An anti-human IL13Ra2 shark single domain antibody or its antigen binding fragment and application

Abstract

The present invention discloses an anti-human IL13Ra2 shark single domain antibody or its antigen-binding fragment and its application, relating to an anti-human IL13Ra2 shark single domain antibody and its amino acid and nucleotide coding sequences, and also to a recombinant plasmid and recombinant expression bacteria comprising the nucleotide coding sequence and its use. It has higher affinity and other pharmacological properties, contributes to the improvement of medical level, is suitable for the preparation of drugs, and has broad market prospects.

Description

Technical Field

[0001] The present invention relates to anti-human IL13Ra2 shark single-domain antibodies, and more specifically, to anti-human IL13Ra2 shark single-domain antibodies and nucleotides thereof. The present invention also relates to recombinant plasmids and recombinant expression bacteria comprising the nucleotide coding sequence, and belongs to the field of single-domain antibody technology development. Background Art

[0002] Antibodies are immunoglobulins produced by B cells that differentiate into plasma cells when stimulated by antigenic substances and can specifically bind to corresponding antigens. Traditional antibodies have two variable domains, called VH and VL, which provide stability and binding specificity to each other.

[0003] In 1993, Hamers Casterman and others from the Free University of Brussels accidentally discovered in their experiments that IgG1 and IgG2 antibodies, which are different from traditional antibodies, exist in the serum of dromedary camels (Camelus dromedarius). This antibody is composed of only two heavy chains, and the stable region lacks the heavy chain constant region 1 of traditional antibodies. It can complete the specific binding with antigens without the help of light chains, and is called heavy-chain antibodies (HCAbs). Later, Andrew et al. also found heavy-chain antibodies with similar structures to HCAbs in nurse sharks in 1995, called (Immunoglobulin new antigen recepter, IgNAR). IgNAR is different from IgW and IgM commonly found in fish in structure. It naturally lacks Ig light chain structure and has only two heavy chain structures. Each heavy chain consists of 5 constant regions, 1 hinge region and 1 variable region. After screening, the smallest unit antibody that can specifically bind to the antigen on IgNAR can be obtained - the heavy chain variable region antibody (VNAR), whose molecular weight is only 12kDa, so this antibody is also called a single-domain antibody or nanobody. The difference in structure between single-domain antibodies and traditional antibodies determines the difference in physical and chemical properties between the two. Compared with traditional antibodies, single-domain antibodies have the characteristics of small molecular weight, strong solubility, high stability, low immunogenicity, and high antigen binding activity. Therefore, the application field of single-domain antibodies is also wider than that of traditional antibodies.

[0004] Glioma is a common malignant tumor of the central nervous system, and its main characteristics are high malignancy, rapid progression, and diffuse infiltration. Despite the continuous improvement of treatment methods, the median survival after comprehensive treatment with surgery as the main method is only 14.6 months. Studies have found that in gliomas, the expression of IL-13Rα2 is positively correlated with the grade of gliomas. High-grade gliomas have high IL-13Rα2 expression, low-grade or normal brain tissues have low or no IL-13Rα2 expression, and high IL-13Rα2 expression is closely related to poor prognosis. In addition, in gliomas, high expression of IL-13Rα2 promotes the proliferation, migration and invasion of glioma cells, and this effect can be achieved by activating the SRC / PI3K / ATK / mTOR pathway. At the same time, IL-13 acts on IL-13Rα2 to prevent 15-LOX-1 from activating PPARγ by inhibiting STAT6 phosphorylation, inhibiting glioma cell apoptosis, and promoting glioma proliferation and growth. IL-13Rα2 can also inhibit tumor apoptosis by upregulating STAT3, but it is not a direct interaction between IL-13Rα2 and STAT3, and its specific mechanism remains to be studied. Based on the above findings, targeted therapy and immunotherapy for IL-13Rα2 have been launched. In terms of conventional targeted therapy, IL-13 fusion chimeric proteins or labeled liposomes targeting IL-13Rα2 have great market potential, but still need further development; in terms of immunotherapy, antibodies against IL-13Rα2 occupy its binding site with IL-13, preventing it from exerting its effect. IL-13Rα2 monoclonal antibodies have been prepared in vivo or in vitro, but their affinity with IL-13Rα2 is lower than that of IL-13PE ligand, and they need intracranial injection. Therefore, antibodies with higher affinity, smaller molecular weight, and blood-brain barrier penetration are urgently needed to be developed. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a single-domain antibody against human IL13Ra2, and at the same time provide the coding sequence of the single-domain antibody, a plasmid containing the coding sequence, and a recombinant VNAR protein having binding activity with the human IL13Ra2 protein.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0007] The present invention discloses an anti-human IL13Ra2 shark single domain antibody or an antigen-binding fragment thereof, wherein the anti-human IL13Ra2 shark single domain antibody or the antigen-binding fragment thereof comprises a variable region VNAR of an IgNAR protein, and the amino acid sequence of the variable region VNAR comprises at least one of SEQ ID: 20530 (1), SEQ ID: 23530 (2), and SEQ ID: 23547 (3).

[0008] Preferably, an anti-human IL13Ra2 shark single domain antibody or an antigen-binding fragment thereof, wherein the sequence of SEQ ID: 20530(1) is:

[0009] AWVEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYIGHSWDAPCDTPLIGDIRYYEGGGTILTVK;

[0010] The SEQ ID: 23530(2) sequence is:

[0011] DLVYQWPRQVITEKGKSVEIHCYQNRTVQDNMLWYLQRPRQAIILIGYFYSGGKPEYGEGFKNRFDISRTRNDRMSKLTVETVQLTDMAVYHCGTGDSAHNDKLIWHLDKKDNNCN;

[0012] The SEQ ID: 23547(3) sequence is:

[0013] GWVDQTPKTIIRGATESLTINCVLRDPSYAFYSTYWYLTKLGATNKESMSIGGRYAETVNKGSNSFSLRITDLRVEDSGTYQCGACDGYVTCNTWSEGSGTILTVK.

[0014] The above (1), (2), and (3) are alternative identifiers for 20530, 23530, and 23547 and should not be understood as restrictions on the sequence.

[0015] The present invention provides a nucleotide sequence encoding the anti-human IL13Ra2 shark single domain antibody or its antigen-binding fragment, comprising a nucleotide encoding a polypeptide, wherein the polypeptide comprises the shark single domain antibody variable region VNAR; the variable region VNAR comprises at least one of SEQ ID: 20530, SEQ ID: 23530, and SEQ ID: 23547, and the SEQ ID: 20530, SEQ ID: 23530, and SEQ ID: 23547 sequences are capable of binding to human IL13Ra2.

[0016] A nucleotide sequence, wherein the nucleotide sequence is at least one of SEQ ID: GENE20530, SEQ ID: GENE23530 and SEQ ID: GENE23547.

[0017] A nucleotide sequence, which is SEQ ID: GENE20530, SEQ ID: GENE23530 and SEQ ID: GENE23547;

[0018] The SEQ ID: GENE20530 sequence is:

[0019] ATGGCATGGGTCGAGCAAACGCCAACAACCACCACGAAAGAGGCTGGAGAGTCCTTGACCATTAATTGCGTCTTGAAGGGTTCATCTTATGCACTTTGCGACACTTACTGGTACTTCACGAAAAAAGGCGCGACCAAGAAAGAATCTCTTTCGAATGGAGGCCGCTACGCTGAAACCGTCAATAAGGCTTCTAAATCGTTTTCATTACGCATCTCTGATCTGCGCGTGGAGGATAGTGGGACGTACTACTGTAAGGCGTATATTGGACACAGTTGGGACGCGCCCTGCGATACGCCCCTTATCGGGGATATTCGCTATTACGAGGGCGGCGGCACAATCTTAACCGTAAAACCGGGCTAA;

[0020] The SEQ ID: GENE23530 sequence is:

[0021] ATGGATCTGGTTTATCAGTGGCCGCGCCAGGTTATTACCGAAAAAGGTAAAAGTGTTGAAATCCATTGCTATCAGAATCGTACCGTTCAGGATAATATGCTGTGGTATCTGCAGCGCCCGCGCCAGGCAATTATTCTGATTGGCTATTTTTATAGCGGCGGTAAACCGGAATATGGCGAAGGCTTTAAAAATCGTTTTGATATTAGCCGTACCCGCAATGATCGCATGAGCAAACTGACCGTTGAAACCGTTCAGCTGACCGATATGGCAGTGTATCATTGCGGTACCGGTGACAGTGCCCATAATGATAAACTGATTTGGCATCTGGATAAAAAAGATAATAACTGCAAC;

[0022] The SEQ ID: GENE23547 sequence is:

[0023] ATGGGTTGGGTTGATCAGACCCCGAAAACCATTATTCGCGGTGCAACCGAAAGTCTGACCATTAATTGTGTGCTGCGCGATCCGAGCTATGCCTTTTATAGCACCTATTGGTATCTGACCAAACTGGGTGCCACCAATAAGGAAAGCATGAGTATTGGTGG TCGCTATGCAGAAACCGTGAATAAGGGCAGTAATAGCTTTAGTCTGCGTATTACCGATCTGCGCGTTGAAGATAGCGGCACCTATCAGTGTGGCGCCTGCGATGGTTATGTGACCTGCAATACCTGGAGCGAAGGCAGCGGTACCATTCTGACCGTGAAA.

[0024] The invention discloses a recombinant plasmid containing the nucleotide sequence.

[0025] The invention discloses a recombinant expression bacterium containing the recombinant plasmid.

[0026] The present invention discloses an application of an anti-human IL13Ra2 shark single-domain antibody in the preparation of a therapeutic antibody drug for brain glioma.

[0027] The present invention discloses an application of a nucleotide sequence in the preparation of a human IL13Ra2 therapeutic antibody drug.

[0028] Regarding: the amino acid sequence of the variable region VNAR comprises SEQ ID: 20530, SEQ ID: 23530, SEQ ID: 23547, and the nucleotide sequence is SEQ ID: GENE20530, SEQ ID: GENE23530 and SEQ ID: GENE23547, the present invention comprises three methods for preparing anti-human IL13Ra2 shark single domain antibodies, and the preparation method comprises the following steps:

[0029] Step 1: preparing an expression vector containing a nucleotide sequence (SEQ ID: GENE20530, SEQ ID: GENE23530 and SEQ ID: GENE23547) for expressing the anti-human IL13Ra2 shark single domain antibody or its antigen-binding fragment;

[0030] Step 2: construct a recombinant expression bacterium containing the expression vector of step 1;

[0031] Step 3: Cultivate the recombinant expression bacteria constructed in step 2;

[0032] Step 4: Separate and purify to obtain the anti-human IL13Ra2 shark single domain antibody or its antigen-binding fragment.

[0033] Beneficial effects: The VHH fragment of the heavy-chain antibody is different from the antigen-binding domain fragment of the traditional antibody. It has the characteristics of small molecular weight, stable physical and chemical characteristics, high affinity and easy recombinant expression and preparation. However, the recombinantly cloned and expressed single-domain antibody has the same structural stability and antigen-binding activity as the heavy-chain antibody, and is the smallest unit known to bind to the target antigen. Glioma is one of the most destructive malignant tumors in the brain, but the median survival after comprehensive treatment with surgery as the main treatment is only 14.6 months, and the prognosis is poor. Studies have shown that IL-13Rα2 can promote the proliferation and invasion of glioma cells and inhibit glioma cell apoptosis after binding to IL-13. IL-13Rα2 exists in the form of monomers on the cell surface, inside the cell and outside the cell, and the receptors in these three places are interconnected and can be converted to each other. Therefore, targeted therapy and immunotherapy of glioma with IL-13Rα2 as the target has been continuously developed. Intracranial injection of IL-13Rα2 antibody or liposomes or chimeric disease labeled with IL-13Rα2 Targeting brain glioma by methods such as toxins has a certain effect, but the antibody affinity is low, and the safety and effectiveness of other methods cannot be controlled. CAR-T cell therapy targeting IL-13Rα2 has also received much attention, but its efficacy is unknown and has other risks. The single-domain antibody of the present invention has a small molecular weight and strong tissue permeability. It can still pass through the blood-brain barrier after recombinant expression. At the same time, since normal cells express low or no IL-13Rα2, it has little effect on normal cells. This is one of its advantages. In addition, the single-domain antibody has a high affinity, and its affinity is in the range of nanomolar to picomolar. Its binding kinetics are comparable to those of conventional antibodies. Highly permeable nanoantibodies can enter dense tissues and have good effects. The single-domain antibody of the present invention provides a new direction for the research and treatment of solid tumors in the brain, and the single-domain antibody against IL-13Rα2 is very likely to correctly and quickly target brain gliomas, inhibit the invasion and spread of gliomas, and promote their apoptosis. It is a new idea and new hope for the treatment of brain glioma-related diseases, suitable for the preparation of drugs, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 SDS-PAGE electrophoresis of negative control, supernatant and precipitate after ultrasonic disruption of three bacterial sludge expression bacteria.

[0035] Figure 2 SDS-PAGE electrophoresis of protein 20530 under nickel column purification conditions.

[0036] Figure 3 SDS-PAGE electrophoresis of protein 23530 under nickel column purification conditions.

[0037] Figure 4 SDS-PAGE electrophoresis of protein 23547 under nickel column purification conditions.

[0038] Figure 5 SDS-PAGE electrophoresis after protein purification and concentration.

[0039] Figure 6 SDS-PAGE electrophoresis of protein purification and concentration after tag cleavage.

[0040] Figure 7 Diagram of in vitro activity detection using double antibody sandwich Elisa using checkerboard titration method. DETAILED DESCRIPTION

[0041] The effective amount described in the present invention refers to the amount or dose of the product described in the present invention (preferably an anti-human IL13Ra2 antibody or an antigen-binding fragment thereof) that provides the desired treatment after being administered to a patient, organ or individual in a single or multiple doses.

[0042] The term "diagnosis" as used herein refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease, or to evaluating a patient's response to treatment.

[0043] The term "treatment" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic interventions that improve the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0044] The "include" or "comprising" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that do not substantially affect the technical effect. When it is used in this application to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] The present invention is described in detail below in conjunction with specific embodiments, but the present invention is not limited to these implementations, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0047] Example 1

[0048] Obtaining the sequence of human IL13Ra2 single domain antibody

[0049] Sequence screening:

[0050] Sharks were immunized with human IL13Ra2 antigen, and the spleen and lymphocytes of the immunized sharks were sequenced for transcriptome, the serum of the immunized sharks was tested by mass spectrometry, and the RNA of the spleen and lymphocytes of the immunized sharks was extracted for RT-PCR to obtain a high-throughput database containing only the shark VNAR region. The obtained transcriptome high-throughput sequencing results were compared with the high-throughput database of the VNAR region, and sequences that were contained in both databases and had a high content were screened; on the other hand, the mass spectrometry results were compared with the high-throughput database of the shark VNAR region, and sequences that were contained in both databases and had a high content were screened. The VNAR variable region sequences obtained by combining the two screening results were SEQ ID: 20530, 23530 and 23547; IgNAR protein is a dimer, and each chain contains 1 variable region (VNAR) and 5 constant regions (CNAR);

[0051] Gene sequence synthesis:

[0052] Three IgNAR protein variable region VNAR amino acid sequences after screening were selected, and the corresponding encoded nucleotide sequences were codon-optimized for Escherichia coli expression. The preferred sequences were SEQ ID: GENE20530, GENE23530 and GENE23547. Restriction sites (preferably Not I and Bham I) were added to both ends of the optimized gene for artificial synthesis. The expression vector pET-32a was selected, in which the TrxA tag can promote the correct folding of the protein. The synthetic nucleotide sequence was as described above. After the protein was expressed, the tag protein could be removed using recombinant enterokinase to obtain the final purified recombinant VNAR.

[0053] The nucleotide sequence of the recombinant IgNAR protein is artificially synthesized, the sequence is accurate, and it can be obtained in large quantities through fermentation. It has low production costs, is environmentally friendly, and can be prepared in large quantities. The recombinant IgNAR protein can effectively bind to antigen proteins and successfully replace or partially replace immunoglobulin IgG. The recombinant IgNAR protein has a small molecular weight. Compared with traditional antibodies, the V region of the recombinant single-domain antibody has a molecular weight of only about 12kDa due to the lack of CDR2. This feature enables it to carry drugs into cells or penetrate the blood-brain barrier.

[0054] Implementation 2

[0055] Construction of recombinant expression plasmid:

[0056] The gene sequence was inserted into the expression vector pET-32a by double enzyme digestion to obtain the pET-32a-IgNAR recombinant plasmid.

[0057] Example 3

[0058] Construction of recombinant expression bacteria:

[0059] 1) Prepare competent E. coli cells. The strain can be E. coli BL21 (DE3), Rosetta (DE3) or other expression bacteria, preferably Rosetta (DE3).

[0060] 2) Slowly add 2 μL of recombinant plasmid pET-32a-IgNAR (no more than 50 ng) into the clean bench, and stir slowly with a pipette tip to mix evenly, and place on ice for 30 minutes.

[0061] 3) Heat shock in a 42°C constant temperature water bath for 90 seconds. Do not touch the competent cells during the heat shock process. After the heat shock, quickly transfer to ice and incubate for 5 minutes.

[0062] 4) Add 1 mL of LB liquid culture medium preheated at 37°C to the clean bench, place in a 37°C shaker, and shake and culture at 220 rpm for 1 hour to revive the competent cells.

[0063] 5) After recovery, centrifuge at room temperature at 10,000 rpm for 5 minutes.

[0064] 6) Discard 1 mL of supernatant in a clean bench, mix the remaining supernatant and precipitate, and aspirate 50 μL to spread on an ampicillin-resistant plate.

[0065] 7) Place the plate in a 37°C biochemical incubator and incubate upside down for about 12 hours.

[0066] 8) Use an inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid culture medium. Then add 5 μL Amp and place it in a shaker at 37°C and 220 rpm for 12 h.

[0067] 9) Sequencing confirmed that the transformation was successful and the recombinant expression bacteria Rosetta-pET-32a-IgNAR were obtained.

[0068] Example 4

[0069] Inducible expression of recombinant proteins:

[0070] 1) The recombinant expression bacteria Rosetta-pET-32a-IgNAR was inoculated into LB medium containing Amp resistance at a ratio of 1:100, and cultured at 37°C and 220 rpm / min for 4 hours.

[0071] 2) IPTG is then added to make the final concentration of IPTG 0.1-0.5 mM, preferably 0.5 mM, and the culture is continued for 4-6 h (preferably 5 h) to obtain the recombinant expression protein.

[0072] Example 5

[0073] 1. The bacterial sludge collected by centrifugation was ultrasonically disrupted in PBS (PBS: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2MmK2HPO4, pH7.4). The disrupted bacterial solution was centrifuged and the precipitate and supernatant were collected for protein gel electrophoresis. All recombinant IgNARs can be expressed in the supernatant, such as Figure 1 shown.

[0074] 2. Collect the supernatant after bacterial cell disruption, filter it with a 0.22 μm filter membrane, add it to a nickel column and incubate it on ice overnight.

[0075] 3. Purification: Follow the instructions for nickel column purification and use elution buffer containing 10 / 50 / 100 / 150 / 200 / 250 / 300 / 350 / 400 / 500mM Imidazole for elution (Elution buffer: 50mM Na2HPO4, 300mM NaCl,

[0076] 10 / 50 / 100 / 150 / 200 / 250 / 300 / 350 / 400 / 500mM Imidazole, pH8.0), the elution results of the three recombinant proteins are as follows Figure 2 , 3 As shown in Figure 4, the results show that the elution effect of the eluent containing 300mM imidazole is better. Subsequently, the eluent containing 300mM imidazole was collected, and the elution was repeated 10 times for each sample.

[0077] 4. Dialysis (dialysis conditions: molecular weight of dialysis bag is 3.4 kDa, 4°C, liquid is changed every 2 hours) Dialysised Buffer: PBS, pH 8.0.

[0078] 5. After the concentration and dialysis are completed, use a 10kDa ultrafiltration tube to ultrafilter and concentrate. The protein results after concentration are as follows Figure 5 shown.

[0079] 6. After the enzyme digestion and concentration is completed, use recombinant enterokinase for enzyme digestion (enzyme cutting buffer: 25mM Tris-HCl, pH8.0). After enzyme digestion, purify the concentrated protein sample as follows Figure 6 shown.

[0080] Lyophilize to obtain recombinant IgNAR protein, i.e., anti-human IL13Ra2 single domain antibody

[0081] Example 6

[0082] In vitro activity assay:

[0083] Absorbance = OD450

[0084] Absorbance of experimental group = experimental group (OD450)

[0085] Blank group absorbance = blank group (OD450)

[0086] The recombinant VNAR protein was diluted to 100ug / mL, 10ug / mL, and 1ug / mL concentrations using sodium carbonate-sodium bicarbonate buffer (CBS, pH 9.6), and 100uL was plated per well. The control was sodium carbonate-sodium bicarbonate buffer. The plates were plated at 4°C overnight. The next day, the plate was patted dry, washed once with TBST (pH 7.5), incubated with 5% BSA solution at 37°C for 1 hour, patted dry, and washed three times with TBST; for IL13Ra2 protein binding, IL13Ra2 protein was diluted to 2ug / mL with 5% BSA solution, 100uL was added to each well, and bound at 37°C for 2h; HRP-labeled rabbit anti-IL13Ra2 protein was used as the secondary antibody, diluted 1:1000 with TBST, incubated at 37°C for 1h, patted dry, washed three times with TBST, 100uL of TMB colorimetric solution was added to each well, and the reaction was carried out in the dark at room temperature for 15min. After the reaction, 2M H2SO4 was added to each well, and the absorbance of the sample at 450nm was measured by an enzyme reader.

[0087] The results are as follows Figure 7 As shown, when the concentration of the single-domain antibody VNAR was as low as 1ug / mL and the concentration of the antigen PD-1 protein was 2ug / mL, the absorbance OD450 of the experimental group was >1.0, the absorbance OD450 of the blank group was <0.5, and the absorbance OD450 of the experimental group / the absorbance OD450 of the blank group was >2.1. The test results were obvious and the test differences were significant, indicating that the single-domain antibody VNAR and the antigen IL13Ra2 protein had good binding properties.

[0088] Example 7

[0089] The present invention provides an application of an anti-human IL13Ra2 single-domain antibody or an antigen-binding fragment thereof in antibody-drug conjugates and pharmaceutical carriers.

[0090] Example 8

[0091] The present invention provides an application of an anti-human IL13Ra2 single domain antibody or an antigen-binding fragment thereof in a pharmaceutical composition.

[0092] Example 9

[0093] The present invention provides an anti-human IL13Ra2 single-domain antibody or an antigen-binding fragment thereof for use in preparing a product for treating diseases related to brain glioma.

[0094] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention. Invention Name: An anti-human IL13Ra2 shark single domain antibody or its antigen binding fragment and application Applicant: Zhejiang Sci-Tech University The SEQ ID: 20530 sequence is: AWVEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESLSNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYIGHSWDAPCDTPLIGDIRYYEGGGTILTVK; The SEQ ID: 23530 sequence is: DLVYQWPRQVITEKGKSVEIHCYQNRTVQDNMLWYLQRPRQAIILIGYFYSGGKPEYGEGFKNRFDISRTRNDRMSKLTVETVQLTDMAVYHCGTGDSAHNDKLIWHLDKKDNNCN; The SEQ ID: 23547 sequence is: GWVDQTPKTIIRGATESLTINCVLRDPSYAFYSTYWYLTKLGATNKESMSIGGRYAETVNKGSNSFSLRITDLRVEDSGTYQCGACDGYVTCNTWSEGSGTILTVK; The SEQ ID: GENE20530 sequence is: ATGGCATGGGTCGAGCAAACGCCAACAACCACCACGAAAGAGGCTGGAGAGTCCTTGACCATTAATTGCGTCTTGAAGGGTTCATCTTATGCACTTTGCGACACTTACTGGTACTTCACGAAAAAAGGCGCGACCAAGAAAGAATCTCTTTCGAATGGAGGCCGCTACGCTGAAACCGTCAATAAGGCTTCTAAATCGTTTTCATTACGCATCTCTGATCTGCGCGTGGAGGATAGTGGGACGTACTACTGTAAGGCGTATATTGGACACAGTTGGGACGCGCCCTGCGATACGCCCCTTATCGGGGATATTCGCTATTACGAGGGCGGCGGCACAATCTTAACCGTAAAACCGGGCTAA; The SEQ ID: GENE23530 sequence is as follows: ATGGATCTGGTTTATCAGTGGCCGCGCCAGGTTATTACCGAAAAAGGTAAAAGTGTTGAAATCCATTGCTATCAGAATCGTACCGTTCAGGATAATATGCTGTGGTATCTGCAGCGCCCGCGCCAGGCAATTATTCTGATTGGCTATTTTTATAGCGGCGGTAAACCGGAATATGGCGAAGGCTTTAAAAATCGTTTTGATATTAGCCGTACCCGCAATGATCGCATGAGCAAACTGACCGTTGAAACCGTTCAGCTGACCGATATGGCAGTGTATCATTGCGGTACCGGTGACAGTGCCCATAATGATAAACTGATTTGGCATCTGGATAAAAAAGATAATAACTGCAAC; The SEQ ID: GENE23547 sequence is as follows: ATGGGTTGGGTTGATCAGACCCCGAAAACCATTATTCGCGGTGCAACCGAAAGTCTGACCATTAATTGTGTGCTGCGCGATCCGAGCTATGCCTTTTATAGCACCTATTGGTATCTGACCAAACTGGGTGCCACCAATAAGGAAAGCATGAGTATTGGTGGTCGCTATGCAGAAACCGTGAATAAGGGCAGTAATAGCTTTAGTCTGCGTATTACCGATCTGCGCGTTGAAGATAGCGGCACCTATCAGTGTGGCGCCTGCGATGGTTATGTGACCTGCAATACCTGGAGCGAAGGCAGCGGTACCATTCTGACCGTGAAA。

Claims

1. An anti-human IL13Ra2 shark single domain antibody, characterized in that: The anti-human IL13Ra2 shark single domain antibody comprises the variable region VNAR of the IgNAR protein, and the amino acid sequence of the variable region VNAR is shown in SEQ ID: 20530 or SEQ ID: 23530 or SEQ ID: 23547; The SEQ ID: 20530 sequence is: AWVEQTPTTTTKEAGESLTINCVLKGSSYALCDTYWYFTKKGATKKESL SNGGRYAETVNKASKSFSLRISDLRVEDSGTYYCKAYIGHSWDAPCDTPLIGD IRYYEGGGTILTVK; The SEQ ID: 23530 sequence is: DLVYQWPRQVITEKGKSVEIHCYQNRTVQDNMLWYLQRPRQAIILIGYF YSGGKPEYGEGFKNRFDISRTRNDRMSKLTVETVQLTDMAVYHCGTGDSAH NDKLIWHLDKKDNNCN; The SEQ ID: 23547 sequence is: GWVDQTPKTIIRGATESLTINCVLRDPSYAFYSTYWYLTKLGATNKESMS IGGRYAETVNKGSNSFSLRITDLRVEDSGTYQCGACDGYVTCNTWSEGSGTIL TVK.

2. A polynucleotide encoding the anti-human IL13Ra2 shark single domain antibody according to claim 1.

3. A recombinant plasmid containing the polynucleotide sequence of claim 2.

4. A recombinant expression bacterium containing the recombinant plasmid according to claim 3.

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