Humanized antibody targeting DLL3 and application thereof

Abstract

The invention discloses a humanized antibody targeting DLL3 and an application of the humanized antibody. The amino acid sequence of a heavy chain variable region of the antibody is designed to be any one shown in SEQ ID NO: 1-SEQ ID NO: 9, the amino acid sequence of a light chain variable region of the antibody is designed to be any one shown in SEQ ID NO: 10-SEQ ID NO: 18, and the amino acid sequence of any heavy chain variable region and the amino acid sequence of any light chain variable region are paired and combined in pairs. The humanized antibody targeting the DLL3 target spot has strong persistence and immune memory, can survive in a body for a long time, continuously monitors and removes residual or recurrent tumor cells, reduces the recurrence risk, mainly aims at advanced cancer patients, has obvious killing effect on lung cancer tumors, and has a good clinical effect on treatment of advanced cancers.

Description

Technical Field

[0001] The present invention relates to a humanized monoclonal antibody, and in particular to a humanized antibody targeting DLL3 and application of the antibody in the preparation of immunotherapy drugs. Background Art

[0002] Delta-Like Ligand 3 (DLL3) is a member of the Delta-like ligands (DLLs) family. DLLs are single-pass transmembrane proteins that attach to the cell surface. Human DLL3 protein consists of 619 amino acids and is characterized by a 40-amino acid N-terminal conserved DSL (Delta, Serrate, Lag2) domain, six EGF-like repeats, and a transmembrane domain. The DSL domain is highly conserved within the ligand family and is essential for binding to the Notch receptor.

[0003] DLL3 is highly conserved throughout evolution, with human and mouse DLL3 protein sequences sharing up to 82% identity. DLL3 RNA is primarily expressed in the brain, endocrine tissues, and blood; DLL3 alone is barely expressed in normal tissues. Studies have found that DLL3 is highly expressed in approximately 80% of small cell lung cancers (SCLC) and other neuroendocrine tumors. This differential expression of DLL3 in normal and tumor cells makes it an attractive target for tumor-selective therapeutics.

[0004] Small cell lung cancer (SCLC) is a poorly differentiated neuroendocrine tumor and the most aggressive type of lung cancer. The development and progression of SCLC is closely linked to the Notch signaling pathway, which regulates many essential processes necessary for normal development. Inactivating mutations in this pathway can induce neuroendocrine differentiation in non-neuroendocrine tumor cells or tumor precursors. DLL3, the sole inhibitory ligand of the Notch signaling pathway, is highly expressed in 85% of SCLCs. This information suggests that DLL3 may be a target for small cell lung cancer and other neuroendocrine tumors. Summary of the Invention

[0005] Based on the above problems, the problem to be solved by the present invention is to provide a monoclonal antibody against human DLL3, which can also be said to be a specific antibody, and the antibody can specifically recognize the DLL3 target.

[0006] The technical solutions of the present invention are as follows:

[0007] A humanized antibody targeting DLL3, comprising a heavy chain variable region (VH) and a light chain variable region (VL); the amino acid sequence of the heavy chain variable region of the antibody is designed as shown in SEQ ID NO: 1 to SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is designed as shown in SEQ ID NO: 11 to SEQ ID NO: 20, and the amino acid sequence of any of the heavy chain variable regions is paired with the amino acid sequence of any of the light chain variable regions.

[0008] In one embodiment, the light and heavy chain variable regions of a humanized antibody can be tandemly connected into an scFv sequence to construct a chimeric antigen receptor targeting human DLL3; the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain; wherein:

[0009] 1) The extracellular domain of the chimeric antigen receptor includes an antigen binding domain; the antigen binding domain includes an scFv, and the scFv is the light and heavy chain variable region of each antibody;

[0010] 2) The transmembrane domain of the chimeric antigen receptor includes the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, the hinge region of CD8 and the transmembrane domain of ICOS, etc.; wherein, the transmembrane domain of the chimeric antigen receptor is the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, or the costimulatory domain of 4-1BB or ICOS;

[0011] 3) The intracellular domain of the chimeric antigen receptor includes a co-stimulatory signal transduction region, a cytokine receptor intracellular region and a CD3ζ chain portion; wherein, the co-stimulatory signal transduction region refers to a portion of the intracellular domain that includes a co-stimulatory molecule; the co-stimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens; the intracellular domain can be a combination of any one of the co-stimulatory domains of CD28, 4-1BB, and ICOS and the intracellular activation signal CD3ζ.

[0012] In one embodiment, the nucleic acid molecule encoding the humanized antibody contains the humanized antibody described above;

[0013] In one embodiment, an expression cassette can be constructed using the gene of a humanized antibody, and this expression cassette is transferred into 293T cells via a delivery system for antibody expression; wherein the delivery system can be one of lentivirus, retrovirus, ordinary plasmid vector, episomal vector, nano-delivery system, electrotransduction and transposon; preferably, the recombinant vector can contain a humanized antibody (e.g., ordinary plasmid vector) or a chimeric antigen receptor (e.g., lentivirus).

[0014] In the present invention, the recombinant cells containing humanized antibodies are immune cells, which can be any one of T cells, NK cells, NKT cells, macrophages, gamma-deltaT cells, TIL cells, and TCR-T cells; specifically, when the immune cells express chimeric antigen receptors CAR, NK cells, NKT cells, TILs, and gamma-deltaT cells are equivalent to T cells (or T cells can replace NK cells).

[0015] The above-mentioned humanized antibodies, nucleic acid molecules, expression cassettes, recombinant vectors and recombinant cells can be used as raw materials or components of biological preparations. Biological preparations, humanized antibodies, nucleic acid molecules, expression cassettes, recombinant vectors and recombinant cells can be widely used in the preparation of drugs for preventing and treating tumors or cancers.

[0016] The humanized antibody targeting DLL3 provided by the present invention has strong persistence and immune memory, can survive in the body for a long time, continuously monitor and eliminate residual or recurrent tumor cells, reduce the risk of recurrence, and is mainly aimed at patients with advanced cancer. It has a more obvious killing effect on lung cancer tumors and has a good clinical effect on the treatment of advanced cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the affinity curve of DLL3;

[0018] Figure 2 This is a growth curve of T cell culture expansion;

[0019] Figure 3 Flow cytometry graph for T cell CAR positivity detection;

[0020] Figure 4 is a graph showing the killing rate of T cells in vitro;

[0021] Figure 5 This is a curve diagram of tumor size changes in mice;

[0022] Figure 6 is the curve of mouse weight changes;

[0023] Figure 7 This is the mouse survival curve. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0025] The humanized antibody provided by the present invention is an antibody or fragment thereof targeting DLL3, which is a monoclonal antibody and can also be called a humanized antibody targeting DLL3.

[0026] This monoclonal antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). To match the DLL3 target, the amino acid sequence of the heavy chain variable region (VH) is designed to be any one of SEQ ID NO:1 to SEQ ID NO:9, and the amino acid sequence of the light chain variable region (VL) is designed to be any one of SEQ ID NO:10 to SEQ ID NO:18. The amino acid sequences corresponding to the heavy and light chain variable regions of these humanized antibodies are paired and used in combination. Specific preferred combinations of heavy and light chains are as follows:

[0027] a1. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 1 and as shown in SEQ ID NO: 10; or

[0028] a2. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 11; or

[0029] a3. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 12; or

[0030] a4. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 13; or

[0031] a5. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 5 and as shown in SEQ ID NO: 14; or

[0032] a6. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 6 and as shown in SEQ ID NO: 15; or

[0033] a7. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 7 and as shown in SEQ ID NO: 16; or

[0034] a8. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 8 and as shown in SEQ ID NO: 17; or

[0035] a9. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 9 and as shown in SEQ ID NO: 18; or

[0036] a10. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 1 and as shown in SEQ ID NO: 13; or

[0037] a11, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 11; or

[0038] a12. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 14; or

[0039] a13. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 12; or

[0040] a14. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 5 and as shown in SEQ ID NO: 17; or

[0041] a15. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 6 and as shown in SEQ ID NO: 15; or

[0042] a16. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 7 and as shown in SEQ ID NO: 18; or

[0043] a17. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 8 and as shown in SEQ ID NO: 16; or

[0044] a18. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 9 and the paired combination as shown in SEQ ID NO: 10.

[0045] In the humanized antibodies targeting the target DLL3, the amino acid sequences of the heavy chain variable region and the light chain variable region can also be used separately; that is, either the amino acid sequence of the heavy chain variable region is used separately, or either the amino acid sequence of the light chain variable region is used separately; or the amino acid sequences of the two heavy chain variable regions can be used in combination, or the amino acid sequences of the two light chain variable regions can be used in combination.

[0046] The amino acid sequences of the heavy chain variable region and light chain variable region designed by the present invention are as follows.

[0047] 1. The amino acid sequence of the heavy chain variable region is as follows:

[0048] As shown in SEQ ID NO: 1:

[0049] EVQLVQPGRSLRLSCAASGFTFDDYAMNWVRQAPGKGLEWVSGISWNSDSIGYAESVKGRFTIS RDNAKNSLYLQMNSLRAEDTAFYYCAKDMEGSGYDSGYFDSWGQGTLVTVSS;

[0050] As shown in SEQ ID NO: 2:

[0051] EVQLVQSGGGLIQPGRSLRLSCKASGFIFRDHIMGWVRQAPGKGLEWVSAISANGGNTFYADSV KGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAKDSGSFDYWGQGTTVTVSS;

[0052] As shown in SEQ ID NO: 3:

[0053] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG II NPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGDILTGPI FDYWGQGTLVTVSS;

[0054] As shown in SEQ ID NO: 4:

[0055] QVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGLEWVGRI IPI LGIANYAQEF QGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDHWGRGTTVTVSS;

[0056] As shown in SEQ ID NO:5:

[0057] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG I IPI FGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASFDYWGQGTLVTVSS;

[0058] As shown in SEQ ID NO:6:

[0059] EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKF QGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAGDLRYSGYDFRRPTRNYFDYWGQGTTVTVSS;

[0060] As shown in SEQ ID NO:7:

[0061] EVQLVESGAEVKKPGSSVKVSCKASGDTFSSYAISWVRQAPGQGLEWVGRI IPI LGIANYAQEF QGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTTVTVSS;

[0062] As shown in SEQ ID NO:8:

[0063] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRI IPI LGIANYAQEF QGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTLVTVSS;

[0064] As shown in SEQ ID NO:9:

[0065] QVQQVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRI IPI LGIANYAQEF QGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTTVTVSS.

[0066] 2. The amino acid sequences of the above light chain variable regions are as follows:

[0067] As shown in SEQ ID NO:10

[0068] NFMLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFS GSKSGTSASLAISGLQSEDEADYYCAAWDDSLGAWVFGGGTKVTVL;

[0069] As shown in SEQ ID NO:11

[0070] EIVLTQSPDFQSATPKEKVI ITCRASQSIGTGLHWYQQKPDQSPKLLIKYASQSISGVPSRFSG SGSGTDFTLTINSLEAEDAAAYYCHQSISLPTFGGGTKVDIK;

[0071] As shown in SEQ ID NO:12

[0072] DIVMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASALQSGVPSRFSG SGSGTDFTLTISSLQPEDFGTYYCQQTYSPPLTFGGGTKVDIK;

[0073] As shown in SEQ ID NO:13

[0074] DIVMTQSPSSLSALVGDRVTITCRTSQSISTLLNWYQQKPGRAPKLLIYAASTLQSGVPSRFSG TGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK;

[0075] As shown in SEQ ID NO:14

[0076] QSALTQPRSVSGSPGQSVTISCAGTSSDVGEYDYVSWYQQHPGKPPKLI IYDVTERPSGVPDRF AGSTSGNTASLTISGLQTEDEADYYCSSYAGSYSHVFGTGTKVTVL;

[0077] As shown in SEQ ID NO:15

[0078] EIVLTQSPSSLSAFVGDRVTITCRASQGIRNDLGWYQQKPGKVPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYTYPRTFGQGTKVEIK;

[0079] As shown in SEQ ID NO: 16:

[0080] DIQLTQSPSSLSASLGDRVTITCRASQSIDSYLNWYQHKPGKAPKLLIYGASTLQSGVPSRFSG TGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK;

[0081] As shown in SEQ ID NO: 17:

[0082] DIVMTQTPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGSDFTLTISSLQPEDFATYYCQQTYSIPTGTFGQGTKVEIK;

[0083] As shown in SEQ ID NO: 18:

[0084] DIVMTQTPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK.

[0085] For each combination when the amino acid sequences of the corresponding complementary determining regions in the heavy and light chain variable regions of the above-mentioned monoclonal antibodies are paired and expressed as humanized antibodies, the present invention also requires designing corresponding protein structures; for example, taking the above-mentioned combinations a1 to a18 as an example, the designed protein structures are as follows:

[0086] SP (signal peptide)-VH-CH (human IgG1)-SP (signal peptide)-VL-CL (human Kappa);

[0087] in,

[0088] The amino acid sequence of SP is shown in SEQ ID NO: 19: MHSSALLCCLVLLTGVRA;

[0089] The amino acid sequence of CH is shown in SEQ ID NO: 20:

[0090] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0091] The amino acid sequence of CL is shown in SEQ ID NO: 21:

[0092] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0093] In the above-designed protein structure, the amino acid sequence of VH is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 9, and the amino acid sequence of VL is selected from any one of SEQ ID NO: 10 to SEQ ID NO: 18; at the same time, the purified humanized antibodies expressed by the corresponding protein structures are named SDL3-1, SDL3-2, SDL3-3, SDL3-4, ..., SDL3-10 (preferably 10 humanizations are named in the present invention, such as the amino acid sequences corresponding to the combinations a1 to a10), and each corresponds to a corresponding amino acid sequence combination; for example, for the above-mentioned a1 combination, the corresponding VH amino acid sequence is shown in SEQ ID NO: 1, the VL amino acid sequence is shown in SEQ ID NO: 10, and the purified humanized antibody is named SDL3-1; for the above-mentioned a2 combination, the corresponding VH amino acid sequence is shown in SEQ ID NO: 2, the VL amino acid sequence is shown in SEQ ID NO: 11, and the purified humanized antibody is named SDL3-2, ..., for the above-mentioned a10 combination, the corresponding VH amino acid sequence is shown in SEQ ID NO: NO:2, the amino acid sequence of VL is as shown in SEQ ID NO:1, the purified humanized antibody is named SDL3-13 and the purified humanized antibody is named SDL3-10, and the same applies to other combinations.

[0094] During the design of protein antibodies, the light and heavy chain variable regions of humanized antibodies can be concatenated into an scFv sequence to construct a chimeric antigen receptor targeting human DLL3; the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain; wherein:

[0095] 1) The extracellular domain of the chimeric antigen receptor includes an antigen binding domain, which includes an scFv;

[0096] 2) The transmembrane domain of the chimeric antigen receptor includes the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, the hinge region of CD8 and the transmembrane domain of ICOS, etc.; wherein, the transmembrane domain of the chimeric antigen receptor is the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, or the costimulatory domain of 4-1BB or ICOS;

[0097] 3) The intracellular domain of the chimeric antigen receptor includes a co-stimulatory signal transduction region, a cytokine receptor intracellular region and a CD3ζ chain portion; wherein, the co-stimulatory signal transduction region refers to a portion of the intracellular domain that includes a co-stimulatory molecule; the co-stimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens; the intracellular domain can be a combination of any one of the co-stimulatory domains of CD28, 4-1BB, and ICOS and the intracellular activation signal CD3ζ.

[0098] After constructing a plasmid expression cassette for the humanized antibody gene, the expression cassette is transferred into 293T cells via a delivery system for antibody expression. The delivery system can be a lentivirus, a retrovirus, a conventional plasmid vector, an episomal vector, a nanoparticle delivery system, electrotransduction, or a transposon. The expression cassette vector is selected from one or more of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, and other gene transfer systems. Preferably, the vector is a viral vector.

[0099] The above-mentioned humanized antibodies can be used to construct recombinant cells by gene modification and insertion, which are generally immune cells, including any of T cells, NK cells, NKT cells, macrophages, gamma-deltaT cells, TIL cells, and TCR-T cells. Specifically, when the immune cells express chimeric antigen receptors CAR, NK cells, NKT cells, TILs, and gamma-deltaT cells are equivalent to T cells (or T cells can replace NK cells).

[0100] Any of the aforementioned humanized antibodies targeting DLL3 of the present invention can be formulated into pharmaceutically acceptable carriers, diluents, or excipients, all of which exhibit good formulation properties while maintaining good efficacy; for example, they can be used in biomaterials and / or biological preparations. For example, the biological preparations comprise an expression cassette (e.g., a gene expression cassette), a recombinant vector (e.g., a plasmid), a recombinant protein (e.g., a fusion protein, an antibody protein, etc.), a recombinant microorganism (e.g., Escherichia coli, a bacteriophage, etc.), or a recombinant cell line (e.g., an immune cell, a CHO cell, etc.) constructed from the aforementioned nucleic acid sequence or amino acid sequence, wherein the nucleic acid sequence or amino acid sequence is derived from the aforementioned monoclonal antibody; wherein the recombinant vectors include genetic recombinant expression vectors and chimeric antigen receptors.

[0101] When the humanized antibody is used in a biological preparation, it exists as a component of the biological preparation configuration, and the biological preparation component also includes a reagent for detecting the concentration of human DLL3 protein, a reagent for detecting the expression level of human DLL3 protein on the surface of tumor cells, an antibody-coupled toxin to kill human DLL3-positive cells, an antibody coupled with other antibodies to produce polyclonal antibodies targeting human DLL3 and other antigens, and an antibody coupled with other proteins to produce a functional recombinant protein targeting human DLL3.

[0102] Through Biopanning screening of a fully human library (Order Number: 22000134F; Manufacturer: Pujian Biotech (Wuhan) Technology Co., Ltd.), 100 candidate antibodies that may bind to the DLL3 target antigen were screened from the human library. The specific DLL3 target screening process is as follows.

[0103] Screening was performed in a human library; target proteins were selected, such as DLL3(Mouse)-6His and DLL3(Human)-6His; negative controls were selected, such as DLL1(Mouse)-6His, DLL1(Human)-6His, DLL4(Mouse)-6His, and DLL4(Human)-6His; library information was used, such as a library capacity of 5.37×10 10 pfu's human library.

[0104] The specific process for DLL3 target screening is as follows:

[0105] 1. Immunotube solid phase panning

[0106] 1.1. Coat immunotubes: Antigen protein DLL3 (Mouse)-6His / DLL3 (Human)-6His (50 μg / ml, CBS buffer), 1000 μl per tube, 1 tube in total, incubate at 4°C overnight;

[0107] 1.2. Wash the immunotubes 3 times with 5 ml PBST;

[0108] 1.3. Block the immunotube with 5 ml of 5% skim milk / PBST at 30°C for 1 hour;

[0109] 1.4 Wash the immunotube once with 5 ml PBS;

[0110] 1.5. Add 1000ul 10 11 -10 12 pfu of library phage (or amplified phage screened in the previous round), incubated at 30°C for 2 h;

[0111] 1.6. Wash 4-6 times with 5 ml PBST (the number of washes can be increased in later rounds depending on the degree of phage enrichment);

[0112] 1.7. Add 500 μl of Gly-HCl (pH = 2.2) to the immunotube to elute the phages and incubate at room temperature with shaking for about 6-8 minutes; add 120-130 μl of Tris-HCl (pH = 9.6) to neutralize the solution to pH = 7.0-8.0.

[0113] 1.8. Dilute the eluted phage, infect E. coli TG1 in the logarithmic phase, and plate the resulting solution to determine the titer.

[0114] 2. Amplification of eluted phage

[0115] 2.1. Take the phage infected with TG1 in step 1.8, let it stand at 37°C for 30 minutes, and then incubate it at 220 rpm for 30 minutes to 1 hour;

[0116] 2.2. Replenish the culture medium containing antibiotic Amp and culture at 37°C, 220 rpm until the bacterial solution OD = about 0.4-0.6;

[0117] 2.3. Add helper phage to the bacterial solution; incubate at 37°C for 30 minutes, then incubate at 220 rpm for 45 minutes to 1 hour;

[0118] 2.4. Centrifuge the bacterial solution at 3000-5000 rpm and discard the supernatant. Resuspend the bacteria in an equal volume of 2YT-Amp-Kan medium and incubate at 30°C, 220 rpm overnight.

[0119] 2.5. The next day, centrifuge the bacterial solution at 8000 rpm for 20 minutes at 4°C. Transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5*PEG / NaCl solution. Mix thoroughly and place on ice or at 4°C for 1-2 hours.

[0120] 2.6. Centrifuge the bacterial suspension obtained in step 2.5 at 8000 rpm for 30 minutes at 4°C and discard the supernatant. Resuspend the pellet in approximately 1 ml of PBS. Centrifuge the suspension again at 8000 rpm for 10 minutes and transfer the supernatant to a fresh centrifuge tube.

[0121] 2.7. Dilute the amplified phage, infect TG1 in the logarithmic phase, and plate to determine the titer.

[0122] According to the panning conditions shown in Table 1, the above-mentioned immunotube panning step and the amplified phage step were repeated 4 times. The obtained panning results are shown in Table 2.

[0123] Table 1 Conditions for each round of panning

[0124] Number of rounds Coating antigen concentration (μg / mL) Blocking buffer Elution times Elution product volume (ul) 1 DLL3(Human)-6His(50) 1% Casein / PBST 5 800 2 DLL3(Mouse)-6His(50) 5% milk / PBST 6 700 3 DLL3(Human)-6His(50) 1% Casein / PBST 6 600 4 DLL3(Mouse)-6His(50) 5% milk / PBST 7 600

[0125] Table 2 Panning results

[0126] Number of rounds Protein (ug / ) Input(pfu) Output(pfu) / antigen 1 DLL3(Human)-6His(50) <![CDATA[2×10 12 ]]> <![CDATA[4.7×10 5 <!-- 7 -->]]> 2 DLL3(Mouse)-6His(50) <![CDATA[9.3×10 11 ]]> <![CDATA[5.6×10 5 ]]> 3 DLL3(Human)-6His(50) <![CDATA[7.71×10 11 ]]> <![CDATA[1.1×10 5 ]]> 4 DLL3(Mouse)-6His(50) <![CDATA[1.5×10 12 ]]> <![CDATA[9.9×10 5 ]]>

[0127] Based on the panning results in Table 2, ELISA assays were performed on the selected polyclonal and monoclonal phages to screen for candidate antibodies that bind to the DLL3 target antigen. The ELISA assay steps for the polyclonal and monoclonal phages are as follows.

[0128] 3. Polyclonal phage ELISA test

[0129] 3.1、Coating immunoplate:

[0130] AG1:4μg / ml DLL3(Mouse)-6His;

[0131] AG2:4μg / ml DLL3(Human)-6His;

[0132] NC1: 2μg / mlDLL-1(Human)-6His+2μg / mlDLL-4(Human)-6His+2μg / ml;

[0133] NC2: PBS; buffer PBS, 100ul, 4℃ overnight;

[0134] 3.2. Wash three times with 300 μl PBST;

[0135] 3.3. Block with 300 μl of 5% skim milk / PBST at 30°C for 1 hour, and wash 2-3 times with 300 μl of PBST.

[0136] 3.4. Dilute the phage amplified in step 2.6 with PBS in each round, increasing the dilution factor by 3 times; the initial concentration is 10 12 pfu / ml; add 100 μl of diluted amplified phage to each well; incubate at 30°C for 1 hour, and wash 4-6 times with 300 μl of PBST;

[0137] 3.5. Add 100 μl of secondary antibody (anti-phage M13) dilution, incubate at 30°C for 1 hour, and wash 4-6 times with 300 μl of PBST.

[0138] 3.6. Add 100 μl of TMB colorimetric solution and develop for 3-8 min in the dark. Add 100 μl of 2M HCl to terminate the reaction and read the result on a microplate reader (450 nm-620 nm). The ELISA results of the polyclonal phage were obtained, as shown in Table 3.

[0139] Table 3 Polyclonal phage ELISA results

[0140]

[0141] In Table 3:

[0142] Ag1: DLL3 (Mouse)-6His;

[0143] Ag2: DLL3 (Human)-6His;

[0144] NC1:2μg / ml DLL1(Human)-6His+2μg / ml DLL4(Human)-6His;

[0145] Nc2:PBS.

[0146] Based on the polyclonal ELISA screening results in step 3, monoclonal screening was performed.

[0147] 4. Monoclonal phage ELISA screening

[0148] 4.1. After 4 rounds of screening, dilute the phage eluted in step 2.6 10-fold, infect TG1 cells in the logarithmic phase, and plate for culture;

[0149] 4.2. The next day, 384 single colonies were picked from the plate, plated into a 96-deep-well plate, and cultured at 37°C, 250 rpm, with shaking until the bacterial solution reached an OD value of 0.4-0.6. Helper phage was then added to the 96-deep-well plate medium. The culture was allowed to stand at 37°C for 30 min, and then cultured at 37°C, 250 rpm, with shaking for another 45 min-1 h.

[0150] 4.3. Centrifuge the 96-well plate at 4000 rpm for 5 minutes and discard the supernatant. Resuspend the bacterial solution in each well with 2YT-Amp-Kan medium and incubate with shaking at 30°C and 250 rpm for 24 hours. Centrifuge the 96-well plate at 4000 rpm for 10-15 minutes and collect the supernatant for ELISA.

[0151] 4.4. Coating the immunoplate

[0152] AG:2μg / ml DLL3(Mouse)-6His+2μg / ml DLL3(Human)-6His;

[0153] NC: 2μg / ml DLL1(Human)-6His+2μg / ml DLL4(Human)-6His100 ul, overnight at 4℃;

[0154] 4.5. Wash three times with 300 μl of PBST; then block with 300 μl of 5% skim milk / PBST at 30°C for 1 hour; then wash 2-3 times with 300 μl of PBST;

[0155] 4.6. Add 100 μl of phage supernatant to each well; incubate at 30°C for 1 hour; wash 4-6 times with 300 μl of PBST;

[0156] 4.7. Add 100 μl of secondary antibody (anti-M13) dilution, incubate at 30°C for 1 hour, and wash 4-6 times with 300 μl of PBST.

[0157] 4.8. Add 100 μl of TMB colorimetric solution and develop for 3-8 min in the dark. Add 100 μl of 2M HCl to terminate the reaction. Read the plate reader (450 nm-620 nm) to obtain the ELISA results of the monoclonal antibody, as shown in Tables 4 to 7.

[0158] Table 4 Monoclonal phage ELISA results (R4P1-AG)

[0159]

[0160] In Table 4, Ag: DLL3(Mouse)-6His+DLL3(Human)-6His.

[0161] Table 5 Monoclonal phage ELISA results (R4P1-NC)

[0162]

[0163] In Table 5:

[0164] NC: ectin-1(Human)-6His+Nectin-2(Human)-6His+Nectin-3(Human)-6His.

[0165] Table 6 Monoclonal phage ELISA results (R4P2-AG)

[0166]

[0167] In Table 6, Ag: DLL3(Mouse)-6His+DLL3(Human)-6His.

[0168] Table 7 Monoclonal phage ELISA results (R4P2-NC)

[0169]

[0170] In Table 7, Nc: DLL1(Human)-6His+DLL2(Human)-6His.

[0171] Sequence the positive clones of candidate antibodies that bind to the DLL3 target antigen identified in step 4.8, remove doublets and repetitive sequences, and obtain the final positive clones. These positive clones will be validated by a secondary ELISA assay according to step 5 below to ensure the authenticity of the positive results.

[0172] 5. Secondary ELISA verification of positive clones

[0173] The above-screened antibodies were subjected to a secondary ELISA test evaluation experiment. The operation steps are as follows:

[0174] 5.1. Prepare 100 ng / well of antigen for coating and incubate at 4°C overnight.

[0175] 5. Block with 2.5% BSA for 1.5 h;

[0176] 5.3. Prepare purified antibody with an initial concentration of 10 μg / ml and serially dilute 10 times to a final volume of 100 μl / well. Incubate at 37°C for 2 h.

[0177] 5.4. Add 100 μl of Goat Anti-Human IgG, F(ab)2 (8 ng / ml) and incubate at 37°C for 1.5 h.

[0178] 5.5. After color development for about 15 minutes, stop the test and load the sample for testing; if the secondary ELISA test result is positive, Figure 1 As shown; wherein, curves a to f in Figure 5 represent affinity curves of 6 groups of antibodies against DLL3:

[0179] Depend on Figure 1 a It can be seen that the antibody with the best affinity for DLL3 is Ab19; Figure 1 The most suitable antibody screened in a is Ab19; the corresponding humanized antibody is named SDL3-1;

[0180] Depend on Figure 1 b It can be seen that the antibody with the best affinity for DLL3 is Ab20; Figure 1 The more suitable antibodies screened in b were Ab20 and Ab25; the corresponding humanized antibodies were named SDL3-2 and SDL3-3;

[0181] Depend on Figure 1 c It can be seen that the antibodies with better affinity for DLL3 are Ab42 and Ab45; Figure 1 The more suitable antibodies screened out in c were Ab42 and Ab45; the corresponding humanized antibodies were named SDL3-4 and SDL3-5;

[0182] Depend on Figure 1 d It can be seen that the antibodies with better affinity for DLL3 are Ab84 and Ab89; Figure 1 d, the more suitable antibodies screened were Ab84 and Ab89; the corresponding humanized antibodies were named SDL3-6 and SDL3-7;

[0183] Depend on Figure 1 e It can be seen that the antibody with the best affinity for DLL3 is Ab94; Figure 1 The most suitable antibody screened out in e was Ab94; the corresponding humanized antibody was named SDL3-8;

[0184] Depend on Figure 1 It can be seen from f that the antibodies with better affinity for DLL3 are Ab105 and Ab107; Figure 1 The more suitable antibodies screened out from f were Ab105 and Ab107; the corresponding humanized antibodies were named SDL3-9 and SDL3-10.

[0185] Depend on Figure 1 The 10 humanized antibodies targeting DLL3 screened out are summarized in Table 8.

[0186] Table 8 Humanized antibodies targeting DLL3

[0187] Antibody nomenclature DLL3 EC50 SDL3-1 XCT04Ab19 0.8617 SDL3-2 XCT04Ab20 0.007435 SDL3-3 XCT04Ab25 0.004817 SDL3-4 XCT04Ab42 0.005325 SDL3-5 XCT04Ab45 0.1443 SDL3-6 XCT04Ab84 0.008895 SDL3-7 XCT04Ab89 0.2195 SDL3-8 XCT04Ab94 0.2195 SDL3-9 XCT04Ab105 0.2349 SDL3-10 XCT04Ab107 0.1764

[0188] As shown in Table 8, the ELISA method was used to detect the selected candidate antibodies, allowing them to bind to the DLL3 antigen. The binding properties of the candidate antibodies were then determined by the OD450 luminosity value. This led to a secondary screening, and ultimately 10 suitable antibodies were selected for subsequent experiments.

[0189] The following uses CAR-T cells as an example to experimentally verify the function of the humanized antibody targeting DLL3 of the present invention.

[0190] Example 1

[0191] 1. T cell culture

[0192] Mononuclear cells were isolated from donor peripheral blood and subjected to density gradient centrifugation using Ficol. T cells were enriched using a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043). T cells were activated, cultured, and expanded using magnetic beads coupled to anti-CD3 / anti-CD28. The culture medium used was TexMACS GMP Medium (Mi ltenyi Biotec, 170-076-309) containing 10% FBS, 2 mM L-glutamine, and 100 IU / ml rhIL-2. All cells were cultured in a 37°C, 5% CO2 incubator to obtain T cells.

[0193] 2. CAR structure design and lentiviral packaging

[0194] 2.1. DLL3-CAR structure, i.e., CAR structure targeting DLL3:

[0195] The light and heavy chain variable region scFv sequences of the 10 groups of humanized antibodies targeting DLL3 obtained from the above screening, namely SDL3-1, SDL3-2, SDL3-3, SDL3-4, SDL3-5, SDL3-6, SDL3-7, SDL3-8, SDL3-9, and SDL3-10, were constructed into an expression cassette of the second-generation CAR, respectively, and named CAR-SDL3-1, CAR-SDL3-2, CAR-SDL3-3, CAR-SDL3-4, CAR-SDL3-5, CAR-SDL3-6, CAR-SDL3-7, CAR-SDL3-8, CAR-SDL3-9, and CAR-SDL3-10. The expression cassette of the CAR constructed with the heavy and light chain variable region scFv of the control antibody targeting DLL3 was used as a control and named CAR-SDL3. The control group cells were NT cells. The specific CAR structures corresponding to each group are as follows:

[0196] 1#, CD8 SP / SDL3-1 scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0197] 2#, CD8 SP / SDL3-2scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0198] 3#, CD8 SP / SDL3-3scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0199] 4#, CD8 SP / SDL3-4scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0200] 5#, CD8 SP / SDL3-5scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0201] 6#, CD8 SP / SDL3-6scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0202] 7#, CD8 SP / SDL3-7scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0203] 8#, CD8 SP / SDL3-8scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0204] 9#, CD8 SP / SDL3-9scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta;

[0205] 10#, CD8 SP / SDL3-10scFv / CD8Hinge / CD8TM / 4-1BB / CD3Zeta.

[0206] 2.2 Lentiviral packaging

[0207] The expression cassettes corresponding to the 10 groups of CAR results designed in step 2.1 of this example were cloned into the PHBLV lentiviral vector backbone and placed under the EF1α (EF-1α) promoter to form 10 groups of cell plasmids, namely, PHBLV-EF1α-CAR-SDL3-1, PHBLV-EF1α-CAR-SDL3-2, PHBLV-EF1α-CAR-SDL3-3, PHBLV-EF1α-CAR-SDL3-4, PHBLV-EF1α-CAR-SDL3-5, PHBLV-EF1α-CAR-SDL3-6, PHBLV-EF1α-CAR-SDL3-7, PHBLV-EF1α-CAR-SDL3-8, PHBLV-EF1α-CAR-SDL3-9 and PHBLV-EF1α-CAR-SDL3-10;

[0208] The 10 groups of cell plasmids were mixed with the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid #12259) and the lentiviral packaging plasmid psPAX2 (Addgene Plasmid #12260), and the three plasmids of each combination were transferred into 293T cells using Lipofectamine 3000 to prepare 10 complete lentiviral expression vectors, namely: Lv-CAR-SDL3-1, Lv-CAR-SDL3-2, Lv-CAR-SDL3-3, Lv-CAR-SDL3-4, Lv-CAR-SDL3-5, Lv-CAR-SDL3-6, Lv-CAR-SDL3-7, Lv-CAR-SDL3-8, Lv-CAR-SDL3-9, and Lv-CAR-SDL3-10;

[0209] After the cells were cultured for 48 hours and 72 hours, the 10 groups of complete lentiviral expression vectors were centrifuged separately. After the centrifugation stopped, the viral supernatants were collected and concentrated by ultracentrifugation to obtain 10 groups of concentrated viruses, which can be used to infect T cells.

[0210] 2.3 CAR-T cell preparation

[0211] 2.3.1 Lentivirus infection

[0212] The primary T cells isolated and purified in step 1 of this example were reactivated for 1 day;

[0213] The 10 types of lentiviruses packaged in step 2.2 of this embodiment, namely Lv-CAR-SDL3-1, Lv-CAR-SDL3-2, Lv-CAR-SDL3-3, Lv-CAR-SDL3-4, Lv-CAR-SDL3-5, Lv-CAR-SDL3-6, Lv-CAR-SDL3-7, Lv-CAR-SDL3-8, Lv-CAR-SDL3-9 and Lv-CAR-SDL3-10, were respectively used for lentiviral vector infection culture at an MOI of 1 to 10.

[0214] 2.3.2 Cell Proliferation

[0215] The 10 virus-infected T cells were transferred to cell culture flasks and placed in a 37°C, 5% CO2 constant temperature incubator for subculture for 13 days. After subculture, 10 CAR-T cell lines were successfully constructed, named CAR-SDL3-1, CAR-SDL3-2, CAR-SDL3-3, CAR-SDL3-4, CAR-SDL3-5, CAR-SDL3-6, CAR-SDL3-7, CAR-SDL3-8, CAR-SDL3-9, and CAR-SDL3-10. NT cells (blank control T cells, i.e., T cells not infected with the lentivirus) were also cultured. During the culture period, X-VIVO+5% Human AB+1.5% IL-2 serum complete culture medium was supplemented every 1-2 days for cell subculture.

[0216] 2.3.3 CAR Positive Rate Detection

[0217] In step 2.3.2 of this example, for 11 types of T cells, such as NT cells, CAR-SDL3-1, CAR-SDL3-2, CAR-SDL3-3, CAR-SDL3-4, CAR-SDL3-5, CAR-SDL3-6, CAR-SDL3-7, CAR-SDL3-8, CAR-SDL3-9, and CAR-SDL3-10, samples were taken on the 6th, 8th, 10th, and 13th days of culture to detect the cell number, and the CAR positivity rate of T cells was detected on the 6th day.

[0218] 1) The proliferation and growth of 11 types of cells Figure 2 As shown. Figure 2 It can be seen that under the same culture conditions, there is no significant difference in the proliferation rates of the 11 T cell types;

[0219] 2) CAR positive rate detection of 11 types of T cells Figure 3 As shown; Figures a to k respectively represent the flow cytometry graphs of the CAR positivity rate of NT cells (control T cells), CAR-SDL3-1, CAR-SDL3-2, CAR-SDL3-3, CAR-SDL3-4, CAR-SDL3-5, CAR-SDL3-6, CAR-SDL3-7, CAR-SDL3-8, CAR-SDL3-9, CAR-SDL3-10, etc. T cells tested on the 6th day; Figure 3 In the figure, the right side of the vertical line in each flow cytometry test graph is the CAR positive ratio.

[0220] Depend on Figure 3 As can be seen from the flow cytometry graphs b to k, different scFv fragments have no significant effect on the CAR positive rate (all greater than 91%), indicating that replacing the scFv fragment does not affect the successful preparation of CAR-T cells; that is, combined with Figure 2 and Figure 3 The test results show that antibodies or fragments targeting human DLL3 protein have no effect on the proliferation of CAR-T cells after constructing CAR-T cells.

[0221] Therefore, the proliferation rates of the 10 cell types, CAR-SDL3-1, CAR-SDL3-2, CAR-SDL3-3, CAR-SDL3-4, CAR-SDL3-5, CAR-SDL3-6, CAR-SDL3-7, CAR-SDL3-8, CAR-SDL3-9, and CAR-SDL3-10, containing DLL3 antibodies prepared by the present invention, are consistent with those of the NT cells in the control group; this indicates that antibodies or fragments thereof targeting human DLL3 protein have no effect on the proliferation of constructed chimeric antigen receptor immune T cells.

[0222] 2.4. CAR-T cell killing assay for 293T-hDLL3 cells

[0223] 2.4.1 In vitro killing experiment

[0224] The 10 CAR-T cells and NT cells obtained in step 2.3.2 of this implementation were subjected to an in vitro killing experiment. The killing effect of CAR-T cells was detected using RTCA equipment. The target cells and effector cells were co-incubated for 24 hours, and the effector-target ratio was 1:1. The killing efficiency was obvious. The results are as follows Figure 4 As shown; the results showed that CAR-SDL3-6 exhibited good killing efficiency, so subsequent in vivo experiments in mice were carried out using this cell.

[0225] 2.4.2 In vivo killing experiment

[0226] Purchase immunodeficient mice for modeling, use 293T-hDLL3 cells for modeling, and wait for the tumor to grow to 50mm in size.3 Finally, the CAR-T cells (CAR-SDL3-6) with the best in vitro killing effect obtained in step 2.4.1 were used: CAR-SDL3-6 was divided into 1E7 cells (high-dose group) and 5E6 cells (low-dose group). The two groups of cells were transfused, and a control group (DPBS group) was set up. The survival time, body weight and tumor size of the mice were recorded. When the tumor size reached 3000 mm 3 The endpoint was reached when the tumor was eliminated, and the mice were euthanized. The survival time of the mice whose tumors were eliminated was recorded, and if the weight loss exceeded 20%, they were also euthanized. Figure 5 This is a graph to monitor tumor size in mice; Figure 6 This is a curve diagram for detecting changes in mouse body weight; Figure 7 The figure is the survival curve of the detected mice.

[0227] After modeling the mice, when the tumor grows to the target size, CAR-T cells are reinfused and monitored. The mice are weighed and weighed every 2 days to observe changes in weight and tumor size. Figure 6 Monitoring data showed that the high-dose group was able to effectively inhibit tumor growth in mice, and the low-dose group had an inhibitory effect on tumors compared to the control group, but the tumor still had a tendency to grow. This determined that 1E7 CAR-T cells could effectively inhibit tumor growth in mice, and after the CAR-T cells were transfused, the mice had no adverse reactions, no weight loss, and could survive for more than 40 days, indicating good safety.

[0228] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the appended claims.

Claims

1. A humanized antibody targeting DLL3, characterized in that The amino acid sequence of the heavy chain variable region of the antibody is designed as any one of SEQ ID NO: 1 to SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the antibody is designed as any one of SEQ ID NO: 10 to SEQ ID NO: 18, and any of the amino acid sequences of the heavy chain variable region and any of the amino acid sequences of the light chain variable region are paired and combined in pairs.

2. The humanized antibody according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 1 and as shown in SEQ ID NO: 10; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 11; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 12; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 13; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 5 and as shown in SEQ ID NO: 14; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 6 and as shown in SEQ ID NO: 15; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 7 and as shown in SEQ ID NO: 16; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 8 and as shown in SEQ ID NO: 17; or The amino acid sequences of the heavy chain variable region and the light chain variable region comprised by the monoclonal antibody are shown in SEQ ID NO: 9 and SEQ ID NO: 18, respectively.

3. The humanized antibody according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 1 and as shown in SEQ ID NO: 13; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 11; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 3 and as shown in SEQ ID NO: 14; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 12; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 5 and as shown in SEQ ID NO: 17; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 6 and as shown in SEQ ID NO: 15; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 7 and as shown in SEQ ID NO: 18; or The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 8 and as shown in SEQ ID NO: 16; or The amino acid sequences of the heavy chain variable region and the light chain variable region comprised by the monoclonal antibody are shown in SEQ ID NO: 9 and the paired combination shown in SEQ ID NO: 10, respectively.

4. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the humanized antibody according to any one of claims 1 to 3.

5. A humanized nucleic acid molecule, characterized in that The nucleic acid molecule encodes the humanized antibody according to any one of claims 1 to 3.

6. A plasmid expression cassette, characterized in that The expression cassette contains the humanized antibody according to any one of claims 1 to 3.

7. A recombinant vector, characterized in that The recombinant vector contains the humanized antibody according to any one of claims 1 to 3, or the chimeric antigen receptor according to claim 4.

8. A recombinant cell, characterized in that The recombinant cell contains the humanized antibody according to any one of claims 1 to 3.

9. A microbial preparation, characterized in that The microbial preparation contains the humanized antibody according to any one of claims 1 to 3, or the chimeric antigen receptor according to claim 4, or the nucleic acid molecule according to claim 5, or the expression cassette according to claim 6, or the recombinant vector according to claim 7, or the recombinant cell according to claim 8.

10. A drug for preventing or treating tumors or cancers, characterized in that: The components of the drug contain the humanized antibody according to any one of claims 1 to 3, or the chimeric antigen receptor according to claim 4, or the nucleic acid molecule according to claim 5, or the expression cassette according to claim 6, or the recombinant vector according to claim 7, or the recombinant cell according to claim 8, or the biological preparation according to claim 9.

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