A nanobody against dll3, related products and uses

Abstract

This application discloses an anti-DLL3 nanobody, related products, and uses. The nanobody comprises heavy chain variable regions of CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 5, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 11, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 17. The anti-DLL3 nanobody of this application comprises only the heavy chain variable region, which can specifically recognize the DLL3 antigen on the cell surface. Simultaneously, the constructed CAR-T cells exhibit killing activity against DLL3-positive tumor cells and can efficiently secrete the cytokine IFN-γ, making it effective for immunotherapy and of great significance for the development of tumor therapeutic drugs.

Description

[0001] This application is a divisional application of application number 2022116893025, the original application was filed on December 27, 2022, and the invention is entitled "An anti-DLL3 nanobody, related products and uses". Technical Field

[0002] This application relates to the field of biomedical technology, and in particular to an anti-DLL3 nanobody, related products and uses. Background Technology

[0003] Heavy chain antibodies (HcAbs), discovered in camels and sharks, are naturally occurring antibodies composed only of heavy chains and lacking light chains. Cloning their variable regions yields single-domain antibodies consisting only of the heavy chain variable region, called VHHs (Variable Domain of Heavy Chain), also known as nanobodies. These are the smallest functional antigen-binding fragments. Unlike conventional antibodies, nanobodies are peptide chains containing approximately 110 amino acids, with a molecular weight about 1 / 10 that of conventional antibodies. Compared to conventional antibodies and recombinant single-chain fragment variables (scFvs), nanobodies offer advantages such as small size, good stability, high solubility, ease of expression, and low production cost, showing broad application prospects in immunological experiments, diagnosis, and treatment.

[0004] Lung cancer is reportedly the leading cause of cancer death worldwide, accounting for approximately 1.8 million deaths (18%) in 2020. Lung cancer is pathologically classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which account for approximately 15% and 85% of all lung cancers, respectively. Small cell lung cancer (SCLC) is a highly lethal and aggressive neuroendocrine tumor. Because SCLC is highly sensitive to chemotherapy and radiotherapy, combined chemotherapy and radiotherapy has become the standard treatment for all stages of SCLC, but the prognosis is extremely poor, with a 5-year survival rate of less than 5%.

[0005] In recent years, immunotherapy has achieved great success in the clinical treatment of non-small cell lung cancer and other types of cancer. However, the progress of immunotherapy for small cell lung cancer (SCLC) has not yielded satisfactory results. Therefore, there is an urgent need to find other more effective immunotherapies.

[0006] Delta-like protein 3 (DLL3) is present in the Notch signaling pathway and is an inhibitory ligand and a highly tumor-selective protein. The Notch pathway plays a tumor-suppressive role in small cell lung cancer (SCLC) and is expressed in neuroendocrine tumors such as SCLC. Because delta-like protein 3 (DLL3) is highly expressed on the surface of SCLC tumor cells but almost not expressed in normal adult tissues, DLL3 is considered a very attractive target for SCLC immunotherapy.

[0007] Saunders et al. found that delta-like protein 3 (DLL3) is highly expressed on the surface of small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) tumor cells. Therefore, an antibody-drug conjugate (ADC) targeting delta-like protein 3 (DLL3), SC16LD6.5, can effectively eradicate tumor initiating cells (TICs) expressing DLL3 in SCLC and LCNEC PDX (patient-derived xenograft) tumors. Furuta M et al. demonstrated that DLL3 promotes tumor growth, migration, and invasion in an SCLC model by regulating SNAI1 / Snail. A bispecific antibody targeting DLL3 / CD3, AMG 757, is used to increase immune cells and promote apoptosis in SCLC tumor cells. AMG 757 is currently being evaluated in a phase 1 clinical trial (NCT03319940) in SCLC patients. In conclusion, DLL3 can serve as a target for immunotherapy of SCLC.

[0008] Exploring the use of chimeric antigen receptor T cells (CAR-T) targeting DLL3 for the treatment of small cell lung cancer (SCLC) is expected to bring more treatment options to SCLC and is of great significance for the treatment of SCLC. Summary of the Invention

[0009] In view of the shortcomings of the prior art described above, the purpose of this application is to provide an anti-DLL3 nanobody, related products and uses. The anti-DLL3 nanobody of this application has high affinity and can be used as the antigen-binding domain of a chimeric antigen receptor molecule to prepare CAR-T cells. The resulting CAR-T cells have good application prospects in tumor treatment and are used to solve the problems in the prior art.

[0010] To achieve the above objectives, the present invention specifically adopts the following technical solution.

[0011] A first aspect of this application provides an anti-DLL3 nanobody, said nanobody comprising a heavy chain variable region including CDR1, CDR2 and CDR3.

[0012] The amino acid sequence of CDR1 is as shown in one of SEQ ID No. 1-SEQ ID No. 6;

[0013] The amino acid sequence of CDR2 is as shown in one of SEQ ID No. 7-SEQ ID No. 12;

[0014] The amino acid sequence of CDR3 is shown in one of SEQ ID No. 13-SEQ ID No. 18.

[0015] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 7, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 13.

[0016] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 8, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0017] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0018] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 3, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 10, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 15.

[0019] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 16.

[0020] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 4, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 8, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 16.

[0021] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 5, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 11, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 17.

[0022] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0023] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 6, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 12, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 18.

[0024] In some embodiments, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 13.

[0025] In some embodiments, the system further includes frame regions FR1-FR4: the amino acid sequence of frame region FR1 includes the sequence shown in any one of SEQ ID No. 19-SEQ ID No. 22, the amino acid sequence of frame region FR2 includes the sequence shown in any one of SEQ ID No. 23-SEQ ID No. 27, the amino acid sequence of frame region FR3 includes the sequence shown in any one of SEQ ID No. 28-SEQ ID No. 33, and the amino acid sequence of frame region FR4 includes the sequence shown in either SEQ ID No. 34 or SEQ ID No. 35.

[0026] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 19, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 23, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0027] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0028] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0029] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 22, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 25, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 31, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 35.

[0030] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0031] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0032] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 26, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 32, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0033] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0034] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 27, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 33, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0035] In some specific embodiments, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0036] In some embodiments, the sequence of the heavy chain variable region includes A1) or A2).

[0037] A1) An amino acid sequence as shown in any one of SEQ ID No. 36-SEQ ID No. 45;

[0038] An amino acid sequence that has at least 80% homology with the amino acid sequence shown in A2) and has the function of the amino acid sequence defined in A1).

[0039] A second aspect of this application provides a chimeric antigen receptor comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises a nanobody as described in the first aspect.

[0040] In some embodiments, the transmembrane domain is selected from CD8α, CD28, and DAP10.

[0041] In some embodiments, the intracellular domain includes a signaling domain or a co-stimulatory domain.

[0042] In some specific embodiments, the co-stimulatory domain is selected from one or more of 4-1BB, CD28, OX40, ICOS, and DAP10.

[0043] In some specific implementations, the signal structure domain is selected from CD3ζ.

[0044] In some embodiments, the chimeric antigen receptor further includes a hinge region.

[0045] In some specific embodiments, the hinge region is selected from CD8α.

[0046] In some embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the nanobody, the transmembrane domain, and the intracellular domain α.

[0047] In some embodiments, the chimeric antigen receptor further includes a signal peptide.

[0048] In some specific embodiments, the signal peptide is selected from the CD8α signal peptide.

[0049] A third aspect of this application provides an isolated polynucleotide encoding a nanobody as described in the first aspect or a chimeric antigen receptor as described in the second aspect.

[0050] In some embodiments, the nucleic acid sequence of the isolated polynucleotide includes the sequence shown in one of SEQ ID No. 46-SEQ ID No. 55.

[0051] A fourth aspect of this application provides a construct comprising isolated polynucleotides as described in the third aspect.

[0052] The fifth aspect of this application provides a cell comprising a construct as described in the fourth aspect or a genome in which isolated polynucleotides as described in the third aspect are integrated.

[0053] The sixth aspect of this application provides nanobodies as described in the first aspect, chimeric antigen receptors as described in the second aspect, isolated polynucleotides as described in the third aspect, constructs as described in the fourth aspect, or cells as described in the fifth aspect for the preparation of products for detecting DLL3, products that bind to DLL3, or products for the diagnosis or prevention of tumors expressing DLL3.

[0054] A seventh aspect of this application provides a detection product comprising nanobodies as described in the first aspect, chimeric antigen receptors as described in the second aspect, isolated polynucleotides as described in the third aspect, constructs as described in the fourth aspect, or cells as described in the fifth aspect.

[0055] In some implementations, the detection product is used to detect DLL3 or in combination with DLL3.

[0056] An eighth aspect of this application provides a pharmaceutical composition comprising a nanobody as described in the first aspect, a chimeric antigen receptor as described in the second aspect, a separated polynucleotide as described in the third aspect, a construct as described in the fourth aspect, or a cell as described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0057] In some embodiments, the pharmaceutical composition is used for the diagnosis or prevention of tumor diseases associated with DLL3 expression.

[0058] Compared with the prior art, the beneficial effects of this application are as follows:

[0059] (1) In this application, phage display nanobody library was constructed by immunizing unimmunized alpacas with recombinant DLL3 protein. Anti-DLL3 antibodies were screened based on the phage display nanobody library. The obtained nanobody can specifically bind to DLL3 antigen and has good affinity. The half-maximal effective concentration (EC50) of the antibody was determined to be 15.14 ng / mL, 15.72 ng / mL, 11.49 ng / mL, 8.25 ng / mL, 14.53 ng / mL, 10.81 ng / mL, 11.36 ng / mL, 11.10 ng / mL, 6.87 ng / mL and 8.49 ng / mL.

[0060] (2) The anti-DLL3 nanobody provided in this application has good affinity. It is used as the antigen-binding domain to construct a chimeric antigen receptor, and T cells are prepared using the chimeric antigen receptor. The CAR-T cells have killing activity against DLL3-positive tumor cells, and after co-culturing with DLL3-positive cells, they efficiently secrete the cytokine IFN-γ. This shows that the anti-DLL3 nanobody of this application can be effectively applied to immunotherapy and is of great significance for the development of tumor treatment drugs. Attached Figure Description

[0061] Figure 1A This is the EC50 curve of the anti-DLL3 nanobody DLL3-A3 detected by ELISA in Example 2.

[0062] Figure 1B This is the EC50 curve of the anti-DLL3 nanobody DLL3-A5 detected by ELISA in Example 2.

[0063] Figure 1C This is the EC50 curve of the anti-DLL3 nanobody DLL3-A6 detected by ELISA in Example 2.

[0064] Figure 1D This is the EC50 curve of the anti-DLL3 nanobody DLL3-3 detected by ELISA in Example 2.

[0065] Figure 1E This is the EC50 curve of the anti-DLL3 nanobody DLL3-10 detected by ELISA in Example 2.

[0066] Figure 1F This is the EC50 curve of the anti-DLL3 nanobody DLL3-12 detected by ELISA in Example 2.

[0067] Figure 1G This is the EC50 curve of the anti-DLL3 nanobody DLL3-14 detected by ELISA in Example 2.

[0068] Figure 1H This is the EC50 curve of the anti-DLL3 nanobody DLL3-18 detected by ELISA in Example 2.

[0069] Figure 1I This is the EC50 curve of the anti-DLL3 nanobody DLL3-26 detected by ELISA in Example 2.

[0070] Figure 1J This is the EC50 curve of the anti-DLL3 nanobody DLL3-27 detected by ELISA in Example 2.

[0071] Figure 2 shows the FACS detection results of the anti-DLL3 nanobody recognizing the DLL3 antigen in Example 2.

[0072] Figure 3 This is a plasmid map of the chimeric antigen receptor lentiviral vector targeting DLL3 in Example 4.

[0073] Figure 4 This is a schematic diagram of the structure of the chimeric antigen receptor expressing DLL3 in Example 3.

[0074] Figure 5 This is a flow cytometry result of the chimeric antigen receptor expression rate of T lymphocytes in Example 4.

[0075] Figure 6 This is a graph showing the killing effect of CAR-T cells on 293T cells in Example 5.

[0076] Figure 7 This is a graph showing the killing effect of CAR-T cells on 293T cells overexpressing DLL3 (293T-DLL3) in Example 5.

[0077] Figure 8 This is a bar chart showing the secretion of IFNγ by CAR-T cells in Example 5.

[0078] Figure 9The image shows the spectrum of the HD SIN03 CEA 1A6 CAR plasmid in Example 3. Detailed Implementation

[0079] This application utilizes recombinant protein from the extracellular domain of DLL3 (DLL3) to stimulate alpacas to produce high-titer antibodies. A phage display nanobody library was constructed using phage display technology, and 10 candidate antibodies were obtained through panning and amplification, including DLL3-A3, DLL3-A5, DLL3-A6, DLL3-3, DLL3-10, DLL3-12, DLL3-14, DLL3-18, DLL3-26, and DLL3-27. Their reduced molecular weight was found to be less than 40 kDa, which is 1 / 10 of that of ordinary antibodies. ELISA detection showed that the EC50 of all 10 candidate antibodies was relatively low, especially DLL3-26, which had an EC50 of 6.87 ng / mL, indicating strong binding affinity to the antigen. Flow cytometry analysis revealed that all 10 candidate antibodies specifically recognized the DLL3 antigen on the cell surface. Simultaneously, CAR-T cells containing 10 candidate antibodies were constructed, and it was found that they exhibited cytotoxic activity against DLL3-positive tumor cells and could efficiently secrete the cytokine IFN-γ, with a secretion level exceeding 1600 pg / mL. This indicates that the anti-DLL3 nanobody of this application can be effectively applied to immunotherapy and is of great significance for the development of tumor therapeutic drugs. Based on this, this application was completed.

[0080] This application provides a nanobody against DLL3, the nanobody comprising a heavy chain variable region including CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in one of SEQ ID No. 1-SEQ ID No. 6; the amino acid sequence of CDR2 is shown in one of SEQ ID No. 7-SEQ ID No. 12; and the amino acid sequence of CDR3 is shown in one of SEQ ID No. 13-SEQ ID No. 18. The sequences of CDR1, CDR2, and CDR3 are shown in Table 1.

[0081] The nanobodies of this application, also known as heavy chain antibodies or single-domain antibodies, refer to a class of antibody molecules that lack the antibody light chain and only have the variable region of the heavy chain. They have advantages such as small molecular weight, high affinity, weak immunogenicity to the human body, easier storage and transportation, easier expression, and genetic engineering modification.

[0082] Table 1

[0083]

[0084] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 7, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 13.

[0085] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 8, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0086] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0087] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 3, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 10, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 15.

[0088] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 16.

[0089] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 4, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 8, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 16.

[0090] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 5, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 11, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 17.

[0091] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 1, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 14.

[0092] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 6, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 12, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 18.

[0093] In this application, the amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 2, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 9, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No. 13.

[0094] In this application, the heavy chain variable region includes framework regions FR1-FR4. Preferably, the amino acid sequence of FR1 includes the sequence shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, or SEQ ID No. 22. Preferably, the amino acid sequence of FR2 includes the sequence shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, or SEQ ID No. 27. Preferably, the amino acid sequence of FR3 includes the sequence shown in SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, or SEQ ID No. 33. Preferably, the amino acid sequence of FR4 includes the sequence shown in SEQ ID No. 34 or SEQ ID No. 35. The sequences of FR1, FR2, FR3, and FR4 are shown in Table 2.

[0095] Table 2

[0096]

[0097] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 19, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 23, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0098] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0099] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0100] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 22, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 25, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 31, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 35.

[0101] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0102] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 29, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0103] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 26, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 32, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0104] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0105] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 27, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 33, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0106] Preferably, the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 21, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 24, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 30, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No. 34.

[0107] In this application, the amino acid sequence of the heavy chain variable region includes A1) or A2): A1) is the amino acid sequence shown in any one of SEQ ID No. 36-SEQ ID No. 45; A2) is an amino acid sequence having at least 80% homology with the amino acid sequence shown in A1) and having the function of the amino acid sequence defined in A1). The amino acid sequence in A2) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more of the same sequence as any one of SEQ ID No. 36-SEQ ID No. 45. Sequence identity refers to the percentage of identical residues in the sequences being compared. The identity of two or more sequences can be calculated using computational software known in the art, such as NCBI. The specific sequences of SEQ ID No.36, SEQ ID No.37, SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, SEQ ID No.42, SEQ ID No.43, SEQ ID No.44 and SEQ ID No.45 are shown below.

[0108] SEQ ID No. 36 (italics represent the frame area) Bold underline (For CDR area):

[0109] QVQLVESGGGLVQAGGSLRLSCSAS RRTVNI MGWYRQAPGKQRELVAI ISYDGTT HYAESVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYC IQRGTPLDS WGQGTQVTVSS.

[0110] SEQ ID No. 37 (italics represent the frame area) Bold underline(For CDR area):

[0111] EVQLVESGGGLVQAGGSLRLSCAAS RRTVNI MGWYRQAPGKQRELVAV IGYDGTT YYAESVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC MQRGTPLDS WGQGTQVTVSS.

[0112] SEQ ID No. 38 (italics represent the frame area) Bold underline (For CDR area):

[0113] QVQLVESGGGLVQAGGSLRLSCAAS RRTVNV MGWYRQAPGKQRELVAV IGYDGAT YYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC MQRGTPLDS WGQGTQVTVSS.

[0114] SEQ ID No. 39 (italics represent the frame area) Bold underline (For CDR area):

[0115] QVQLVESGGGSVQAGGSLRLSCAAS GNIASING MGWHRQATGKQRELVAF IRSGGSP NYADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYC GADIYGTGY WAKGTLVTVSS.

[0116] SEQ ID No. 40 (bold italics indicate the frame area) underline is the CDR area): QVQLVESGGGLVQAGGSLRLSCAAS RRTVNI MGWYRQAPGKQRELVAV IGYDGAT YYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NQR GTPLDS WGQGTQVTVSS.

[0117] SEQ ID No. 41 (italics represent the frame area) Bold underline (For CDR area):

[0118] EVQLVESGGGLVQAGGSLRLSCAAS RRTINI MGWYRQAPGKQRELVAV IGYDGTTYYAESVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC NQRGTPLDS WGQGTQVTVSS.

[0119] SEQ ID No. 42 (italics represent the frame area) Bold underline (For CDR area):

[0120] EVQLVESGGGLVQAGGSLRLSCAAS GTISGISI MAWHRQAPGKRRELVAS IRGDGST IYGDSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYC GVSLQDLAY WGQGTQVTVSS.

[0121] SEQ ID No. 43 (italics represent the frame area) Bold underline is the CDR area): QVQLVESGGGLVQAGGSLRLSCAAS RRTVNI MGWYRQAPGKQRELVAV IGYDGAT YYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC MQR GTPLDS WGQGTQVTVSS.

[0122] SEQ ID No. 44 (italics represent the frame area) Bold underline is the CDR area): QVQLVESGGGLVQAGGSLRLSCAAS GSTLTINV VGWSRQVPGKQREAVAT ITRSGTT NYATSVKGRFTISRDNAKNTVDLQINSLKPEDSADYYC Y ARTYDNANY WGQGTQVTVSS.

[0123] SEQ ID No. 45 (italics represent the frame area, Bold underline (For CDR area):

[0124] QVQLVESGGGLVQAGGSLRLSCAAS RRTVNV MGWYRQAPGKQRELVAV IGYDGAT YYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC IQRGTPLDS WGQGTQVTVSS.

[0125] The anti-DLL3 nanobody provided in this application can be derived from alpacas, and its overall molecular weight can be about half that of a ScFv single-chain antibody. Therefore, it can effectively reduce the molecular weight of the overall structure, thereby enhancing its tissue penetration, reaching target tissues and organs more effectively, and improving the therapeutic effect. Moreover, this structure is easier to prepare than a structure with two ScFv tandem structures.

[0126] The anti-DLL3 nanobody provided in this application can typically be a murine antibody.

[0127] In this application, phage display technology was used to screen a DLL3-immunized alpaca VHH library. The resulting nanobodies exhibited high affinity and showed significant application potential in constructing chimeric antigen receptors targeting DLL3. Alternatively, existing techniques can be used to obtain anti-DLL3 nanobodies, such as inserting the gene encoding the nanobodies into an expression vector to obtain a recombinant expression vector, introducing the recombinant expression vector into cells and culturing it, followed by separation and purification.

[0128] Another aspect of this application provides a chimeric antigen receptor comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises the anti-DLL3 nanobody described above.

[0129] Preferably, the chimeric antigen receptor further includes a signal peptide for expressing the chimeric antigen receptor on the cell membrane. In a specific embodiment, this is the CD8α signal peptide.

[0130] CD8α signal peptide: MALPVTLPLALLLHAARP (SEQ ID NO.56)

[0131] Preferably, the chimeric antigen receptor further includes a hinge region. More preferably, the hinge region includes a CD8α hinge region.

[0132] Preferably, the transmembrane domain is used to immobilize the chimeric antigen receptor onto the cell membrane of a T cell, and the transmembrane domain is selected from one or more of CD8α, CD28, and DAP10. In a specific embodiment, it is CD8α.

[0133] The amino acid sequences of the CD8α hinge region and transmembrane region are as follows:

[0134] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 57).

[0135] Preferably, the chimeric antigen receptor further includes an intracellular domain. The intracellular domain is generally divided into two main categories, for example, it may include a co-stimulatory domain or a signaling domain. The co-stimulatory domain typically provides a secondary or co-stimulatory signal for complete cell activation in an antigen-independent manner. It can bind to homologous co-stimulatory ligands on antigen-presenting cells to enhance T cell responses. Preferably, the co-stimulatory domain is selected from any one or a combination of at least two of the following: 4-1BB, CD28 intracellular region, OX40, ICOS, or DAP10 intracellular region. In a specific embodiment, it is 4-1BB. The signaling domain is typically based on an immune receptor tyrosine activation motif, usually present in the cytoplasmic tail of various receptors. In a specific embodiment, it is an immune receptor tyrosine activation motif (CD3ζ).

[0136] CD3ζ:

[0137] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 58).

[0138] 4-1BB: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 59).

[0139] In one specific embodiment, the chimeric antigen receptor may sequentially include, from the N-terminus to the C-terminus, a CD8α signal peptide, the aforementioned anti-DLL3 nanobody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and CD3ζ immune receptor.

[0140] Another aspect of this application provides an isolated polynucleotide encoding an anti-DLL3 nanobody as described above or a chimeric antigen receptor as described above.

[0141] In this application, the nucleic acid sequence of the isolated polynucleotide includes the sequence shown in one of SEQ ID No. 46-SEQ ID No. 55.

[0142] SEQ ID No. 46

[0143] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTAGCGCAAGCAGAAGGACAGTGAACATCATGGGATGGTACAGGCAGGCACCCGGCAAGCAGAGGGAGTTGGTGGCTATTATTAGCTATGACGGCACAACACACTACGCCGAGTCCGTGAAAGGAAGATTCACCATCTCCCGCGACAACGCCAAGAACACAGTCTACCTGCAGATGAACAGCCTGAAGGCAGAAGATACTGCTGTGTATTACTGCATCCAGCGCGGAACACCCCTCGACAGCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0144] SEQ ID No.47

[0145] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGGCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCAGGAGGACAGTGAACATTATGGGGTGGTACAGGCAGGCACCAGGAAAGCAGAGGGAGCTGGTGGCAGTGATCGGCTACGACGGAACAACATACTACGCAGAGAGCGTGAAGGGAAGGTTCACAATCAGCAGGGACAACGCTAAGAACACAGTGTATCTCCAGATGAACAACCTGAAACCCGAAGATACTGCTGTGTATTACTGCATGCAGAGGGGAACACCCCTGGACAGCTGGGGGCAGGGCACCCAGGTGACCGTGTCCTCC

[0146] SEQ ID No.48

[0147] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTGCTGCTAGTAGGAGGACAGTGAACGTGATGGGCTGGTACAGGCAGGCCCCAGGCAAGCAGAGGGAGTTGGTGGCTGTTATTGGGTATGACGGAGCTACATACTACGCCGAGTCCGTGAAAGGAAGGTTCACAATCAGCAGGGACAACGCTAAGAACACCGTCTATCTCCAGATGAACTCATTGAAGCCTGAGGATACCGCCGTGTACTATTGTATGCAGAGGGGAACACCCCTGGACAGCTGGGGACAGGGCACCCAGGTGACCGTGAGCAGC

[0148] SEQ ID No.49

[0149] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCTCCGTCCAGGCAGGAGGAAGTCTCAGGCTCTCCTGCGCCGCAAGCGGCAACATCGCCAGTATCAACGGCATGGGCTGGCACAGGCAGGCCACCGGAAAACAGAGGGAGCTGGTGGCTTTCATTAGGTCCGGAGGGTCCCCAAACTACGCTGACTCCGTGAAGGGCCGCTTTACCATCTCCAGGGATAACGCCAAGAATACAGTCCACCTGCAGATGAACAGCCTGAAACCTGAGGACACCGCAGTCTACTACTGCGGCGCCGACATCTACGGCACCGGCTATTGGGCCAAGGGGACCTTGGTGACCGTGTCCTCC

[0150] SEQ ID No.50

[0151] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTGCTGCTAGTAGGAGGACAGTGAACATCATGGGATGGTACAGGCAGGCTCCCGGCAAGCAGAGGGAGTTGGTGGCTGTGATTGGATATGACGGCGCTACATACTACGCCGAGTCCGTGAAAGGAAGATTCACCATCTCCCGCGACAACGCCAAGAACACCGTCTACCTGCAGATGAACAGCCTGAAGCCTGAAGATACCGCCGTGTACTACTGTAACCAGAGGGGAACACCCCTGGACAGCTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0152] SEQ ID No.51

[0153] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGGCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCAGGAGGACAATTAATATCATGGGGTGGTACAGGCAGGCACCAGGAAAGCAGAGGGAGCTGGTGGCAGTGATCGGCTATGACGGCACAACATACTACGCCGAGTCCGTGAAGGGAAGGTTCACAATCTCCAGGGACAACGCTAAGAACACAGTGTACCTGCAGATGAACAACCTGAAACCCGAGGACACCGCCGTCTATTACTGCAACCAGAGGGGAACACCCCTGGACAGCTGGGGCCAGGGGACCCAGGTGACCGTGAGCTCC

[0154] SEQ ID No.52

[0155] GAGGTGCAGTTGGTGGAGAGCGGGGGGGGGCTGGTGCAGGCTGGAGGAAGTTTGAGGTTGAGCTGTGCCGCAAGCGGGACAATTAGCGGGATTAGTATTATGGCCTGGCACAGGCAGGCACCCGGAAAGAGGAGGGAGCTGGTGGCAAGCATCAGGGGAGACGGAAGCACCATCTATGGAGACAGCGTGAAGGGAAGGTTCACCATTAGCAGGGACAACGCAAAGAACACAGCATACTTGCAGATGAACAGCCTGAAACCTGAGGACACCGCAGTGTACTACTGCGGCGTGAGCTTGCAGGACCTGGCCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC

[0156] SEQ ID No.53

[0157] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTGCTGCTAGTAGGAGGACAGTGAACATCATGGGATGGTACAGGCAGGCTCCCGGCAAGCAGAGGGAGTTGGTGGCTGTGATTGGATATGACGGCGCTACATACTACGCCGAGTCCGTGAAAGGAAGATTCACCATCTCCCGCGACAACGCCAAGAACACCGTCTACCTGCAGATGAACAGCCTGAAGCCTGAAGATACCGCCGTGTACTACTGTATGCAGAGGGGAACACCTCTGGACAGCTGGGGCCAGGGCACCCAGGTGACAGTGAGCAGC

[0158] SEQ ID No.54

[0159] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTGCTGCTAGTGGCAGCACACTGACAATCAACGTGGTGGGATGGTCCAGGCAGGTGCCAGGCAAACAGAGGGAGGCTGTGGCAACCATTACAAGGAGCGGAACCACTAAC TACGCCACCTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAATGCAAAGAACACAGTCGACCTCCAGATCAACAGCTTGAAGCCCGAGGACAGCGCAGACTACTGCTACGCCAGGACCTACGACAACGCCAACTGGGGCCAGGGAACCCAGGTGACCGTGAGCTCC

[0160] SEQ ID No. 55

[0161] CAGGTCCAGCTCGTCGAGTCAGGCGGCGGCCTCGTGCAGGCTGGAGGAAGTTTGAGGTTGAGTTGTGCTGCTAGTAGGAGGACAGTGAACGTGATGGGCTGGTACAGGCAGGCCCCAGGCAAGCAGAGGGAGTTGGTGGCTGTTATTGGGTATGACGGAGCTACATACT ACGCCGAGTCCGTGAAAGGAAGGTTCACAATCAGCAGGGACAACGCTAAGAACACCGTCTATCTCCAGATGAACTCATTGAAGCCTGAGGATACCGCCGTGTACTATTGTATCCAGAGGGGAACACCACTGGACAGCTGGGGCCAGGGCACACAGGTGACCGTCAGCAGC

[0162] Another aspect of this application provides a construct comprising the isolated polynucleotides as described above. The construct is typically obtained by inserting the isolated polynucleotides into a suitable vector, and those skilled in the art can select a suitable expression vector. For example, the type of vector may include, but is not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. As another example, it may be any one of a lentiviral vector, a retroviral vector, or an adeno-associated virus vector, preferably a lentiviral vector.

[0163] Another aspect of this application provides a recombinant lentivirus comprising the construct described above. The recombinant lentivirus is prepared by viral packaging of the construct described above. The recombinant lentivirus can be used to infect host cells to prepare CAR cells. The host cells are selected from one or more of T cells, B cells, NK cells, mast cells, or macrophages.

[0164] Another aspect of this application provides a cell comprising the construct described above or with the isolated polynucleotides described above integrated into its genome, thereby enabling the expression of the anti-DLL3 nanobody or chimeric antigen receptor. Methods for introducing the construct into host cells should be known to those skilled in the art, such as microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, etc. Preferably, the host cell is selected from one or more of T cells, B cells, NK cells, mast cells, or macrophages. For example, the T cells are cytotoxic T cells, helper T cells, γδ cells, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4 / CD8 double-negative T cells, CD3+ T cells, naive T cells, effector T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell memory TSCM cells, central memory TCM cells, effector memory TEM cells, or effector memory TEMRA cells.

[0165] Another aspect of this application provides nanobodies as described above, or chimeric antigen receptors as described above, or isolated polynucleotides as described above, or constructs as described above, or cells as described above, for the preparation of products for detecting DLL3, or products that bind to DLL3, or products for the diagnosis or prevention of tumors expressing DLL3.

[0166] Preferably, the product is used to treat tumors expressing DLL3.

[0167] More preferably, the tumors expressing DLL3 include small cell lung cancer.

[0168] Another aspect of this application provides a pharmaceutical composition comprising a nanobody as described above, a chimeric antigen receptor as described above, an isolated polynucleotide as described above, a construct as described above, or a cell as described above, and a pharmaceutically acceptable carrier. The pharmaceutical composition is used for the diagnosis of tumor diseases associated with DLL3 expression. Preferably, the tumors associated with DLL3 expression include small cell lung cancer.

[0169] Preferably, the pharmaceutically acceptable carrier includes any one or a combination of at least two of the following: carrier, surfactant, disintegrant, coating material, excipient, solubilizer, diluent, pH adjuster, binder, wetting agent, colorant, emulsifier, antibacterial agent, cosolvent, osmotic pressure regulator, filler, antioxidant, or buffer.

[0170] Another aspect of this application provides a detection product comprising, as described above, a nanobody, a chimeric antigen receptor, an isolated polynucleotide, a construct, or a cell. The detection product is used to detect DLL3 or binding to DLL3.

[0171] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0172] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0173] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0174] In the following embodiments of this application, pShort phage particles, SU320+Hφ competent cells and NEB alpha 5 F' cells were provided by Chengdu Shengshi Junlian Biotechnology Co., Ltd.; 293T cells were purchased from ATCC.

[0175] Example 1: Obtaining anti-DLL3 nanobodies

[0176] In this embodiment, a phage nanobody library was constructed and panned, and preliminary screening was performed using ELISA. The specific steps are as follows:

[0177] 1.1 Construction of phage nanobody library

[0178] Alpaca were immunized with recombinant protein of the extracellular domain of DLL3 (DLL3) to stimulate B cells to express antigen-specific nanobodies. After serum titers were detected by ELISA, peripheral blood was collected. Lymphocytes were isolated, total RNA was extracted, and then reverse transcribed into cDNA. The VHH gene was then amplified by nested PCR. The VHH gene was inserted into pShort phage particles, electroporated into SU320+Hφ competent cells, and amplified. The phage particles were then separated and purified by PEG8000 / NaCl precipitation to obtain a phage nanobody library. The concentration was adjusted, aliquoted, and stored at -80℃ for later use.

[0179] 1.2 Screening of phage nanobody libraries

[0180] First, 293T cells were co-incubated with the phage nanobody library constructed in step 1.2 for negative screening. Then, the supernatant was collected and incubated with 293T-DLL3 cells (DLL3 positive, see Example 4 for details) and 293T cells, respectively. The cells were washed four times with PT buffer pre-cooled at 4°C. The cells were then infected with NEB alpha 5F. ’ Cells were cultured overnight after the addition of helper phages; the cells were plated using the Drop method, and the enrichment level was calculated the next day; the phages were isolated and purified by PEG8000 / NaCl precipitation and entered the next round of screening; after enrichment, the gene sequence of the nanobody VHH was amplified using the obtained phages as templates, and second-generation sequencing was performed to obtain 10 anti-DLL3 nanobodies, named DLL3-A3, DLL3-A5, DLL3-A6, DLL3-3, DLL3-10, DLL3-12, DLL3-14, DLL3-18, DLL3-26 and DLL3-27, respectively, and their corresponding amino acid sequences are shown in SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44 and SEQ ID NO.45, respectively.

[0181] The nucleotide sequences of the 10 anti-DLL3 nanobodies DLL3-A3, DLL3-A5, DLL3-A6, DLL3-3, DLL3-10, DLL3-12, DLL3-14, DLL3-18, DLL3-26, and DLL3-27 are shown in SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, and SEQ ID No. 55, respectively.

[0182] Example 2: Expression and purification of anti-DLL3 nanobodies

[0183] In this embodiment, the 10 candidate antibodies obtained in Example 1 were expressed, purified, and their EC50 was detected by ELISA, and their specificity was detected by flow cytometry. The steps included:

[0184] 2.1 Expression and purification of VHH Fc nanobodies

[0185] The 10 candidate antibodies obtained in Example 1 were cloned into the eukaryotic expression plasmid pcDNA3.4 with a mouse Fc tag, expressed in CHO cells, and purified by Protein A / G affinity chromatography.

[0186] Ten purified anti-DLL3 VHH antibodies with mouse Fc tags were obtained, including anti-DLL3-A3 VHH Fc antibody, anti-DLL3-A5 VHH Fc antibody, anti-DLL3-A6 VHH Fc antibody, anti-DLL3-3 VHH Fc antibody, anti-DLL3-10 VHH Fc antibody, anti-DLL3-12 VHH Fc antibody, anti-DLL3-14 VHH Fc antibody, anti-DLL3-18 VHH Fc antibody, anti-DLL3-26 VHH Fc antibody, and anti-DLL3-27 VHH Fc antibody. The OD280 absorbance was measured by an ELISA reader and the concentration was calculated. The purity and molecular weight were determined by SDS-PAGE gel chromatography.

[0187] The quality test results of 10 anti-DLL3 VHH antibodies with mouse Fc tags are shown in Table 3.

[0188] Table 3 Protein expression levels and other relevant information

[0189] protein name Concentration (mg / mL) Reduced molecular weight Kda Non-reduced molecular weight Kda Purity (SDS-PAGE) Anti-DLL3-A3 VHH Fc antibody 1.87 39Kda 78Kda >95% Anti-DLL3-A5 VHH Fc antibody 2.46 39Kda 78Kda >95% Anti-DLL3-A6 VHH Fc antibody 2.18 39Kda 78Kda >95% Anti-DLL3-3 VHH Fc antibody 2.70 38Kda 76Kda >95% Anti-DLL3-10 VHH Fc antibody 2.88 39Kda 78Kda >95% Anti-DLL3-12 VHH Fc antibody 2.64 39Kda 78Kda >95% Anti-DLL3-14 VHH Fc antibody 2.26 38Kda 76Kda >95% Anti-DLL3-18 VHH Fc antibody 2.60 39Kda 78Kda >95% Anti-DLL3-26 VHH Fc antibody 2.16 39Kda 78Kda >95% Anti-DLL3-27 VHH Fc antibody 2.37 39Kda 78Kda >95%

[0190] 2.2 ELISA detection of EC50

[0191] The 10 anti-DLL3 VHH antibodies with mouse Fc tags obtained in step 2.1 of this embodiment were subjected to enzyme-linked immunosorbent assay (ELISA) to determine the median effective concentration (EC50) to reflect the antibody affinity for the DLL3 antigen.

[0192] The principle is to use enzyme-labeled anti-antibodies (anti-human immunoglobulin antibodies) to detect the test antibody bound to the solid-phase antigen. The antigen-antibody complex formed on the solid-phase support is separated from other substances in the liquid by washing. Then, enzyme-labeled antigen or antibody is added, which also binds to the solid-phase support through a reaction. At this point, the amount of enzyme on the solid phase is in a certain proportion to the amount of the test substance in the sample. After adding the substrate for the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. The amount of product is directly related to the amount of the test substance in the sample; therefore, qualitative or quantitative analysis can be performed based on the intensity of the color. Because the enzyme's catalytic efficiency is very high, it indirectly amplifies the results of the immunoreaction, enabling the assay method to achieve very high sensitivity. The assay results are shown in Table 4 and... Figure 1A-1J .

[0193] from Figures 1A-1J As shown in Table 3, the EC50 values ​​(ng / mL) of the 10 antibodies are 15.14, 15.72, 11.49, 8.25, 14.53, 10.81, 11.36, 11.10, 6.87 and 8.49, respectively.

[0194] Table 4

[0195] Nanobody EC50 ng / mL Anti-DLL3-A3 VHH Fc antibody 15.14 Anti-DLL3-A5 VHH Fc antibody 15.72 Anti-DLL3-A6 VHH Fc antibody 11.49 Anti-DLL3-3 VHH Fc antibody 8.25 Anti-DLL3-10 VHH Fc antibody 14.53 Anti-DLL3-12 VHH Fc antibody 10.81 Anti-DLL3-14 VHH Fc antibody 11.36 Anti-DLL3-18 VHH Fc antibody 11.10 Anti-DLL3-26 VHH Fc antibody 6.87 Anti-DLL3-27 VHH Fc antibody 8.49

[0196] As shown in Table 4, these 10 nanobodies have a high binding capacity to the DLL3 antigen. Among them, the EC50 of the anti-DLL3-26VHH Fc antibody is the lowest at 6.87 ng / mL, followed by the anti-DLL3-3 VHH Fc antibody and the anti-DLL3-27 VHH Fc antibody, which are 8.25 ng / mL and 8.49 ng / mL, respectively.

[0197] 2.3 Specificity of Flow Cytometry Detection

[0198] 293T cells (purchased from ATCC) and 293T-DLL3 cells (a 293T cell line overexpressing DLL3, see Example 4 for construction process details) were mixed with the purified anti-DLL3 VHH antibody with mouse Fc tag obtained in step 2.1 of this example, incubated on ice for 30 min, and then incubated with APC-labeled goat anti-mouse IgG antibody for 30 min. The results were detected by flow cytometry and are shown in the figure below. Figure 2A and Figure 2B .

[0199] From Figure 2A and Figure 2B It can be seen that the 10 candidate antibodies did not bind to DLL3-negative 293T cells. Figure 2A ), and its binding to DLL3 in the DLL3-overexpressing cell line 293T-DLL3 is very significant ( Figure 2B This indicates that the anti-DLL3 nanobody of this application can specifically recognize the DLL3 antigen on the cell surface.

[0200] Example 3: Construction and Packaging of Lentiviral Vectors

[0201] In this embodiment 3, a lentivirus vector is constructed and packaged to obtain a lentivirus.

[0202] First, a chimeric antigen receptor (i.e., a chimeric antigen receptor carrying a DLL3 CAR) was formed using the purified anti-DLL3 VHH antibody with a mouse Fc tag obtained in step 2.1 of Example 2. A schematic diagram of the chimeric antigen receptor is shown below. Figure 4 As shown, it includes CD8α signal peptide, anti-DLL3 nanobody (anti-DLL3 VHH), CD8α hinge region, transmembrane region, co-stimulatory molecule and immune receptor tyrosine activation motif (CD3ζ).

[0203] The amino acid sequences of the anti-DLL3 nanobodies are shown in SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, and SEQ ID No. 45.

[0204] The amino acid sequence of the signal peptide is as follows:

[0205] MALPVTALLLPLALLLHAARP (SEQ ID NO. 56).

[0206] The amino acid sequences of the CD8α hinge region and transmembrane region are as follows:

[0207] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 57).

[0208] The amino acid sequence of the intracellular region of 4-1BB is as follows:

[0209] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 59).

[0210] The CD3ζ amino acid sequence is:

[0211] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 58).

[0212] Secondly, the lentiviral vector HD SIN03 DLL3 CAR expressing the chimeric antigen receptor was constructed, and its vector map is shown below. Figure 3 As shown.

[0213] The specific steps are as follows:

[0214] 3.1 Construction of lentiviral vectors

[0215] 1) Obtaining fragments of anti-DLL3 nanobodies

[0216] Using the anti-DLL3 VHH cDNA clone (synthesized by Shanghai Baiying Biotechnology Co., Ltd.) as a template, anti-DLL3 nanobodies (anti-DLL3 VHH) were amplified using the primers in Table 5. The PCR reaction system was prepared according to Table 6 (the reagents in the table are from TOYOBO Inc.), and the reaction was carried out according to the PCR procedure shown in Table 7 to obtain 10 PCR amplification products, which are fragments of 10 anti-DLL3 nanobodies.

[0217] Table 5

[0218]

[0219] Table 6

[0220] reagents Volume (µL) 10×buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM upstream primer (primer F in Table 5) 1 10 µM downstream primer (primer R in Table 5) 1 Template DNA (cDNA clone) for each of the 10 anti-DLL3 nanobodies. 1 Deionized water (PCR grade water) 33 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0221] Table 7

[0222]

[0223] 2) Obtaining fragments of CD8α singal DLL3 VHH

[0224] Using the PCR amplification product obtained in step 1) as a template, and BamH-CD8α sig-F and CD8H-R as primers, the PCR reaction system was prepared according to Table 8, and the reaction was carried out according to the PCR procedure shown in Table 7 to obtain the CD8α singal DLL3 VHH fragment.

[0225] BamH-CD8α sig-F:

[0226] gctgcaggtcgactctagaggatcccgccaccatggccttaccagtgaccgccttgctcctgccgctggccttgc (SEQ ID NO. 67).

[0227] CD8H-R: gtcgcggcgctggcgtcgtggt (SEQ ID NO. 68).

[0228] Table 8

[0229] reagents Volume (µL) 10×buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM upstream primer (BamH-CD8α sig-F) 1 10 µM downstream primer (CD8H-R) 1 The PCR amplification products obtained in step 1) 2 Sterile deionized water (PCR grade water) 32 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0230] After PCR, 1% agarose gel electrophoresis was performed to recover fragments of about 700 bp, which were then quantified by UV absorption.

[0231] 3) Obtaining the CD8α hinge-TM-41BB-CD3Z fragment

[0232] Using HD SIN03 CEA1A6 CAR plasmid (plasmid map see...) Figure 9 Using CD8H2-F and Vector-R as primers, the PCR reaction system was prepared according to Table 9. After preparation, the PCR reaction was carried out according to the PCR procedure shown in Table 7.

[0233] CD8H2-F: cgacgccagcgccgcgaccacc (SEQ ID NO. 69).

[0234] Vector-R:tcgataagcttgatatcg (SEQ ID NO. 70).

[0235] Table 9

[0236] reagents Volume (µL) 10×buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM upstream primer (CD8H2-F) 1 10 µM downstream primer (Vector-R) 1 Template DNA (HD SIN03 CEA 1A6 CAR) 1 Sterile deionized water (PCR grade water) 33 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0237] After PCR, 1% agarose gel electrophoresis was performed to recover a fragment of about 700 bp. The fragment was then quantified by UV absorption to obtain the CD8α hinge-TM-41BB-CD3Z fragment.

[0238] 4) Enzyme digestion

[0239] 10 μg of the laboratory-constructed HD SIN03 CEA 1A6 CAR plasmid (plasmid image shown) was added. Figure 9 The vector was digested with BamHI and EcoRI, reacted in a water bath at 37°C for 2 h, and then recovered.

[0240] 5) Connection and Transformation

[0241] The CD8α singal DLL3 VHH fragment from step 2) and the CD8α hinge-TM-41BB-CD3Z fragment from step 3) were respectively ligated with the recovered vector from step 4) using recombinase. The recombinant reaction system is shown in Table 10. After preparation, the reaction was carried out in a water bath at 37°C for 30 min and transformed into Escherichia coli stbl3 competent cells according to conventional methods.

[0242] Table 10

[0243] reagents Usage Step 4) HD SIN03 CEA 1A6 CAR 154.2ng Step 2) CD8α singal DLL3 VHH 10ng Step 3) CD8α hinge-TM-41BB-CD3Z 14ng 5×CE Multis buffer 2µL ExnaseMultis 1.2µL Sterile deionized water (PCR grade water) To 12µL

[0244] Single clones were selected from solid culture medium and inoculated into liquid culture medium, cultured overnight, and then subjected to PCR identification. The PCR reaction system is shown in Table 11, and the PCR program is shown in Table 12. After PCR, positive clones were selected and sent to a sequencing company for further sequencing identification. The sequencing results were as expected. The primer sequences used for sequencing are as follows:

[0245] LV-F2: tcttggttcattctcaagcctc (SEQ ID NO. 71).

[0246] LV-R: gcaacatagttaagaatacc (SEQ ID NO. 72).

[0247] Table 11

[0248] reagents Volume (µL) Taq PCR Master Mix 10 10 µM upstream primer (LV-F2) 1 10 µM downstream primer (LV-R) 1 The bacterial solution in step 5) 1 Sterile deionized water (PCR grade water) 7

[0249] Table 12

[0250]

[0251] The sequencing results were as expected, yielding positive clones expressing the chimeric antigen receptor (collectively referred to as HDSIN03-DLL3 CAR).

[0252] 3.2 Lentiviral Packaging

[0253] 1) with 1.6×10 6 293T cells were seeded in 10cm culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM containing 10% fetal bovine serum.

[0254] 2) Dissolve 5.4 μg of the lentiviral vector HD SIN03-DLL3 CAR obtained in step 3.1 of this embodiment, 6.2 μg of the helper plasmid pMDlg-pRRE, 6.2 μg of the helper plasmid pRSV-REV, and 2.4 μg of the envelope plasmid VSVg in 0.8 mL of serum-free DMEM culture medium, mix well, and obtain a DNA mixture.

[0255] 3) Dissolve 60.6 μg PEI (1 μg / μL) in 0.8 mL of serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25 °C for 5 min to obtain PEI mixture;

[0256] 4) Add the PEI mixture obtained in step 3) to the DNA mixture obtained in step 2). Vortex or gently mix immediately after adding. Incubate at 25°C for 25 minutes to obtain the transfection complex.

[0257] 5) Add 1.6 mL of the transfection complex obtained in step 4) to the 293T cell culture in step 1). After 6 hours, replace with fresh culture medium. After 48 hours, collect the viral supernatant, filter it through a 0.45 μm filter membrane, aliquot it, and store it at -80℃.

[0258] Example 4 Construction of overexpression cell lines

[0259] In this embodiment, the construction of 293T cells overexpressing DLL3 (293T-DLL3) includes the following:

[0260] DLL3-GFP lentivirus was obtained by co-transfection with 14.5 μg of DLL3-GFP plasmid (purchased from Yunzhou Biotechnology), 16.7 μg of helper plasmid pMDlg-pRRE, 16.7 μg of helper plasmid pRSV-REV, and 6.5 μg of envelope plasmid VSVg, following the same procedure as in Example 3.3. 6 Cells were seeded per well into 6-well plates, and 1 mL of DLL3-GFP lentivirus was added to obtain 293T cells overexpressing DLL3 (abbreviated as 293T-DLL3).

[0261] Example 5: Construction of CAR-T cells

[0262] In this embodiment, the lentivirus prepared in step 3.2 of Example 3 is used to transfect T lymphocytes, including the following steps:

[0263] 1) Adjust the density of human PBMCs to 1.5 × 10⁻⁶ using T cell culture medium (X-VIVO + 10% FBS + 300 U / mL IL-2). 6 T cells / mL were activated by adding T Cell TransAct (commercially available magnetic beads coupled with CD3 and CD28) at a volume ratio of 1:100 for 24 h.

[0264] 2) Collect activated T cells and adjust the cell density to 9 × 10⁶ cells / year. 5 The lentivirus obtained in step 3.2 of Example 3 was added at a cell / mL ratio according to a multiplicity of infection (MOI) of 10, and polybrene was added to a final concentration of 8 μg / mL. The culture was incubated overnight at 37°C and 5% CO2, and then replaced with fresh medium. The culture was passaged every 3 days.

[0265] 3) Five days after T-cell infection, 3 × 10⁻⁶ cells were collected. 5 T cells were centrifuged at 500 g for 5 min at 4 °C, the supernatant was discarded, and the cells were washed once with flow cytometry buffer. The cells were resuspended in 50 μL buffer, and 0.5 μg of Rabbit Anti-Camelid VHH Antibody (iFluor488) antibody was added. The cells were incubated on ice for 30 min. After washing once with buffer, the cells were resuspended in 300 μL buffer.

[0266] The expression rate of chimeric antigen receptors in T lymphocytes was detected by flow cytometry. The results are shown in [Figure number missing]. Figure 5 .

[0267] Uninfected T cells served as a control and were treated in the same way as above.

[0268] from Figure 5 The infection efficiencies of the CAR-T cells in each group were 64.4%, 61%, 56%, 67.9%, 56.8%, 59.4%, 68%, 53.8%, 72.3%, and 62.5%, respectively, indicating that CAR-T cells were successfully constructed.

[0269] Example 6: In vitro toxicity assay of CAR-T cells and IFN-γ secretion assay

[0270] In this embodiment, CAR-T cells prepared in Example 4 were used to conduct in vitro toxicity experiments and IFN-γ secretion experiments, including the following steps:

[0271] 6.1 In vitro toxicity test

[0272] 1) Add 50 µL of culture medium to the wells of E-Plate 96.

[0273] 2) Place the E-Plate 96 on the RTCA Station.

[0274] 3) The RTCA system performs a scan (“Scan Plate”) to detect the baseline (Background).

[0275] 4) Take out E-Plate 96 and add 100µL of well-mixed target cell suspension to each well. 293T (DLL3 negative tumor cells) and 293T-DLL3 (Example 4) are used as target cells, so that the number of cells in each well is 15,000 cells / 100µL.

[0276] 5) Place E-Plate 96 in a clean bench at room temperature for 30 minutes.

[0277] 6) Place the E-Plate 96 on the RTCA Station in the incubator.

[0278] 7) Run the program and monitor the resistance value of the cells in each well.

[0279] 8) After 24 hours, pause the procedure and add 50 μL of the CAR-T cells constructed in Example 5 to E-Plate 96 at effector-target ratios of 1:1 and 3:1, respectively. The experimental groups and control groups are as follows:

[0280] Experimental group: target cells + effector cells;

[0281] Control group: Target cells were cultured alone;

[0282] 9) Continue running the program.

[0283] The formula for calculating CAR-T lethality is:

[0284] Kill rate (%) = (1 - CI of target cells in experimental group / CI of target cells in control group) * 100%

[0285] In this experiment, the CI value was inversely proportional to the amount of cell death; the higher the amount of cell death, the lower the CI value. Once the number of dead cells in the wells reached a certain level, i.e., CAR-T cell killing reached a plateau, the CI value no longer decreased. The results are shown below. Figure 6 and Figure 7 .

[0286] from Figure 6 and Figure 7It is known that the CAR-T cells constructed in this application have no significant killing effect on DLL3-negative 293T cells, but have killing activity on DLL3-positive tumor cells (293T-DLL3), indicating that the CAR-T cells constructed in this application not only have efficient tumor killing ability, but also have high specificity.

[0287] 6.2. IFN-γ secretion experiment

[0288] In this embodiment, the secretion of CAR-T cytokine IFN-γ was detected using the HumanIFN-γ ELISA Kit (Lianke Biotechnology, catalog number: EK180-96).

[0289] 6.2.1 Cell Culture Supernatant

[0290] Centrifuge the cell culture with an effect-to-target ratio of 1:1 at 400×g for 10 min to remove the precipitate, and collect the supernatant for testing.

[0291] 6.2.2 Reagent Preparation

[0292] Before testing, restore all reagents and samples to 25°C. If concentrated reagents crystallize, incubate at 37°C until all crystals dissolve. Prepare 1× washing solution and 1× test buffer according to the instructions.

[0293] 6.2.3 Preparation of Standards and Samples

[0294] Standards: The stock solution of the standard was diluted twice with 10% 1640 medium, with a total of 8 dilution gradients, including zero concentration.

[0295] Samples: Dilute the samples using 10% 1640 medium.

[0296] 6.2.4 Detection Procedure

[0297] 1) Soaking the microplate: Add 300 μL of 1× washing buffer and let it stand for 30 seconds. Discard the washing buffer and pat the microplate dry on absorbent paper.

[0298] 2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard well, and add 100 μL of standard diluent to the blank well;

[0299] 3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0300] 4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0301] 5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0302] 6) Washing: Discard the liquid, add 300μL of washing solution to each well and wash the plate 6 times. After each wash, pat the plate dry on absorbent paper.

[0303] 7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well.

[0304] 8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, incubate at 25°C for 45 min, then wash;

[0305] 9) Add substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 20 min;

[0306] 10) Add stop solution: Add 100 μL of stop solution to each well;

[0307] 11) Detection Reading: Within 30 minutes, use a microplate reader to perform dual-wavelength detection, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength; the calibrated OD value is the measured value at 450 nm minus the measured value at the reference wavelength. IFN-γ factor secretion results are as follows: Figure 8 .

[0308] from Figure 8 It was found that the spontaneous MOCK group, being a CAR-T cell-only group, showed almost no detectable cytokine release; the co-culture group of CAR-T cells and 293T cells also showed no significant cytokine release; and after co-culturing with target cells overexpressing DLL3, the IFN-γ secreted by CAR-T cells exceeded 1600 pg / mL. The CAR-T cells constructed in this application released cytokines on DLL3-positive tumor cells, but showed no significant cytokine secretion on DLL3-negative cells.

[0309] In summary, this application immunized alpacas with recombinant protein of the extracellular domain of DLL3 (DLL3) to construct a phage-display nanobody library. Based on this phage-display nanobody library, anti-DLL3 nanobodies were screened to obtain anti-DLL3 nanobodies with CDR amino acid sequences as shown in SEQ ID NO. 36-45 of the heavy chain variable region. ELISA detection of EC50 demonstrated that the anti-DLL3 nanobodies of this application have a high binding capacity to the DLL3 antigen. Flow cytometry detection demonstrated that the anti-DLL3 nanobodies of this application did not bind to DLL3-negative cells, but strongly bound to DLL3-positive cells, and could specifically recognize the DLL3 antigen on the cell surface. A chimeric antigen receptor was constructed using the anti-DLL3 nanobody of this application as the antigen-binding domain, and CAR-T cells were prepared using this chimeric antigen receptor. Experiments verified that the constructed CAR-T cells had killing activity against DLL3-positive tumor cells, and after co-culturing with DLL3-positive cells, they efficiently secreted the cytokine IFN-γ. This indicates that the anti-DLL3 nanobody, its chimeric antigen receptor, and CAR-T cells can be effectively applied to immunotherapy, which is of great significance for the development of tumor therapeutic drugs.

[0310] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. A nanobody against DLL3, characterized in that, The nanobody includes a heavy chain variable region comprising CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 includes the sequence shown in SEQ ID No. 5, the amino acid sequence of CDR2 includes the sequence shown in SEQ ID No. 11, and the amino acid sequence of CDR3 includes the sequence shown in SEQ ID No.

17.

2. The nanobody as described in claim 1, characterized in that, It also includes frame regions FR1-FR4: the amino acid sequence of frame region FR1 includes the sequence shown in SEQ ID No. 20, the amino acid sequence of frame region FR2 includes the sequence shown in SEQ ID No. 26, the amino acid sequence of frame region FR3 includes the sequence shown in SEQ ID No. 32, and the amino acid sequence of frame region FR4 includes the sequence shown in SEQ ID No.

34.

3. The nanobody as described in claim 1, characterized in that, The sequence of the heavy chain variable region includes A1) or A2). A1) The amino acid sequence as shown in SEQ ID No. 42; An amino acid sequence that has at least 80% homology with the amino acid sequence shown in A2) and has the function of the amino acid sequence defined in A1).

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises a nanobody as described in any one of claims 1-5; Preferably, the transmembrane domain is selected from one of CD8α, CD28, and DAP10; Preferably, the intracellular domain includes a signaling domain or a co-stimulatory domain; the co-stimulatory domain is selected from one or more of 4-1BB, CD28, OX40, ICOS, and DAP10; more preferably, the signaling domain is selected from CD3ζ. Preferably, the chimeric antigen receptor further includes a hinge region; more preferably, the hinge region is selected from CD8α. Preferably, the chimeric antigen receptor further includes a signal peptide; more preferably, the signal peptide is selected from the CD8α signal peptide. Preferably, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the nanobody, the transmembrane domain, and the intracellular domain.

5. An isolated polynucleotide, characterized in that, Encoding the nanobody as described in any one of claims 1-3 or the chimeric antigen receptor as described in claim 4; Preferably, the nucleic acid sequence of the polynucleotide includes the sequence shown in SEQ ID No.

52.

6. A construct, characterized in that, It contains the isolated polynucleotide as described in claim 5.

7. A cell, characterized in that, It comprises a construct as described in claim 6 or a genome in which the isolated polynucleotide as described in claim 5 is integrated.

8. The nanobody as described in any one of claims 1-3, or the chimeric antigen receptor as described in claim 4, or the isolated polynucleotide as described in claim 5, or the construct as described in claim 6, or the cell as described in claim 7, in the preparation of products for detecting DLL3, products binding to DLL3, or products for diagnosing or preventing tumors expressing DLL3.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, an isolated polynucleotide as described in claim 5, a construct as described in claim 6, or a cell as described in claim 7, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is used for the diagnosis of or in connection with tumors expressing DLL3.

10. A testing product, characterized in that, The detection product comprises a nanobody as described in any one of claims 1-3, a chimeric antigen receptor as described in claim 4, an isolated polynucleotide as described in claim 5, a construct as described in claim 6, or a cell as described in claim 7; preferably, the detection product is used to detect DLL3 or to bind to DLL3.

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