Immune globulin specifically aiming at type IV collagen alpha3 chain

Abstract

The invention discloses immunoglobulin specifically aiming at an IV type collagen alpha3 chain, belongs to the field of antibodies and related technologies, and can be applied to diagnosis of Alport syndrome.

Description

Technical Field

[0001] The invention belongs to the field of antibodies and related technologies, and in particular relates to an immunoglobulin specifically targeting type IV collagen α3 chain. Background Art

[0002] Alport syndrome is also known as hereditary progressive nephritis. Its clinical features are hematuria, proteinuria and progressive renal function decline. Some patients may have extrarenal manifestations such as sensorineural deafness, eye abnormalities, and esophageal leiomyoma. The disease is caused by mutations in the gene COL4An (n=3, 4, 5) encoding the α3-α5 chain of type IV collagen in the glomerular basement membrane. About 85% of Alport syndrome patients are X-linked dominant Alport syndrome caused by mutations in COL4A5 or COL4A5 and COL4A6 genes; male patients have more severe conditions, with 90% of them developing renal failure before the age of 40, while female patients have relatively milder conditions. About 15% of Alport syndrome patients are autosomal hereditary Alport syndrome caused by mutations in the COL4A3 or COL4A4 genes, of which autosomal recessive Alport syndrome patients are the majority, and almost all of them develop renal failure before the age of 30.

[0003] Immunofluorescence examination and immunohistochemistry are common disease diagnosis methods with the advantages of high sensitivity and convenient detection. This method relies on highly specific antibodies to recognize antigens. In the treatment of Alport syndrome, the use of type IV collagen α3 chain antibodies to detect the expression of type IV collagen in the kidney and skin basement membrane is the most ideal diagnostic method. Therefore, it is necessary to develop an immunoglobulin specific for type IV collagen α3 chain. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides an immunoglobulin specific for type IV collagen α3 chain, which solves the problems raised in the above background technology.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] An immunoglobulin specific for type IV collagen α3 chain, the immunoglobulin comprising a heavy chain variable region and a light chain variable region,

[0009] The heavy chain variable region:

[0010] CDR1 comprises the amino acid sequence DSNIH (SEQ ID NO: 1); CDR2 comprises the amino acid sequence WIDPENGDTEYAPKFQG (SEQ ID NO: 2); CDR3 comprises the amino acid sequence VNWDYFDYW (SEQ ID NO: 3);

[0011] The light chain variable region:

[0012] CDR1 comprises the amino acid sequence RSSQSLVHNNGNTYLH (SEQ ID NO: 4); CDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 5); COR3 comprises the amino acid sequence SQSTHIPIT (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] Furthermore, the immunoglobulin is an antibody, and the antibody is a monoclonal antibody, a single-chain antibody or a single-domain antibody.

[0015] Furthermore, the antibody is a mouse antibody or a humanized antibody.

[0016] Furthermore, the heavy chain of the immunoglobulin includes the signal peptide sequence shown in SEQ ID NO:9, and the light chain of the immunoglobulin includes the signal peptide sequence shown in SEQ ID NO:10.

[0017] Furthermore, the nucleotide sequence of the immunoglobulin is encoded by a nucleic acid molecule.

[0018] Furthermore, the nucleic acid molecule has an expression vector that matches it.

[0019] Furthermore, the expression vector grows in a host cell.

[0020] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0021] The monoclonal antibody provided by the present invention specifically binds to the human type IV collagen α3 chain antibody, and can be used to diagnose Alport syndrome in kidney disease diagnosis. The monoclonal antibody provided by the present invention does not bind to α5, α1 or other collagen chains, and can diagnose specific types of Alport syndrome, such as X-linked dominant hereditary male and female Alport syndrome and autosomal recessive hereditary Alport syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The results of the mouse serum titer test after immunizing mice with type IV collagen polypeptide are shown.

[0023] Figure 2 The results of EC50 determination of the antibodies in the Examples are shown.

[0024] Figure 3 The results of the antibody heat acceleration test in the Examples are shown. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The term "antibody" as used herein is used in its broadest sense, including immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and is a protein or polypeptide that exhibits binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, etc. Classical antibody molecules are generally tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. According to the conservative differences in the amino acid sequence, the heavy chain and the light chain are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is used to recognize and bind to antigens. In the variable region of the heavy chain and the light chain, the amino acid composition and arrangement order of the three local regions have a higher degree of variation, which is the key position for the antibody to bind to the antigen, and is therefore also referred to as a complementary determining region (CDR). The amino acid sequence of the CDR can be easily determined using a numbering scheme recognized in the art, such as Kabat, Chothia, IMGT, AbM or Contact. In a specific embodiment, the CDR of the antibody described herein has been determined according to the IMGT numbering scheme. Herein, the three heavy chain complementarity determining regions are referred to as CDR1, CDR2 and CDR3, respectively, and the three light chain complementarity determining regions are referred to as CDR1, CDR2 and CDR3, respectively.

[0027] As used herein, 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 a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0028] For antibodies or antigen-binding fragments thereof, "targeting", "directed against" or "specific binding" means that one molecule (such as an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (such as an antigen) relative to other molecules present in the environment at the same time. A molecule can target, direct against or specifically bind to more than one molecule, for example, a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding affinity of an antibody to an antigen can be measured by some parameters, such as the EC50 value or KD value of the antibody binding to the antigen.

[0029] An immunoglobulin specific for type IV collagen α3 chain or an antigen-binding fragment of type IV collagen α3 chain, comprising a heavy chain variable region and a light chain variable region,

[0030] The heavy chain variable region:

[0031] CDR1 comprises the amino acid sequence DSNIH (SEQ ID NO: 1); CDR2 comprises the amino acid sequence WIDPENGDTEYAPKFQG (SEQ ID NO: 2); CDR3 comprises the amino acid sequence VNWDYFDYW (SEQ ID NO: 3);

[0032] The light chain variable region:

[0033] CDR1 comprises the amino acid sequence RSSQSLVHNNGNTYLH (SEQ ID NO: 4); CDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 5); COR3 comprises the amino acid sequence SQSTHIPIT (SEQ ID NO: 6).

[0034] 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.

[0035] The immunoglobulin is an antibody, and the antibody is a monoclonal antibody, a single-chain antibody or a single-domain antibody.

[0036] The antibody is a mouse antibody or a humanized antibody.

[0037] The heavy chain of the immunoglobulin or the antigen-binding fragment of type IV collagen α3 chain comprises the signal peptide sequence shown in SEQ ID NO:9, and / or the light chain of the immunoglobulin comprises the signal peptide sequence shown in SEQ ID NO:10.

[0038] The nucleic acid molecule encodes the nucleotide sequence of the immunoglobulin or antigen-binding fragment thereof.

[0039] The nucleic acid molecule has an expression vector that matches it.

[0040] The expression vector grows inside the host cell.

[0041] The detection kit contains the above-mentioned immunoglobulin specific for type IV collagen α3 chain.

[0042] The antibodies or antigen-binding fragments thereof provided herein and other forms of molecules including the antibodies or antigen-binding fragments thereof (e.g., immunoconjugates or bispecific antibody molecules) can be placed in containers to form detection or diagnostic kits. These containers can be in the form of boxes, ampoules, vials, tubes, bags, or suitable containers known in the art. These containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for preserving proteins. If necessary, instructions for use are also provided with the container. The instructions can generally include information about how to use antibodies or antigen-binding fragments thereof for immunoassays, for example, instructions for how to quantify the α3 chain of type IV collagen in a sample; instructions for how to diagnose Alport syndrome based on the test results. The instructions can be printed directly on the container (if present), or as a label attached to the container, or as a separate paper, booklet, card, or folded printed matter provided in or with the container.

[0043] Example 1: Animal immunization and serum titer detection

[0044] PBS dissolved 50μg hemocyanin (KLH) coupled human type IV collagen α3 chain polypeptide and Freund's adjuvant (antigen: adjuvant = 1:1) were emulsified by stirring, and 100μl / mouse were used for subcutaneous immunization of 3 female Balb / c mice aged 6-8 weeks. Subsequently, the immunization was repeated every 2 weeks for booster immunization for a total of 3 times. The first blood was collected one week after the second immunization, and the second blood was collected one week after the third immunization, and the serum was tested by ELISA. The mouse with the highest serum titer was selected for shock immunization in the third week after the third immunization. The spleen was taken on the 4th day after immunization for plasma cell enrichment and single antigen-specific B cell screening.

[0045] Serum titer test is as follows Figure 1 shown.

[0046] Example 2: Screening of mouse single B cells against human type IV collagen α3 chain

[0047] A1: Mouse plasma cell enrichment: The spleen of the immunized mouse was taken and homogenized to produce a single cell suspension and counted. The number of cells was 1.5*10 ^8 , adjust the cell density to 1*10^8 / ml and place at 4℃ for later use.TM Mouse CD138 Positive Selection Kit (Catalog #18957) was used to enrich mouse plasma cells according to the kit instructions. After enrichment, the cell count was 1.8*10 ^6 , adjust the cell density to 1*10^7 / ml and place at 4℃ for later use.

[0048] A2: Beacon single cell light guide system antigen chronic plasma cell screening: Use the Beacon light guide platform for antigen-specific B cell screening. Turn on the machine according to the machine operating instructions, put in the OptoSelect Chip 14K chip, first rinse with water 3 times, then rinse with load medium 3 times, take the prepared mouse plasma cells, gently blow and mix with a pipette, turn on the Beacon loading program to absorb 5ul cells into the chip, and start the import program to import single cells into the small chamber of the chip. After the import program is completed, start the flushing program and rinse with load medium 3 times. Turn on the Beacon loading program to absorb 15ulCapture Beads+biotin-labeled antigen+fluorescently labeled anti-mouse IgG secondary antibody mixture (1ug / ml antigen, 5ul+1:500 diluted Anti-mouse FITC, 5ul+20ul Capture Beads) into the chip, observe the uniform distribution of Capture Beads in each field of view of the chip under white light, and set the analysis and shooting program. After shooting, use loadmedium to rinse 3 times to rinse the Capture Beads mixture clean. At the same time, analyze the positive clones on the machine and mark them. A total of 12 positive cells were obtained. Set up the export program and export the positive clones to a 96-well microplate with 5ul of cell lysis solution (Buffer TCL, 2x, cat no: 1070498) added to each well in advance. After the export is completed, take the 96-well plate out of the machine, centrifuge it instantly in a centrifuge and put it in a -80℃ refrigerator.

[0049] Example 3: Single B cell antibody variable region sequencing

[0050] Single B cell RNA extraction and cDNA synthesis: Take out the 96-well plate from the -80°C refrigerator, return to room temperature, add 10ul of RNAClean XP (BECKMAN COULTER, A63987) to each well to extract total RNA of the cells. Then use Opto PlasmaB Discovery cDNA Synthesis Kit (cat no: 750-02030, Berkeley Lights) for cDNA synthesis, and add 30ul of AMPure XP (BECKMAN COULTER, A63881) to each well to purify cDNA.

[0051] Amplification and sequencing of antibody variable region sequences: After PCR amplification (Genscript) to obtain the antibody heavy chain and light chain variable region sequences, insert them into the pcDNA3.1 expression plasmid and transform them into DH5α competent cells. Shake the bacteria for 3 to 4 hours, directly aspirate 100 μL of bacterial solution into the plate, cover the plate, and take it to the desktop shaker for coating for 3 minutes. Take the coated plate back to the clean bench, and then place the plate in a 37°C water-insulated constant temperature incubator for overnight culture for 12 to 16 hours. Pick a single colony, shake the bacteria, and sequence it. The sequencing results are as follows:

[0052] Heavy chain variable region (including signal peptide) (136aa):

[0053] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4:

[0054] MKCSWVIFFLMAVVIGINSEVQLQQSGAELVRSGASVKLSCTASGFNIKDS NIHWLIQRPEQDLEWIGWIDPENGDTEYAPKFQGEATMTADTSSNTAYLQLSSL TSEDTAVYFCNAVNWDYFDYWGQGTTLTVSS.

[0055] Light chain variable region (including signal peptide) (131aa):

[0056] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-COR3-FR4

[0057] MKLPVRLLLVLMFWIPVSSSDVVVTQTPLSLPVSLGDQASISCRSSQSLVHNN GNTYLHWYLQRPGQPKLLIYKVSNRFSGVPDRVSGSGSGTDFTLKISRVEAEDLG VYFCSQSTHIPITFGSGTKLEIK.

[0058] Example 4: Recombinant expression of antibodies

[0059] The antibody heavy chain and light chain were transfected into suspended CHO-K1 cells at a ratio of 1:1. After 16 hours, the culture medium was replaced with new culture medium and selection antibiotics were added. After the cells were cultured in a 37°C shake flask for 6 days, the supernatant was collected for antibody purification. The column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and filtered for sterilization. The filtered supernatant and protein A column were incubated at room temperature for 2 hours, and after washing the column with 1× the above buffer, IgG was eluted with sterile 0.1M sodium citrate (pH3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH9.0).

[0060] Under sterile conditions, the product buffer was exchanged into PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

[0061] The purified antibody was analyzed by SDS-PAGE using a 10% precast gel using 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 to be approximately 150 KD under non-reducing conditions and 2 bands of approximately 50 KD and 25 KD under reducing conditions, respectively.

[0062] Example 5: Indirect ELISA detection of recombinant antibodies

[0063] Indirect ELISA was used to evaluate the binding capacity of antibodies in serum and supernatant to peptides. ELISA plates were coated with 100 μl / well of 1 μg / ml type IV collagen α3 chain peptide in PBS at 4°C overnight. The plates were washed with PBS-T (0.05% Tween) and incubated with 200 μl / well of PBST containing 1% BSA at 37 ℃ Block for 2 hours. Then discard the blocking solution, add 100 μl of cell culture supernatant to each well, and then incubate at room temperature for 1 hour. Wash the plate three times with PBST and incubate with 100 μl / well of goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson, catalog number 115-035-071) at 37°C for 0.5 hour. Wash the plate five times with PBST, then add TMB colorimetric solution and incubate in the dark at room temperature for 13 minutes. Terminate the reaction by adding 50 μl of 1M HCl stop solution (Sigma). Read the plate at 450nm using a microplate reader. 15 positive clone wells were obtained, and the OD 450 Greater than 0.5.

[0064] Example 6: Monoclonal Antibody EC 50 Determination of

[0065] EC50 (concentration for 50% of maximal effect) refers to the concentration that causes 50% of the maximum effect. In enzyme-linked immunosorbent assay (ELISA), when used to indicate the binding ability of an antibody molecule to the corresponding antigen, it can refer to the concentration of the antibody molecule that produces half of the maximum detection signal (such as colorimetric or fluorescence intensity). 50 The lower the value, the greater the binding affinity to the antigen.

[0066] Indirect ELISA is used to evaluate the binding ability of purified antibodies to polypeptides. The ELISA plate was coated with 0.5 μg / ml of polypeptide in 100 μl / well PBS at 4°C overnight. The plate was washed with PBS-T (0.05% Tween) and blocked with 250 μl / well of PBST containing 1% BSA at 37°C for 2 hours. The blocking solution was then discarded, and 100 μl of 1 μg / ml of purified antibody produced by the hybridoma was added to the first well, and diluted in a 3-fold gradient, for a total of 11 test concentration gradients and 1 zero well. Then incubate at room temperature 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 conjugated to horseradish peroxidase (Jackson, Cat. No. 115-035-071).

[0067] The plate was washed 4 times with PBST, then TMB colorimetric solution was added and incubated for 13 minutes at room temperature in the dark. The reaction was terminated by adding 50 μl of 1 M HCl stop solution. The plate was read at 450 nm using a microplate reader. Figure 2 As shown, the EC50 of 20-8 is 4.931 ng / ml.

[0068] Example 7: The thermal acceleration stability experiment is used to determine the storage time of the antibody at a certain temperature, and the shelf life of the antibody is determined according to the Arrhenius equation. One day of thermal acceleration in a 37°C constant temperature incubator is equivalent to 1.5 months of storage at 4°C; 20-8 purified antibodies are divided into 20μl / tube and then stored at -20°C for future use.

[0069] 21 days, 14 days, and 7 days before the test, take out the samples and store them at 4°C and 37°C respectively. Finally, on the day of the test, take out the antibodies to be tested under different conditions (including those stored at -20°C) and uniformly use the indirect ELISA method to determine the binding ability of the purified antibodies to the polypeptide after heat acceleration to evaluate the stability of the antibodies. Coat the ELISA plate with 1 μg / ml polypeptide in 100 μl / well PBS at 4°C overnight. Wash the plate once with PBS-T (0.05% Tween) and block it with 250 μl / well PBST containing 1% BSA at 37°C for 2 hours. Then discard the blocking solution, add 100 μl of 1 μg / ml purified antibody to the first well, and dilute it in a 3-fold gradient, with a total of 11 test concentration gradients and 1 zero well. Then incubate at 37°C for 1 hour. The plate was washed three times with PBST and incubated with 100 μl / well of goat anti-mouse IgG antibody (Jackson, Cat. No. 115-035-071) at 37°C for 0.5 hours. The plate was washed four times with PBST, then TMB colorimetric solution 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 plate was read at 450 nm using a microplate reader. According to the Arrhenius equation, the antibody can be stably stored at 37°C for 21 days. According to the Arrhenius equation, the antibody can be stably stored at 4°C for 31.5 months. The results are as follows: Figure 3 shown.

[0070] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An immunoglobulin specific for type IV collagen α3 chain, characterized in that: The immunoglobulin comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region: CDR1 comprises the amino acid sequence DSNIH (SEQ ID NO: 1); CDR2 comprises the amino acid sequence WIDPENGDTEYAPKFQG (SEQ ID NO: 2); CDR3 comprises the amino acid sequence VNWDYFDYW (SEQ ID NO: 3); The light chain variable region: CDR1 comprises the amino acid sequence RSSQSLVHNNGNTYLH (SEQ ID NO: 4); CDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO: 5); COR3 comprises the amino acid sequence SQSTHIPIT (SEQ ID NO: 6).

2. The immunoglobulin specific for type IV collagen α3 chain according to claim 1, characterized in that: 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; The SEQ ID NO:7 is as follows: EVQLQQSGAELVRSGASVKLSCTASGFNIKDSNIHWLIQRPEQ DLEWIGWIDPENGDTEYAPKFQGEATMTADTSSNTAYLQLSSLTSE DTAVYFCNAVNWDYFDYWGQGTTLTVSS; The SEQ ID NO: 8 is as follows: DVVVTQTPLSLPVSLGDQASISCRSSQSLVHNNGNTYLHWYL QRPGQSPKLLIYKVSNRFSGVPDRVSGSGSGTDFTLKISRVEAEDL GVYFCSQSTHIPITFGSGTKLEIK.

3. The immunoglobulin specific for type IV collagen α3 chain according to claim 1, characterized in that: The immunoglobulin is an antibody, and the antibody is a monoclonal antibody, a single-chain antibody or a single-domain antibody.

4. The immunoglobulin specific for type IV collagen α3 chain according to claim 1, characterized in that: The antibody is a mouse antibody or a humanized antibody.

5. The immunoglobulin specific for type IV collagen α3 chain according to any one of claims 1 to 4, characterized in that: The heavy chain of the immunoglobulin includes the signal peptide sequence shown in SEQ ID NO:9: MKCSWVIFFLMAVVIGINS.

6. The immunoglobulin specific for type IV collagen α3 chain according to any one of claims 1 to 4, characterized in that: The light chain of the immunoglobulin includes the signal peptide sequence shown in SEQ ID NO: 10: MKLPVRLLVLMFWIPVSSS.

7. The immunoglobulin specific for type IV collagen α3 chain according to claim 1, characterized in that: The nucleotide sequence of the immunoglobulin is encoded by a nucleic acid molecule.

8. The immunoglobulin specific for type IV collagen α3 chain according to claim 7, characterized in that: The nucleic acid molecule has an expression vector matching it.

9. The immunoglobulin specific for type IV collagen α3 chain according to claim 8, characterized in that: The expression vector grows within the host cell.

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