Antibodies or antigen-binding fragments thereof targeting the alpha5 chain of collagen type IV

Abstract

This invention discloses an antibody or its antigen-binding fragment that targets the α5 chain of type IV collagen, belonging to the field of antibody and related technologies, and can be applied to the diagnosis of Alport syndrome.

Description

Technical Field

[0001] This invention belongs to the field of antibody and related technologies, specifically relating to antibodies or antigen-binding fragments of type IV collagen α5 chain. Background Technology

[0002] Alport syndrome, also known as hereditary progressive nephritis, is clinically characterized by hematuria, proteinuria, and progressive renal function decline. Some patients may also have extrarenal manifestations such as sensorineural hearing loss, ocular abnormalities, and esophageal leiomyoma. This disease is caused by mutations in the COL4An (n=3, 4, 5) gene, which encodes the α3-α5 chains of type IV collagen in the glomerular basement membrane. Approximately 85% of Alport syndrome patients have an X-linked dominant inheritance pattern due to mutations in either the COL4A5 or both COL4A5 and COL4A6 genes. Male patients tend to have a more severe condition, with up to 90% experiencing renal failure before age 40, while female patients generally experience milder symptoms. About 15% of Alport syndrome patients have an autosomal recessive inheritance pattern due to mutations in the COL4A3 or COL4A4 genes, with the latter being the most common type, and almost all of these patients develop renal failure before age 30.

[0003] Immunofluorescence and immunohistochemical antigen detection are common diagnostic methods for diseases, offering advantages such as high sensitivity and ease of detection. These methods rely on highly specific antibodies to recognize antigens. Type IV collagen α5 chain staining in kidney or skin tissue can diagnose not only Alport syndrome but also autosomal dominant Alport syndrome. In patients with autosomal dominant Alport syndrome, type IV collagen α5 chain staining in kidney tissue is negative in both the glomeruli and tubules; type IV collagen α5 chain staining in the skin basement membrane is also negative. Therefore, using type IV collagen α5 chain antibody to detect the expression of type IV collagen in the kidney and skin basement membrane is the most ideal method for diagnosing Alport syndrome. In conclusion, it is necessary to develop an immunoglobulin specifically targeting the type IV collagen α5 chain. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides an antibody or antigen-binding fragment thereof targeting the α5 chain of type IV collagen, thus solving the problems mentioned in the background section.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An antibody or antigen-binding fragment thereof targeting the α5 chain of type IV collagen, wherein the antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region:

[0010] CDR1 is the amino acid sequence TSGMGVG (SEQ ID NO:1); CDR2 is the amino acid sequence HIWWDDDKHYNPSLKS (SEQ ID NO:2); CDR3 is the amino acid sequence RGDYFDY (SEQ ID NO:3).

[0011] The variable region of the light chain:

[0012] CDR1 is the amino acid sequence KSSQSLFDSGTRKNYLT (SEQ ID NO:4); CDR2 is the amino acid sequence WASTRES (SEQ ID NO:5); COR3 is the amino acid sequence CKQSYNLFT (SEQ ID NO:6).

[0013] Furthermore, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:8;

[0014] The SEQ ID NO:7 is as follows:

[0015] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKHYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCTRGDYFDYWGQGTTLTVSS;

[0016] The SEQ ID NO:8 is as follows:

[0017] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLFDSGTRKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

[0018] Furthermore, the antibody is a monoclonal antibody, a single-chain antibody, or a single-domain antibody.

[0019] Furthermore, the antibody is a mouse antibody or a humanized antibody.

[0020] Furthermore, the heavy chain variable region includes the signal peptide sequence shown in SEQ ID NO:9: MDRLTSSFLLLIVPAYVLS.

[0021] Furthermore, the light chain variable region includes the signal peptide sequence shown in SEQ ID NO:10: MDSQAQVLILLLLWVSGTCG.

[0022] Furthermore, the nucleotide sequence of the antibody or its antigen-binding fragment is encoded by a nucleic acid molecule.

[0023] Furthermore, the nucleic acid molecule has a matching expression vector.

[0024] Furthermore, the expression vector grows within host cells.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0026] The antibody or antigen-binding fragment targeting the α5 chain of type IV collagen provided by this invention can be used to diagnose Alport syndrome in the treatment of kidney disease. Furthermore, the monoclonal antibody provided by this invention does not bind to α3, α1, or other collagen chains, and can diagnose specific types of Alport syndrome, such as X-linked dominant male and female Alport syndrome, and autosomal recessive Alport syndrome. Attached Figure Description

[0027] Figure 1 The results of serum titer assays in mice immunized with a peptide targeting type IV collagen are shown.

[0028] Figure 2 The EC50 assay results for the antibody targeting the α5 chain of type IV collagen are shown.

[0029] Figure 3 The results of the thermo-accelerated antibody assay targeting the α5 chain of type IV collagen are shown. Detailed Implementation

[0030] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] As used herein, the term "antibody" is used in its broadest sense to include immunoglobulins or other types of molecules containing one or more antigen-binding domains that specifically bind to antigens, and proteins or peptides that exhibit binding specificity to a particular antigen. Specific examples of antibodies may include intact antibodies (e.g., classic tetrachain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, etc. Classic antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains linked together by disulfide bonds. Based on the conservation of amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind antigens. Within the variable regions of the heavy and light chains, there are three local regions with a higher degree of variability in the amino acid composition and sequence, which are critical sites for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequence of the CDR can be readily determined using numbering schemes recognized in the art, such as Kabat, Chothia, IMGT, AbM, or Contact. In one specific embodiment, the CDR of the antibody described herein has been determined according to the IMGT numbering scheme. In this paper, the three heavy chain complementarity determination regions are referred to as CDR1, CDR2 and CDR3, respectively, and the three light chain complementarity determination regions are referred to as CDR1, CDR2 and CDR3, respectively.

[0032] In this document, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably to refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include (but are not limited to) DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0033] For antibodies or their antigen-binding fragments, "targeting," "specifically binding," or "specifically binding" refers to the fact that one molecule (e.g., an antibody or its antigen-binding fragment) has a higher binding affinity for another molecule (e.g., an antigen) compared to other molecules present in the environment. A molecule can target, bind to, or specifically bind to more than one molecule; for example, a bispecific antibody can have a higher binding affinity for two different antigens compared to other molecules. The binding affinity of an antibody to an antigen can be measured using parameters such as the EC50 value or KD value of antibody-antigen binding.

[0034] An antibody or antigen-binding fragment thereof targeting the α5 chain of type IV collagen, wherein the antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region;

[0035] Heavy chain variable region:

[0036] CDR1 is the amino acid sequence TSGMGVG (SEQ ID NO:1); CDR2 is the amino acid sequence HIWWDDDKHYNPSLKS (SEQ ID NO:2); CDR3 is the amino acid sequence RGDYFDY (SEQ ID NO:3).

[0037] Light chain variable region:

[0038] CDR1 is the amino acid sequence KSSQSLFDSGTRKNYLT (SEQ ID NO:4); CDR2 is the amino acid sequence WASTRES (SEQ ID NO:5); COR3 is the amino acid sequence CKQSYNLFT (SEQ ID NO:6).

[0039] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:8;

[0040] SEQ ID NO:7 is as follows:

[0041] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKHYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCTRGDYFDYWGQGTTLTVSS;

[0042] SEQ ID NO:8 is as follows:

[0043] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLFDSGTRKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

[0044] In some embodiments, the antibody targeting the α5 chain of type IV collagen is a monoclonal antibody, a single-chain antibody, or a single-domain antibody.

[0045] In some embodiments, the antibody targeting the α5 chain of type IV collagen is a mouse antibody or a humanized antibody.

[0046] The heavy chain variable region includes the signal peptide sequence shown in SEQ ID NO:9: MDRLTSSFLLLIVPAYVLS.

[0047] The light chain variable region includes the signal peptide sequence shown in SEQ ID NO:10: MDSQAQVLILLLLWVSGTCG.

[0048] In some embodiments, the nucleotide sequence of an antibody or its antigen-binding fragment targeting the α5 chain of type IV collagen is encoded by a nucleic acid molecule. The nucleic acid molecule has a matching expression vector. The expression vector grows within a host cell.

[0049] The antibodies or antigen-binding fragments thereof described herein, as well as other forms of molecules including such antibodies or antigen-binding fragments (e.g., immunoconjugates or bispecific antibody molecules), may be placed in containers to form a detection or diagnostic kit. These containers may be boxes, ampoules, vials, tubes, bags, or other suitable forms of containers known in the art. These containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for preserving proteins. Instructions for use may be provided with the container, if desired. Instructions for use may typically include information on how to perform immunoassays using the antibody or its antigen-binding fragment, such as instructions on how to quantify type IV collagen α5 chains in a sample; and instructions on how to diagnose Alport syndrome based on the test results. Instructions for use may be printed directly on the container (if present), as a label affixed to the container, or as a separate paper, booklet, card, or folded printed material provided in or with the container.

[0050] Animal immunization and serum titer testing:

[0051] Human type IV collagen α5 chain polypeptide containing 50 μg of hemocyanin (KLH) conjugated with Freund's adjuvant (antigen:adjuvant = 1:1) was emulsified by stirring in PBS and administered subcutaneously to three female Balb / c mice aged 6-8 weeks at a dose of 100 μl / mouse. Booster immunizations were then performed every two weeks for a total of three immunizations. Blood samples were collected one week after the second immunization and one week after the third immunization for ELISA analysis. The mouse with the highest serum titer was selected for a pulse immunization three weeks after the third immunization. Spleen samples were harvested on day 4 post-immunization for plasma cell enrichment and single antigen-specific B cell screening.

[0052] Serum titer test results are as follows: Figure 1 As shown.

[0053] Screening of mouse single B cells against human type IV collagen α5 chain:

[0054] A1: Enrichment of mouse plasma cells: Spleens from immunized mice were harvested and homogenized to produce a single-cell suspension, which was then counted, yielding a cell count of 1.5*102. ^8The cell density was adjusted to 1*10^8 / ml and stored at 4℃ for later use. Mouse plasma cells were enriched using the EasySep™ Mouse CD138 Positive Selection Kit (Catalog # 18957) according to the kit instructions. The cell count after enrichment was 1.8*10^8 / ml. ^6 Adjust the cell density to 1*10 ^7 / ml, store at 4℃ for later use.

[0055] A2: Antigen-based Chronic Plasma Cell Screening using the Beacon Single-Cell Photoconductive System: Antigen-specific B cell screening was performed using the Beacon photoconductive platform. The machine was powered on according to the operating instructions. An OptoSelect Chip 14K was placed in the chip. The chip was first rinsed three times with water, then three times with load medium. Prepared mouse plasma cells were collected and gently mixed using a pipette. The Beacon loading program was activated, and 5 μL of cells were transferred into the chip. The loading program was then activated to transfer single cells into the chip chamber. After the loading program was completed, the rinsing program was activated, and the cells were rinsed three times with load medium. The Beacon loading program was activated again, and 15 μL of a mixture of Capture Beads + biotin-labeled antigen + fluorescently labeled anti-mouse IgG secondary antibody (1 μg / ml antigen, 5 μL + 1:500 diluted Anti-mouse FITC, 5 μL + 20 μL Capture Beads) was transferred into the chip. The uniform distribution of Capture Beads in each field of view was observed under white light before setting the analysis and imaging program. After the capture was completed, the mixture was rinsed three times with loadmedium to remove the Capture Beads mixture. Positive clones were analyzed and labeled on the machine, yielding a total of 12 positive cells. The export program was set up to export the positive clones into 96-well microplates with 5 μL of cell lysis buffer (Buffer TCL, 2x, cat no: 1070498) added to each well. After the export was completed, the 96-well plates were removed from the machine, briefly centrifuged, and then placed in a -80°C freezer.

[0056] Sequencing of the variable regions of the screened single B cell antibodies:

[0057] RNA extraction and cDNA synthesis from single B cells: The 96-well plates were removed from the -80°C freezer and allowed to reach room temperature. 10 μL of RNAClean XP (BECKMAN COULTER, A63987) was added to each well to extract total RNA from the cells. Then, cDNA synthesis was performed using the Opto PlasmaB Discovery cDNA Synthesis Kit (cat no: 750-02030, Berkeley Lights). 30 μL of AMPure XP (BECKMAN COULTER, A63881) was added to each well to purify the cDNA.

[0058] Antibody variable region sequence amplification and sequencing: After obtaining the antibody heavy and light chain variable region sequences through PCR amplification (Genscript), the sequences were inserted into the pcDNA3.1 expression plasmid and transformed into DH5α competent cells. The cells were shaken for 3-4 hours, and 100 μL of bacterial culture was directly aspirated into a plate. The plate was covered and spread on a benchtop shaker for 3 minutes. The spread plate was then returned to a clean bench and incubated overnight at 37°C in a water-jacketed incubator for 12-16 hours. Single colonies were picked, shaken, and sequenced. The sequencing results are as follows:

[0059] Heavy chain variable region (containing signal peptide) (135 aa):

[0060] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0061] MDRLTSSFLLLIVPAYVLSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKHYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCTRGDYFDYWGQGTTLTVSS.

[0062] Light chain variable region (containing signal peptide) (132 aa):

[0063] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0064] MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLFDSGTRKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

[0065] Antibody recombinant expression:

[0066] The heavy and light chains of the single B cell antibody were transfected into suspended CHO-K1 cells at a 1:1 ratio. After 16 hours, the medium was replaced with fresh culture medium and selection antibiotics were added. The cells were cultured in shake flasks at 37°C for 6 days, and the supernatant was collected for antibody purification. The column was reequilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× of the above buffer and filtered for sterilization. The filtered supernatant was incubated with a Protein A column at room temperature for 2 hours. After washing the column with 1× of the above buffer, IgG was eluted with sterile 0.1M sodium citrate (pH 3.5). The eluent was collected and neutralized with 1 / 9 volume of sterile 1M Tris-HCl (pH 9.0).

[0067] Under aseptic conditions, the product buffer was replaced with PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibody was quantified at OD 280 nm using an extinction coefficient Ec (0.1%) of 1.43.

[0068] The purified antibody was analyzed by SDS-PAGE using a 10% precast gel via a BioRad electrophoresis system. The gel was stained with Coomassie Brilliant Blue, and the molecular size was estimated by comparing the stained bands with protein markers. The molecular size was approximately 150 kDa under non-reducing conditions and approximately 50 kDa and 25 kDa under reducing conditions, respectively.

[0069] Indirect ELISA detection of recombinant antibodies:

[0070] Indirect ELISA was used to assess the binding ability of antibodies to peptides in serum and supernatant. The ELISA plate was coated overnight at 4°C with 100 μl / well of PBS containing 1 μg / ml of type IV collagen α5 chain peptide. The plate was washed with PBS-T (0.05% Tween) and then incubated with 200 μl / well of PBST containing 1% BSA at 37°C. ℃ Block for 2 hours. Then discard the blocking solution, add 100 μl of cell culture supernatant to each well, and incubate at room temperature for 1 hour. Wash the plate three times with PBST and incubate at 37°C for 0.5 hours with 100 μl / well of horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson, catalog number 115-035-071). Wash the plate five times with PBST, then add TMB chromogenic solution and incubate in the dark at room temperature for 13 minutes. Stop the reaction by adding 50 μl of 1M HCl stop solution (Sigma). Read the plate at 450 nm using a microplate reader. 15 wells yielded positive clones, with OD... 450 Greater than 0.5.

[0071] Monoclonal antibody EC 50 Measurement:

[0072] EC50 (concentration for 50% of maximal effect) refers to the concentration at which the antibody produces a 50% maximum effect. In enzyme-linked immunosorbent assays (ELISA), when used to express the binding ability of antibody molecules to their corresponding antigens, it can refer to the antibody molecule concentration at which half of the maximum detection signal (such as colorimetric or fluorescence intensity) is produced. 50 The lower the value, the greater the affinity for binding with the antigen.

[0073] Indirect ELISA was used to assess the binding affinity of purified antibodies to peptides. ELISA plates were coated overnight at 4°C with 100 μl / well of 0.5 μg / ml peptide in PBS. The plates were washed with PBS-T (0.05% Tween) and blocked at 37°C for 2 hours with 250 μl / well of PBST containing 1% BSA. The blocking solution was then discarded, and 100 μl of purified hybridoma-produced antibody (1 μg / ml) was added to the first well. This was followed by 3-fold serial dilutions, resulting in 11 test concentration gradients and one zero well. The plates were then incubated at room temperature for 1 hour. The plates were washed three times with PBST and incubated at 37°C for 0.5 hours with 100 μl / well of horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson, catalog number 115-035-071).

[0074] Wash the plate four times with PBST, then add TMB chromogenic buffer and incubate in the dark at room temperature for 13 minutes. Stop the reaction by adding 50 μl of 1M HCl stop solution. Read the plate at 450 nm using a microplate reader. Figure 2 As shown, the EC50 of 20-8 is 25.34 ng / ml.

[0075] The thermally accelerated stability assay is used to determine the storage time of antibodies at a certain temperature, and the shelf life of the antibodies is determined according to the Arrhenius equation. One day of thermal acceleration in a 37°C incubator is equivalent to 1.5 months of storage at 4°C. The purified antibody (20-8) is aliquoted into 20 μl tubes and then stored at -20°C for later use.

[0076] 21 days, 14 days, and 7 days before the test, samples were taken and stored at 4℃ and 37℃, respectively. On the day of the test, all antibodies stored under different conditions (including those stored at -20℃) were taken out and tested using an indirect ELISA method to determine the binding ability of the heat-accelerated purified antibody to the peptide, assessing antibody stability. The ELISA plate was coated overnight at 4℃ with 100 μl / well of PBS containing 1 μg / ml peptide. The plate was washed once with PBS-T (0.05% Tween) and blocked at 37℃ for 2 hours with 250 μl / well of PBST containing 1% BSA. The blocking solution was then discarded, and 100 μl of purified antibody (1 μg / ml) was added to the first well, followed by 3-fold serial dilutions, resulting in 11 test concentration gradients and 1 zero well. The plates were then incubated at 37℃ for 1 hour. The plate was washed three times with PBST and incubated at 37°C for 0.5 hours with 100 μl / well of goat anti-mouse IgG antibody (Jackson, catalog number 115-035-071). The plate was then washed four times with PBST, and TMB chromogenic buffer was added and incubated in the dark at 25°C for 15 minutes. The reaction was terminated by adding 50 μl of 1M HCl stop solution. The plates were read at 450 nm using a microplate reader. According to the Arrhenius equation, the antibody was stable for 21 days at 37°C; based on the Arrhenius equation, it was stable for 31.5 months at 4°C. The results are as follows: Figure 3 As shown.

[0077] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof targeting the alpha 5 chain of collagen type IV, characterized in that: The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises a signal peptide sequence as shown in SEQ ID NO: 9: MDRLTSSFLLLIVPAYVLS. CDR1 is an amino acid sequence TSGMGVG (SEQ ID NO: 1); CDR2 is an amino acid sequence HIWWDDDKHYNPSLKS (SEQ ID NO: 2); and CDR3 is an amino acid sequence RGDYFDY (SEQ ID NO: 3); The light chain variable region comprises a signal peptide sequence as shown in SEQ ID NO: 10: MDSQAQVLILLLLWVSGTCG. CDR1 is an amino acid sequence KSSQSLFDSGTRKNYLT (SEQ ID NO: 4); CDR2 is an amino acid sequence WASTRES (SEQ ID NO: 5); and CDR3 is an amino acid sequence CKQSYNLFT (SEQ ID NO: 6); The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7; The SEQ ID NO: 7 is as follows: QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKHYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCTRGDYFDYWGQGTTLTVSS. The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8; The SEQ ID NO: 8 is as follows: DIVMSQSPSSLAVSAGEKVTMSCKSSQSLFDSGTRKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

2. The antibody or antigen-binding fragment thereof targeting the alpha 5 chain of collagen type IV according to claim 1, characterized in that: The antibody is a monoclonal antibody or a single-chain antibody.

3. The antibody or antigen-binding fragment thereof targeting the alpha 5 chain of collagen type IV according to claim 1, characterized in that: The antibody is a murine antibody or a humanized antibody.

4. The antibody or antigen-binding fragment thereof targeting the alpha5 chain of collagen type IV according to any one of claims 1 to 3, characterized in that: The heavy chain variable region comprises a signal peptide sequence as shown in SEQ ID NO: 9: MDRLTSSFLLLIVPAYVLS.

5. The antibody or antigen-binding fragment thereof targeting the alpha 5 chain of collagen type IV according to any one of claims 1 to 3, characterized in that: The light chain variable region comprises a signal peptide sequence as shown in SEQ ID NO: 10: MDSQAQVLILLLLWVSGTCG.

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