047] Homologous recombination of the target gene fragment and the double-digested plasmid fragment was performed using a seamless cloning kit, following the instructions of the Nearshore Seamless Cloning Kit (catalog number: NR005-01A). After seamless recombination, the cells were transformed into DH5α competent cells and screened using ampicillin-resistant LB agar plates. The cells were incubated at 37°C for 16–18 h, and five single clones from each plate were selected for gene sequencing. Colonies with correct sequencing results were expanded and plasmids were extracted.

[0048] 2. Plasmid transfection High-concentration, high-purity antibody heavy and light chain recombinant plasmids obtained after expanded culture were transfected into CHO / HEK293 cells according to the PEI transfection reagent (Kyfora Bio, 26008-5) instructions. The antibody heavy and light chains were pre-mixed at a molar ratio of 1:2. The plasmid was diluted with CHO / HEK293 cell basal medium, and an equal volume of PEI was diluted with the same medium (plasmid:PEI = 1:3, w / w). This solution was then transfected into CHO / HEK293 cells cultured in the logarithmic growth phase. Forty-eight hours after transfection, the antibody titer secreted in the supernatant was detected using an indirect ELISA method to identify cell lines with relatively high expression levels.

[0049] 3. Antibody engineered expression and purification The selected cell line was expanded to a volume of 100 mL, and the cell growth state was adjusted to the logarithmic growth phase. Plasmids were transfected, and feed was added every other day. Cell supernatant was collected on day 6 post-transfection. The supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a Protein G column. The bound antibody was eluted with citrate buffer at pH 6.0, and the eluent was collected. The eluent was rapidly neutralized to pH 7.2–7.4 using Tris-HCl solution at pH 8.8. The purified antibody was then concentrated by ultrafiltration and centrifugation to a concentration above 1 mg / mL.

[0050] Example 3: Application of anti-human DLL3 recombinant monoclonal antibody in the preparation of DLL3 in vitro detection reagents or kits This embodiment provides the application of anti-human DLL3 recombinant monoclonal antibody in the preparation of DLL3 in vitro detection reagents or kits, as detailed below: This embodiment provides a DLL3 detection reagent, which includes the working solution of the anti-human DLL3 recombinant monoclonal antibody from Example 1, with an antibody working concentration of 0.2 μg / mL.

[0051] Based on the above-mentioned anti-human DLL3 recombinant monoclonal antibody working solution, a human DLL3 immunohistochemical detection kit is constructed by combining antigen EDTA retrieval solution, peroxidase blocking agent, horseradish peroxidase-labeled goat anti-mouse / rabbit secondary antibody, DAB and its buffer, hematoxylin and other supporting reagents.

[0052] The above-mentioned detection reagents or kits were used to stain small cell lung cancer tissue, as detailed below: 1. Paraffin-embedded tissue sections of small cell lung cancer, 3μm / section, baked at 65℃ for 2 hours.

[0053] 2. Dewaxing and hydration: Paraffin sections were treated as follows: xylene for 15 min; xylene for 15 min; anhydrous ethanol for 5 min; anhydrous ethanol for 5 min; 90% ethanol for 5 min; 80% ethanol for 5 min; 70% ethanol for 5 min; and purified water for 5 min.

[0054] 3. Antigen retrieval: After heating and boiling the EDTA antigen retrieval solution at pH 9.0, place the tissue slide in the solution, adjust to a gentle boiling mode, time for 20 minutes, allow to cool naturally for 5 minutes, rinse with running water to cool to room temperature, remove the slide, soak in purified water for 5 minutes, and rinse and soak in TBST for 5 minutes.

[0055] 4. Add peroxidase blocking agent, 100 μL / sheet, incubate at room temperature for 5 min, rinse and soak with TBST for 5 min / time, for a total of 2 times.

[0056] 5. Primary antibody incubation: Add 100 μL of anti-human DLL3 recombinant monoclonal antibody working solution to each sheet and incubate at 37°C for 30 min. Rinse and soak with TBST for 5 min each time, for a total of 2 times.

[0057] 6. Secondary antibody incubation: Add 100 μL of horseradish peroxidase-labeled goat anti-mouse / rabbit secondary antibody per sheet, incubate at room temperature for 30 min, rinse and soak with TBST for 5 min each time, for a total of 2 times.

[0058] 7. Color development: Add 100 μL of DAB color development solution to each sheet, incubate at room temperature for 4 min, and soak twice in purified water for 5 min each time.

[0059] 8. Counterstaining: Add 100 μL of hematoxylin staining solution to each sheet, incubate at room temperature for 4 min, and rinse thoroughly with purified water.

[0060] 9. Dehydrated and transparent: Dehydrated with conventional gradient ethanol, and transparent with xylene.

[0061] 10. Mount the slide with neutral resin and observe under a microscope.

[0062] The staining results of the anti-human DLL3 recombinant monoclonal antibody (3B8) prepared in this invention are as follows: Figure 1 As shown, the staining results of CellSignaling Technology's DLL3 antibody E3J5R (antibody concentration of 0.3 μg / mL) are as follows. Figure 2 As shown.

[0063] Depend on Figure 1 and Figure 2 It is known that the 3B8 antibody of the present invention can achieve a strong staining effect and a better background at a concentration of 0.2 μg / mL. The staining effect is better than that of the E3J5R antibody, and it has better specificity and sensitivity. It can be used as a substitute for the E3J5R antibody.

[0064] In summary, this invention provides the variable region amino acid sequences of the heavy and light chains of the recombinant anti-human DLL3 monoclonal antibody. Based on these sequences, the monoclonal antibody of this invention can be obtained using conventional antibody engineering methods, as detailed in Example 2. Example 3 demonstrates that the recombinant anti-human DLL3 monoclonal antibody of this invention can specifically recognize human DLL3 protein, and its staining effect is better than that of the E3J5R antibody, making it suitable for immunohistochemical detection.

[0065] Compared with traditional monoclonal antibody preparation methods, the anti-human DLL3 recombinant monoclonal antibody prepared by the genetic engineering method of this invention has advantages such as known sequence, stable antibody properties, and good reproducibility. The standardized antibody production process avoids the risk factors that occur during the production and storage of traditional monoclonal antibodies. Based on the antibody amino acid sequence provided by this invention, modifications such as the addition, deletion, or substitution of one or more amino acids can be used to obtain its active fragment or conserved variant, laying the foundation for further improving the specificity and affinity of the antibody.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant monoclonal antibody against human DLL3, characterized in that, The anti-human DLL3 recombinant monoclonal antibody comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO.1~3, and VLCDR1, VLCDR2, and VLCDR3 with amino acid sequences as shown in SEQ ID NO.4~6.

2. The anti-human DLL3 recombinant monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region of the anti-human DLL3 recombinant monoclonal antibody has the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

8.

3. The use of the anti-human DLL3 recombinant monoclonal antibody as described in claim 1 or 2 in the preparation of DLL3 in vitro detection reagents or kits.

4. A detection reagent or kit comprising the anti-human DLL3 recombinant monoclonal antibody as described in claim 1 or 2.

5. The detection reagent or kit according to claim 4, characterized in that, The detection reagents or kits include immunohistochemical detection reagents or kits.

6. A nucleic acid molecule encoding the anti-human DLL3 recombinant monoclonal antibody as described in claim 1 or 2.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleotide sequence of the heavy chain variable region gene of the anti-human DLL3 recombinant monoclonal antibody is shown in SEQ ID NO.20; the nucleotide sequence of the light chain variable region gene of the anti-human DLL3 recombinant monoclonal antibody is shown in SEQ ID NO.

21.

8. A method for preparing a recombinant monoclonal antibody against human DLL3, characterized in that, The nucleic acid molecule as described in claim 6 or 7 is introduced into a host cell, the cell supernatant is collected, and purified by ultrafiltration.

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