A single-domain antibody targeting NUPR1, its screening method, and its application.

Abstract

This invention discloses a single-domain antibody targeting NUPR1, its screening method, and its application. Fusing the single-domain antibody with the C-degron degradation element cOdc1 enables targeted degradation of NUPR1, effectively inhibiting tumor progression. When fused with the TAT transmembrane peptide, the single-domain antibody can easily enter cells, minimizing immunogenicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a single-domain antibody targeting nucleoprotein 1 (NUPR1), its screening method, and its application. Background Technology

[0002] Nucleoprotein 1 (NUPR1), also known as p8 or Com1 protein, is an intrinsic nuclear disorder and stress protein. NUPR1 is transcriptionally activated under stress to protect cells from stress damage. Studies have shown that NUPR1 expression is significantly upregulated in tumor tissues such as pancreatic cancer, breast cancer, lung cancer, and colon cancer, and it participates in tumor cell migration and invasion by regulating cellular activities such as autophagy, cell cycle, apoptosis, and DNA damage. Silencing NUPR1 or inhibiting its expression with antibodies may be a potential new approach for cancer treatment. Given the limitations of traditional monoclonal antibodies, such as low stability, complex preparation processes, and high costs, there is an urgent need to develop novel antibodies targeting NUPR1.

[0003] Single-domain antibodies (sdAbs, also known as nanobodies) are naturally occurring heavy-chain-only antibodies (HcAbs) found in the serum of camels, lacking the light chain. The variable domain (VHH) of the heavy chain of HcAbs has a molecular weight of approximately 15 kDa. sdAbs are structurally stable, possess high affinity and antigen-binding capacity, have a small relative molecular mass, easily penetrate tissue barriers, and are readily produced and genetically engineered. Due to their unique advantages, sdAbs have received widespread research and attention, and their application in clinical diagnosis and treatment is actively being explored.

[0004] Traditional methods for preparing sdAbs involve immunizing alpacas with antigens, isolating alpaca plasma cells, preparing hybridoma cells, and screening for hybrid cells that can produce sdAbs that specifically recognize the antigen. These hybrid cells are then used for subsequent sdAb isolation, purification, and functional verification. The entire process is time-consuming, requires a large amount of experimental materials, is expensive, and has an extremely low success rate in obtaining high-affinity sdAbs. Furthermore, some studies have reported on engineered sdAbs based on known amino acid sequences of antibody antigen-determining regions, but these studies only supplement existing antibody functions; attempts to find new, more specific sdAbs from scratch still face significant challenges. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a single-domain antibody targeting NUPR1 and a screening method thereof, and also provides the gene encoding the single-domain antibody, expression vector, host cell, and related applications.

[0006] On one hand, the present invention provides a single-domain antibody against NUPR1, the single-domain antibody comprising three complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3, respectively. Preferably, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0007] Furthermore, the single-domain antibody also comprises four backbone regions, FR1, FR2, FR3, and FR4, alternately linked to three complementarity-determining regions. The amino acid sequences of FR1, FR2, FR3, and FR4 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 7, respectively. Preferably, the amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NO. 4 to SEQ ID NO. 7, respectively.

[0008] Preferably, the amino acid sequence of the single-domain antibody against NUPR1 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 8. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO. 8, and for ease of description in this application, this amino acid sequence is also referred to as #07.81.

[0009] In another aspect, the present invention provides a fusion protein comprising the single-domain antibody against NUPR1. According to the present invention, the single-domain antibody against NUPR1 is as defined above.

[0010] In some embodiments of the present invention, the fusion protein further comprises a degradation element cOdc1; preferably, the fusion protein further comprises a cell-penetrating peptide.

[0011] In one embodiment of the present invention, the fusion protein comprises ABCDEF, wherein A is a secretory peptide or is absent, B is the single-domain antibody against NUPR1, C is a first tag sequence or is absent, D is a degradation element cOdc1 or is absent, E is a cell-penetrating peptide or is absent, F is a second tag sequence or is absent, and there may be or may not be a flexible linker between A, B, C, D, E and / or F.

[0012] According to the present invention, the amino acid sequence of the degradation element cOdc1 is: SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV.

[0013] According to the present invention, the cell-penetrating peptide can be an amino acid sequence known in the art that has the function of penetrating cell membranes, including but not limited to: TAT-derived peptides, antennal leader peptides, antennal peptides, polyarginine peptides, etc. For example, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, GRKKRRQRRR, KKWKMRRNQFWVKVQRG, RQIKIWFQNRRMKWKK, or a polyarginine peptide containing 6-9 arginine residues (e.g., RRRRR, or RRRRRRRRR). In a specific embodiment of the present invention, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR.

[0014] According to the present invention, the first tag sequence and the second tag sequence may be the same or different, and are selected from commonly used tag sequences in the art, including but not limited to: His tag, Myc tag, HA tag, GST tag, mCherry, GFP, Flag tag, etc. In a specific embodiment of the present invention, the first tag sequence and the second tag sequence are different, and are His tag and Flag tag, respectively. For example, the first tag sequence is HHHHHH, and the second tag sequence is DYKDHDGDYKDHDIDYKDDDDK; or, the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK, and the second tag sequence is HHHHHH.

[0015] According to the present invention, the flexible linker can be an amino acid sequence known in the art for connecting two peptide fragments, such as (Gm S) n Connection sequences, DDK connection sequences, etc. In a specific embodiment of the present invention, the sequence of the flexible joint is GSG.

[0016] Preferably, the fusion protein is ABCDEF, wherein A is a secretory peptide or is absent, B is the single-domain antibody against NUPR1, C is a first tag sequence or is absent, D is the degradation element cOdc1, E is a cell-penetrating peptide, and F is a second tag sequence or is absent; a flexible linker may or may not exist between A, B, C, D, E, and / or F. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO. 8, and / or, the amino acid sequence of the degradation element cOdc1 is SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV, and / or, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or, the first tag sequence and the second tag sequence are different and selected from HHHHHH and DYKDHDGDYKDHDIDYKDDDDK. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is MGQVQLVESGGGSVQAGGSLRLSCTASGGSEYSYSTFSLGWFRQAPGQEREAVAAIASMGGLTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAALSGPSWDWECLSAIVASGELNWGQGTQVTVSSGSGDYKDHDGDYKDHDIDYKDDDDKGSGSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINVYGRKKRRQRRRHHHHHH.

[0017] Preferably, the fusion protein is ABCDEF, wherein A is a secreted peptide or is absent, B is the single-domain antibody against NUPR1, C is the first tag sequence, D is absent, E is a cell-penetrating peptide or is absent, F is absent, and there may or may not be a flexible linker between A, B, C, and / or E. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO. 8, and / or the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK.

[0018] Preferably, the fusion protein is ABCDEF, wherein A is a secreted peptide or is absent, B is the single-domain antibody against NUPR1, C is absent, D is absent, E is a cell-penetrating peptide or is absent, and F is a second tag sequence, with or without flexible linkers between A, B, E, and / or F. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO. 8, and / or the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or the second tag sequence is selected from HHHHHH and DYKDHDGDYKDHDIDYKDDDDK.

[0019] In another aspect, the present invention provides an antibody formulation comprising the single-domain antibody against NUPR1 or the fusion protein. Furthermore, the antibody formulation further comprises a pharmaceutically acceptable carrier, including but not limited to: buffer solutions, sterile water, surfactants, etc.

[0020] In another aspect, the present invention provides a kit comprising the single-domain antibody against NUPR1 or the fusion protein. The single-domain antibody against NUPR1 or the fusion protein can quantitatively or qualitatively detect the presence of NUPR1 protein in a test sample via an antigen-antibody binding reaction, thereby the kit can be used to detect the presence of NUPR1 protein in a test sample.

[0021] As those skilled in the art will know, methods for detecting antigen-antibody binding reactions include, but are not limited to: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme matrix staining, and antigen-antibody aggregation assay. Therefore, depending on the method employed, the kit may further include other chemical substances.

[0022] For example, the method for quantitative or qualitative detection via antigen-antibody binding reaction is ELISA, and the kit contains the single-domain antibody against NUPR1 of the present invention as the first antibody. The first antibody is adsorbed onto the surface of a solid-phase support. The solid-phase support can be any material conventionally used in the art, including but not limited to: polyvinyl chloride, polystyrene, polyacrylamide, or cellulose. The form of the solid-phase support includes, but is not limited to: multi-well plates, test tubes, beads, etc. The kit may further contain a second antibody bound to an indicator molecule, which specifically binds to the NUPR1 protein, and the second antibody binds to a different antigenic determinant than the first antibody. When the indicator molecule is an enzyme, the kit may further contain an enzyme substrate.

[0023] For example, the method for quantitative or qualitative detection via antigen-antibody binding reaction is immunoblotting, and the kit may contain the single-domain antibody against NUPR1 of the present invention as a first antibody. The first antibody may further bind to an indicator molecule. When the first antibody does not bind to the indicator molecule, the kit may further contain a second antibody bound to the indicator molecule, which may bind to the first antibody; or, the kit may further contain an IgG-binding protein bound to the indicator molecule, such as protein A. The kit may also further contain reagents for electrophoretic separation of proteins.

[0024] According to the present invention, the indicator molecule may be, for example, a fluorescent substance, a radioactive substance, and / or an enzyme. The enzyme may be a commonly used enzyme in the art, including but not limited to: peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, glucose oxidase, etc. The enzyme may be labeled onto the antibody using cross-linking methods known in the art, including but not limited to: glutaraldehyde method and periodate oxidation method, etc. The fluorescent substance may be a variety of fluorescent substances known and commonly used in the art, including but not limited to: fluorescein isothiocyanate, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, phycoerythrin, Cy3, Cy5, DyLight405, DyLight488, DyLight550, DyLight594, DyLight633, DyLight650, DyLight680, DyLight755, DyLight800, SYBR Green I, etc.

[0025] The sample being tested can be a living organism or a substance that was previously a living organism, including but not limited to: blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid; cells such as endothelial cells, leukocytes, and monocytes; organs (heart, kidneys, spleen, lungs, etc.); and tissues (bone marrow, lymph nodes, connective tissue, adipose tissue, etc.).

[0026] In another aspect, the present invention provides an isolated nucleic acid molecule that encodes the single-domain antibody against NUPR1 or the fusion protein.

[0027] Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.12 or SEQ ID NO.18.

[0028] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

[0029] Preferably, the expression vector can be a plasmid, bacteriophage, or virus.

[0030] In another aspect, the present invention provides a host cell whose genome integrates the aforementioned nucleic acid molecules; or, the host cell contains the aforementioned expression vector. Preferably, the host cell is a eukaryotic cell or a prokaryotic cell.

[0031] In another aspect, the present invention provides a pharmaceutical composition comprising the single-domain antibody against NUPR1 or the fusion protein. Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" includes, but is not limited to, any and all solvents, dispersion media or other liquid carriers, dispersing or suspending agents, diluents, isotonic agents, preservatives, colorants, sweeteners or flavorings, stabilizers, antioxidants, antimicrobial or antifungal agents, molar osmolality adjusters, pH adjusters, buffers, chelating agents, cryoprotectants, and / or fillers, as suitable for the desired specific dosage form. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzyl chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, and combinations thereof. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and combinations thereof. Exemplary diluents include, for example, lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol, etc. Exemplary adhesives include: starch, gelatin, sodium carboxymethyl cellulose, polyvinylpyrrolidone, etc. Exemplary antioxidants include: vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, etc. Exemplary lubricants include: magnesium stearate, micronized silica gel, talc, etc. Exemplary disintegrants include: starch, methyl cellulose, xanthan gum, croscarmellose sodium, etc.

[0032] The dosage form of the drug of this invention can be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; or an injectable dosage form, such as an injection solution, powder for injection, etc., administered via intravenous, intraperitoneal, subcutaneous, or intramuscular routes. All dosage forms used are well known to those skilled in the art of pharmacy.

[0033] In another aspect, the present invention provides the use of the single-domain antibody against NUPR1 or the fusion protein in the preparation of reagents or kits for detecting NUPR1 in samples.

[0034] In another aspect, the present invention provides the use of the single-domain antibody against NUPR1 or the fusion protein in the preparation of medicaments for the prevention and / or treatment of cancer.

[0035] The cancers mentioned include, but are not limited to: pancreatic cancer, breast cancer, liver cancer, lung cancer, colorectal cancer, oral cancer, bladder cancer, myeloma, basal cell carcinoma, bile duct cancer, bone cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, leukemia, lymphoma, melanoma, neuroblastoma, ovarian cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancers, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancers, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, and chronic myeloid leukemia.

[0036] In another aspect, the present invention provides a method for screening single-domain antibodies against NUPR1, the method comprising:

[0037] 1) Replace the CDR3 domain on the sdAb backbone with a first nucleotide sequence encoding 21 random amino acids, and link a nucleotide sequence encoding a first expression tag to the 3' end of the sdAb backbone to obtain the nucleotide sequence of the sdAb library, and then insert it into the first expression vector to obtain the first recombinant expression vector of the sdAb library.

[0038] Preferably, the sdAb backbone is cAbBCII10, and its amino acid sequence is shown in SEQ ID NO.9;

[0039] 2) The 3' end of the second nucleotide sequence encoding the NUPR1 protein is joined with a nucleotide sequence encoding the second expression tag, and then inserted into the second expression vector to obtain the second recombinant expression vector expressing the NUPR1 protein;

[0040] The first expression tag is different from the second expression tag, and the first expression vector is the same as or different from the second expression vector; preferably, the first expression tag and the second expression tag are selected from Flag or HA; the first expression vector and the second expression vector are selected from pcDNA3.1;

[0041] 3) The first recombinant expression vector and the second recombinant expression vector were co-transfected into cells. After culturing for a period of time, the cells were fixed. The fixed cells were subjected to in situ proximity ligation assay (isPLA), and then isPLA positive cells were sorted.

[0042] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 4) using the lysed isPLA positive cells obtained in step 3) as a template for PCR, designing primers based on the upstream and downstream sequences of the CDR3 domain insertion site of the first recombinant expression vector for PCR amplification, and replacing the CDR3 domain on the sdAb backbone with the amplified DNA fragment to obtain a NUPR1-specific sdAb.

[0043] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 5) verifying the NUPR1-specific sdAb obtained in step 4) using the NanoBiT method.

[0044] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 6) linking the nucleotide sequence of the positive sdAb from step 5) to a C-degron(cOdc1) degradation element for further verification.

[0045] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 7) expressing, purifying and functionally validating the sdAb that was verified as positive in step 6).

[0046] Sequence list of the present invention:

[0047]

[0048]

[0049]

[0050] The beneficial effects of this invention are:

[0051] This invention obtains a #07.81 single-domain antibody targeting NUPR1 through screening, which can specifically bind to NUPR1; after fusing the single-domain antibody with the C-degron degradation element cOdc1, targeted degradation of NUPR1 can be achieved, while effectively inhibiting tumor progression; further fusing the TAT transmembrane domain can make the single-domain antibody easily enter cells and minimize immunogenicity.

[0052] This invention develops a simple, easy-to-operate, and visualized single-domain antibody screening method for NUPR1 at the single-cell level by combining isPLA technology with next-generation DNA sequencing technology. This method is low in cost and highly efficient.

[0053] In this invention, "and / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0055] In this invention, the terms "single-domain antibody," "sdAb," and "VHH" are used interchangeably to refer to an antibody having a single monomeric domain antigen-binding / recognition domain. Such antibodies include camel antibodies or shark antibodies. In some embodiments, the VHH comprises three CDRs and four framework regions, referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH may be truncated at the N-terminus or C-terminus so that it contains only a portion of FR1 and / or FR4, or lacks one or two of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0056] As used herein, the "percentage of amino acid sequence identity (%)" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignments for determining the percentage of amino acid sequence identity can be performed in various ways within this art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIG NTM (DNASTAR) software. Attached Figure Description

[0057] Figure 1 The screening process for anti-NUPR1 sdAbs includes:

[0058] A: Screening workflow for anti-NUPR1 sdAbs: 1) Screen for CDR3 sequences using isPLA; 2) Validate the CDR3 sequences screened by isPLA using NanoBiT (in vivo live-cell interaction) technology; 3) Further validate the positive CDR3 sequences screened by NanoBiT using the C-degron assay; 4) Finally, express and purify the positive sdAbs, and perform functional validation on the anti-NUPR1 sdAbs.

[0059] B: The NUPR1 CDR3 sequence that was positive for isPLA screening was verified by NanoBiT experiment: LgBiT-CDR3 and SmBiT-NUPR1 were co-transfected in HEK293T cells, and their luciferase reporter activity was detected to verify the interaction between different CDR3 and NUPR1.

[0060] C: Western blot analysis of C-degron (cOdc1) assay results: HEK293T cells were transfected with different doses of Flag-sdAb#07.32-cOdc1 (left), Flag-sdAb#07.81-cOdc1 (middle), and Flag-sdAb#31.89-cOdc1 (right) expression plasmids, and compared with the control group (Flag-sdAb-Con) to detect the endogenous NUPR1 protein level, with ACTB as an internal control.

[0061] D: Different doses of the expression plasmids Flag-sdAb#07.32-cOdc1 (left), Flag-sdAb#07.81-cOdc1 (middle), and Flag-sdAb#31.89-cOdc1 (right) were transfected into MDA-MB-231 cells, and compared with the control group (Con or Flag-sdAb-Con) to detect the endogenous protein level of NUPR1, with ACTB as an internal control.

[0062] E: Different doses of Flag-sdAb#07.81-cOdc1 expression plasmid were transfected into MDA-MB-468 cells and compared with the control group (Flag-sdAb-Con) to detect the endogenous protein level of NUPR1, with ACTB as an internal control.

[0063] F: 4T1 cells were transfected with expression plasmids Flag-sdAb#07.81-cOdc1, Flag-sdAb#07.32-cOdc1, and Flag-sdAb#31.89-cOdc1, respectively, and compared with control groups (Con and Flag-sdAb-Con) to detect the endogenous protein level of NUPR1, with ACTB as an internal control.

[0064] Figure 2 The in vivo and in vitro functional validation results of anti-NUPR1 sdAbs include:

[0065] A: Purification results of anti-NUPR1 sdAb protein: The protein was purified by overexpressing sdAb-Con-3×Flag-cOdc1-Tat-6×His, sdAb#07.81-3×Flag-cOdc1-Tat-6×His, and sdAb#31.89-3×Flag-cOdc1-Tat-6×His in HEK293F cells. The purification effect was detected using Coomassie blue staining, with BSA as a control. S: Supernatant; P: Purified sdAb protein.

[0066] B: Immunofluorescence (IF) colocalization analysis results of anti-NUPR1 sdAb in 4T1 cells: 4T1 cells were stained with triple immunofluorescence using anti-Flag mouse antibody (green), anti-NUPR1 rabbit antibody (red), and DAPI (blue), and imaged using confocal microscopy. White arrows indicate the locations of the yellow dots indicating colocalization. 4T1 cells were inoculated with purified sdAb-Con, sdAb#07.81, and sdAb#31.89 proteins and cultured for 48 hours before immunofluorescence detection. Scale bar: 20 μm.

[0067] C: GST pull-down experiment to verify the interaction between anti-NUPR1 sdAbs and NUPR1: GST pull-down experiment was performed using purified sdAb-Con or sdAb#07.81, which were incubated overnight at 4°C with purified GST or GST-NUPR1, respectively. The bound proteins were separated by SDS-PAGE electrophoresis and analyzed by Coomassie brilliant blue staining.

[0068] D: Surface Plasmon Resonance (SPR) Analysis: The left figure shows the SPR sensing curve between sdAb#07.81 and GST, and the right figure shows the SPR sensing curve between sdAb#07.81 and GST-NUPR1.

[0069] E: isPLA assay to verify the function of anti-NUPR1 sdAb#07.81: 48 hours after transfection, the interaction between anti-NUPR1 and sdAb was detected by isPLA assay. White arrows indicate isPLA signal points. Nuclear staining was performed using DAPI (blue). Scale bar: 10 μm.

[0070] FI: Immunoblot analysis of NUPR1 in MDA-MB-231 and 4T1 cells: After treating MDA-MB-231(FG) and 4T1(HI) cells with anti-NUPR1 sdAb#07.81, sdAb#31.89, and sdAb#31.89+#07.81 for 48 hours, the protein expression levels of NUPR1 and Flag-sdAb were detected, with ACTB as an internal control and Con as the control group.

[0071] Figures 3A-3E Results of the experiment on the inhibition of 4T1 cell progression by anti-NUPR1 sdAb#07.81, including:

[0072] Figure 3A Allogeneic transplantation experiments were conducted on BALB / c mice. Each mouse was injected with 4 T1 cells (1×106 cells). The experiment was divided into three groups: control group (Con, n=7), single-domain antibody control group (sdAb-Con, n=7), and sdAb#07.81 treatment group (n=7). Purified sdAb protein was injected into the tumor once every 3 days.

[0073] Figure 3B : Tumor images taken from mice in each group.

[0074] Figure 3C Tumor weight was measured and statistical analysis was performed using a t-test. **** indicates p < 0.0001.

[0075] Figure 3D Representative immunohistochemical (IHC) staining images show the expression of Ki67 in subcutaneous tumor tissue. Scale bar: 200 μm.

[0076] Figure 3E The protein levels of Flag-sdAb and NUPR1 in tumors were detected by Western blot, with ACTB used as an internal control. Detailed Implementation

[0077] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0078] Materials and methods:

[0079] Reagents: DAPI (D9542, Sigma-Aldrich), BCA protein assay kit (23250, Thermo Scientific), BSA (New England BioLabs), ECL (enhanced chemiluminescence) assay kit (32106, Thermo Fisher Scientific), EB (ethidium bromide, E1385, Sigma-Aldrich), GSH (G2451, Sigma-Aldrich), imidazole (I5513, Sigma-Aldrich), Tris (T1503, Sigma-Aldrich), IgG (AC011, mouse, Abclonal), polyethyleneimine (PEI) (Polysciences, Inc.), Lipo 3000 transfection reagent (Invitrogen, L3000075), Lipo293F transfection reagent (Beyotime, C0518), paraformaldehyde (158127, Sigma-Aldrich), polylysine (P4707, Sigma-Aldrich), protease inhibitor mixed tablets (4693132001, Roche, Basel, Switzerland), tissue cell lysis buffer (Prilex, C1051), restriction endonucleases including EcoRI and BamHI (BioLabs, New England), seamless cloning and assembly kit (CU201, TransGen Biotech), DNA Markers Plus II (BM121, TransGen Biotech) and protein pre-staining marker (26616, Thermo Scientific).

[0080] Antibodies: Flag antibody (F7425, mouse monoclonal, Sigma-Aldrich), HA antibody (#7695, rabbit monoclonal, Cell Signaling Technology), anti-Flag M2 affinity beads (A2220, Sigma-Aldrich), NUPR1 antibody (rabbit polyclonal antibody, self-made antibody).

[0081] Reagent test kit: Protein: Protein Interaction System (N2014, Promega, USA), Duolink In Situ Red Starter Kit Mouse / Rabbit (DUO92101, Sigma-Aldrich, Germany).

[0082] Cells: HEK293T cells, MDA-MB-231 cells and 4T1 cells were purchased from ATCC (Manassas, VA, USA).

[0083] Example 1: sdAb Library Construction

[0084] The nucleotide sequence of camel-derived sdAb (cAbBCII10, amino acid sequence as shown in SEQ ID NO. 9) was synthesized using gene synthesis methods and cloned into the pcDNA3.1 vector. The CDR3 region sequence was replaced with EcoRI, BamHI restriction sites and a 6-base-pair linker DNA sequence (5'-GAATTCGGCAGCGGATCC-3'). The complete nucleic acid sequence is shown in SEQ ID NO. 11. A 3×Flag tag was added to the 3' end of the sdAb gene to obtain the sdAb control (hereinafter referred to as "sdAb Con") plasmid. The sdAb Con plasmid also serves as the backbone sequence for library construction.

[0085] The first nucleotide sequence encoding 21 random amino acids was substituted into the CDR3 region of the sdAb Con plasmid to obtain the first recombinant expression vector expressing the sdAb library. The first nucleotide sequence contains 63 degenerate bases and approximately 20 nucleotides at both ends that match sdAb con. The first nucleotide sequence is: 5'-CTATTTATTATTGTGCTGCT(NNN). 21 TGGGGTCAAGGTACTCAAGTTACT-3'. Simultaneously, a primer complementary to its 3' end was synthesized for completing the DNA double strand via PCR. The primer sequence is: 5'-AGTAACTTGAGTACCTTGACC-3'.

[0086] Example 2: Screening for anti-NUPR1 sdAbs

[0087] 1) isPLA Filtering

[0088] HEK293T cells were co-transfected with a first recombinant expression vector expressing an sdAb library tagged with Flag and a second recombinant expression vector expressing NUPR1 tagged with HA. After 48 h of culture, cells were trypsin-digested and fixed with 1% PFA, and then collected for subsequent isPLA assays. The isPLA assay was performed according to the instructions of the Duolink In Situ Red Starter Kit Mouse / Rabbit (DUO92101, Sigma-Aldrich). Collected cells were permeabilized with PBS containing 0.5% Triton X-100 at room temperature for 10 min, then transferred to 1.5 mL EP tubes and blocked with blocking buffer at 37°C for 1 h. Anti-HA rabbit antibody and anti-Flag mouse antibody were then added, and the cells were incubated at 37°C for 1 h. Finally, MINUS (anti-mouse) and PLUS (anti-rabbit) PLA probes provided in the kit were added, and the cells were incubated at 37°C for 1 h. The hybridization probe was then ligated in Ligation-Ligase solution at 37°C for 30 min, and finally amplified in Amplification-Polymerase solution at 37°C for 100 min.

[0089] Following isPLA, HEK293T cells were washed twice with PBS buffer containing 1% BSA, 2mM EDTA, and 0.1% NaN3 (1500 rpm, 4°C, 5 min), and immediately sorted using a BD FACS Aria II flow cytometer. Data were analyzed using FlowJo V10.0.7 software (Tree Star). The sorted positive cells were collected in nucleic acid-free water and used as PCR templates for cDNA amplification.

[0090] Cells collected in nucleic acid-free water by flow cytometry were incubated in a metal bath at 95°C for 10 min and used as templates for PCR to amplify DNA fragments in the CDR3 region. PCR primers complementary to the upstream and downstream sequences of the CDR3 region, CDR3-forward:5'-ACACCGCCATCTACTACTGC-3' and CDR3-reverse:5'-GCTGCTCACTGTCACTTGTG-3', were synthesized by Invitrogen. Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific) was used according to the manufacturer's instructions. 500 cells were used as templates in a 50 μL reaction system, with a final primer concentration of 0.5 μM. The PCR steps included: pre-denaturation at 98°C for 2 min; amplification for 50 cycles, including denaturation at 98°C for 30 sec, annealing at 62°C for 30 sec, extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR product is used for constructing the sublime library in the next round of screening. If no further screening is required, the PCR product can be directly used for next-generation sequencing. If a single clone is desired, the PCR product can be subcloned into the sdAb Con vector, and then sequenced after obtaining the single clone. The sequencing primer is 5'-GCACCAAAATCAACGGGAC-3'.

[0091] 2) The NUPR1 sdAb sequence that is positive for isPLA screening was validated using NanoBiT experiments.

[0092] NanoBiT is a type of... The NanoBiT system is a two-subunit system for detecting protein-protein interactions within living cells. It consists of two small units: Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acid peptide), each fused to a target protein. When the two target proteins interact, LgBiT becomes structurally complementary to SmBiT, forming a functional luciferase that reacts with the substrate to produce a bright luminescent signal. The empty vector plasmid provided by the PPI System Technical Manual TM461 Promega kit (Promega, Cat#N2014) is used to ligate the nucleic acid sequence encoding sdAb into the LgBiT vector, resulting in the plasmid hereinafter referred to as LgBiT-CDR3. The NUPR1 sequence is then ligated into the SmBiT vector, resulting in the plasmid hereinafter referred to as SmBiT-NUPR1.

[0093] HEK293T cells were seeded in 24-well plates. The next day, they were co-transfected with LgBiT-CDR3 and SmBiT-NUPR1 plasmids. After 6-8 hours, the culture medium was aspirated from the 24-well plates and replaced with phenol red-free medium. Detection was performed after 48 hours. The culture medium was first aspirated, then slowly washed once with PBS. Nano- Live Cell Substrate and Nano- LCSDilution Buffer (Promega, Cat#N2011) was mixed at a ratio of 1:19, then added to the plate, and immediately used a microplate reader to detect luciferase reporter activity. The results are as follows: Figure 1 As shown in Figure B. The results showed that, compared with the control group sdAb sequences (represented as "NAb(Con)" in the figure), sdAb sequences numbered #07.32, #31.53, #31.89, and #07.81 all exhibited significant luciferase activity. These results indicate that sdAb sequences numbered #07.32, #31.53, #31.89, and #07.81 specifically bind to NUPR1 in living cells, with sdAb sequence #07.81 exhibiting the strongest binding specificity.

[0094] 3) C-degron assays were performed to validate the positive sdAb sequences selected by isPLA and NanoBiT.

[0095] HEK293T, MDA-MB-231, MDA-MB-468, and 4T1 cells were transfected with the expression plasmids sdAb#07.32-3×Flag-cOdc1, sdAb#07.81-3×Flag-cOdc1, and sdAb#31.89-3×Flag-cOdc1, respectively, and compared with the control group (sdAb-Con-3×Flag-cOdc1). Endogenous NUPR1 protein levels were detected by Western blot, using ACTB as an internal control. The specific procedure was as follows: 48 hours after transfection, cells were collected and treated in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL CA-630, 0.2 mM EDTA, pH 8.0), and then centrifuged at 12,000 rpm for 15 minutes. Western blotting was performed using the corresponding antibody, followed by development using an ECL detection system (32106, Thermo Scientific), exposure onto X-ray film, and scanning and recording of the exposure bands. The results are as follows: Figure 1The results show that both sdAb#07.32-3×Flag-cOdc1 (represented as "#07.32-cOdc1" in the figure) and sdAb#07.81-3×Flag-cOdc1 (represented as "#07.81-cOdc1" in the figure) can significantly reduce the level of endogenous NUPR1 protein. sdAb #07.81 showed high activity in all cell lines. These results indicate that sdAb #07.81 has a strong specific binding affinity to NUPR1, and that cOdc1 can achieve targeted degradation of NUPR1.

[0096] Example 3: Functional verification of resistance to NUPR1 sdAbs

[0097] 1) Purification of anti-NUPR1 sdAb protein

[0098] The constructed eukaryotic expression plasmids containing secretory peptides, sdAb-Con-3×Flag-cOdc1-Tat-6×His, sdAb#07.81-3×Flag-cOdc1-Tat-6×His, and sdAb#31.89-3×Flag-cOdc1-Tat-6×His, were transiently introduced into HEK293F cells and cultured at 37°C, 130 rpm, and 8% CO2. After 72 h of transfection, the supernatant was collected and filtered through a 0.45 μm filter. The protein was then purified using a nickel affinity chromatography column (Sangon Biotech, C600793). The purified protein was visualized by Coomassie brilliant blue staining, with bovine serum albumin (BSA) as a control. In this embodiment, the corresponding proteins are referred to as "sdAb-Con", "sdAb#07.81", and "sdAb#31.89", respectively. The results are as follows: Figure 2 As shown in Figure A. 2) Immunofluorescence (IF) colocalization analysis of 4T1 cells.

[0099] 4T1 cells were inoculated with purified recombinant proteins sdAb-Con, sdAb#07.81, and sdAb#31.89 and cultured for 48 hours before immunofluorescence detection. Triple immunofluorescence staining of 4T1 cells was performed using anti-Flag mouse antibody, anti-NUPR1 rabbit antibody, and DAPI, and imaging was performed using confocal microscopy. Results are as follows: Figure 2 As shown in Figure B. The results showed that both sdAb#07.81 and sdAb#31.89 co-localized with endogenous NUPR1 in 4T1 cells, and the co-localization effect of sdAb#07.81 was significantly better than that of sdAb#31.89.

[0100] 3) GST pull-down experiment verifies the interaction between sdAbs and NUPR1.

[0101] GST pull-down experiments were performed using purified sdAb-Con or sdAb#07.81, incubated overnight at 4°C with purified GST or GST-NUPR1, respectively. The bound proteins were separated by SDS-PAGE electrophoresis and analyzed by Coomassie Brilliant Blue staining. Results are as follows: Figure 2 As shown in Figure C. The results showed that, in vitro, sdAb#07.81 binds to GST-NUPR1 and interacts directly, but does not interact with GST. These results indicate that a specific binding interaction exists between sdAb#07.81 and NUPR1.

[0102] 4) Surface plasmon resonance (SPR) analysis

[0103] Prepare and purify the GST, GST-NUPR1, sdAb Con, and sdAb#07.81 proteins required for the SPR experiment in advance, ensuring they are all in the same buffer system (1×PBS). The experiment was performed on a Biacore 8K instrument (Cytiva), using the CM5 chip. The CM5 surface was activated using a prepared NHS and EDC mixture (1:1, v / v). sdAb-Con and dAb#07.81 were coupled to the CM5 chip using the standard coupling method at a protein concentration of 20 μg / mL. Before ligand coupling, the ligands were diluted with Cytiva's pH 4.0 solution (pH selection depends on the protein's isoelectric point). All remaining wells were filled with running buffer. Analytes capture: GST and GST-NUPR1 proteins were diluted with running buffer to pre-determined concentrations: 4000 nM, 2000 nM, 1000 nM, 500 nM, and 250 nM. The analytes were bound to the chip at a flow rate of 30 μL / min for 180 s, followed by dissociation with running buffer for 200 s. After each cycle, the chip was regenerated with glycine solution at pH 3.0. A total of 9 cycles were performed. At the end of the experiment, the dissociation constant was calculated and analyzed using Cytiva software. Results are as follows: Figure 2 As shown in Figure D. The results showed that, in vitro, sdAb#07.81 could bind to GST-NUPR1 but not to GST. These results indicate that there is a specific binding interaction between sdAb#07.81 and NUPR1.

[0104] 5) isPLA experimental verification of the function of sdAb in #07.81

[0105] After transfecting HEK293T cells with the plasmid expressing sdAb#07.81-3×Flag, the cells were seeded onto poly-lysine (P4707, Sigma-Aldrich) coated coverslips in 12-well plates. After overnight culture, the cells were fixed with 1% paraformaldehyde (15812, Sigma-Aldrich) for 10 min, washed twice with PBS buffer, and then subjected to isPLA assays using primary antibodies against NUPR1 and Flag. Finally, the cells were mounted with DAPI-containing mounting medium, and the results were analyzed and photographed using a laser scanning confocal microscope. The results are as follows: Figure 2 As shown in Figure E. The results showed that, compared with the negative control sdAb sequence (sdAb-Con), the sdAb of #07.81 could specifically interact with endogenous NUPR1, and the red fluorescent signal (indicated by the white arrow) was the positive signal. The results indicate that the sdAb of #07.81 can specifically bind to endogenous NUPR1.

[0106] 6) Immunoblot analysis of anti-NUPR1 sdAb protein in MDA-MB-231 and 4T1 cells

[0107] The purified anti-NUPR1 sdAb#07.81, sdAb#31.89, and the combination of sdAb#31.89+sdAb#07.81 prepared above were used to treat MDA-MB-231 and 4T1 cells for 48 hours, respectively. The protein expression levels of NUPR1 and anti-NUPR1 sdAb were then detected, with ACTB used as an internal control. The control group was Con. Results are as follows: Figure 2 As shown in Figure FI. The results showed that compared with the sdAb-Con group, sdAb#07.81 could specifically target NUPR1, and its ability to degrade NUPR1 gradually increased with increasing protein concentration. These results indicate that the purified sdAb#07.81 still has the ability to target NUPR1 and still has the function of degrading NUPR1.

[0108] Example 4: Experimental study on the inhibition of 4T1 cells by anti-NUPR1 sdAb#07.81

[0109] The purified protein prepared in Example 3 was used to conduct the experiments in this example.

[0110] Allogeneic transplantation experiments were performed on BALB / c mice. Each mouse received a subcutaneous injection of 4 T1 cells (1 × 10⁶ cells) into the posterior dorsal side of one side. The experiment was divided into three groups: a control group (Con, n = 7), a single-domain antibody control group (sdAb-Con, n = 7), and an sdAb#07.81 treatment group (n = 7). Purified sdAb protein (15 mg / kg) was injected intratumorally on days 5, 8, 11, and 14. Tumors were harvested on day 17 for analysis. Tumor images and weight are shown below. Figure 3B-3C As shown in the figure. The results showed that compared with the Con group and the sdAb-Con group, the tumor volume and weight of mice in the sdAb#07.81 group were significantly reduced, indicating that sdAb#07.81 has a significant inhibitory effect on tumors. Immunohistochemical (IHC) staining images showed the expression of Ki67 in subcutaneous tumor tissue, and the results are shown in the figure. Figure 3D As shown in the figure. The results showed that the positive signal of Ki67 expression in tumor tissues of the sdAb#07.81 group was significantly reduced, indicating that sdAb#07.81 can effectively inhibit the proliferation of tumor cells. The protein levels of anti-NUPR1 sdAb and NUPR1 in tumors were detected by Western blot, with ACTB as an internal control. The results are shown in the figure. Figure 3E As shown in the figure. The results showed that compared with the Con group and the sdAb-Con group, the expression level of NUPR1 protein in the sdAb#07.81 group was significantly decreased, indicating that sdAb#07.81 targets NUPR1 and can degrade NUPR1.

[0111] This invention utilizes isPLA high-throughput screening to obtain an anti-NUPR sdAb library containing a 21-amino acid sequence with randomly arranged CDR3, yielding a single-domain antibody specifically recognizing NUPR1—sdAb number #07.81. Linking the C-degron degradation element cOdc1 to the sequence of the #07.81 single-domain antibody enables specific degradation of NUPR1 and exerts an inhibitory effect on tumor progression.

[0112] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A single-domain antibody targeting NUPR1, characterized in that: The single-domain antibody contains three complementarity-determining regions, namely HCDR1, HCDR2 and HCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The single-domain antibody as described in claim 1, characterized in that: The single-domain antibody further comprises four backbone regions alternately linked to three complementarity-determining regions, namely FR1, FR2, FR3 and FR4, wherein the amino acid sequences of FR1, FR2, FR3 and FR4 have at least 75% sequence identity with the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

3. The single-domain antibody as described in claim 2, characterized in that, The amino acid sequences of FR1, FR2, FR3 and FR4 have at least 80% sequence identity with the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

4. The single-domain antibody as described in claim 2, characterized in that, The amino acid sequences of FR1, FR2, FR3 and FR4 have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

5. The single-domain antibody as described in claim 2, characterized in that, The amino acid sequences of FR1, FR2, FR3 and FR4 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

6. The single-domain antibody as described in claim 2, characterized in that, The amino acid sequences of FR1, FR2, FR3 and FR4 have at least 95% sequence identity with the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

7. The single-domain antibody as described in claim 2, characterized in that, The amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

8. The single-domain antibody as described in claim 1, characterized in that: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.

8.

9. A fusion protein, characterized in that: The fusion protein is composed of ABCDEF, wherein A is a secretory peptide or is absent, B is a single-domain antibody according to any one of claims 1-8, C is a first tag sequence, D is a degradation element cOdc1, E is a cell-penetrating peptide, and F is a second tag sequence, and there may or may not be a flexible linker between A, B, C, D, E and / or F; the amino acid sequence of the degradation element cOdc1 is: SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV; The cell-penetrating peptide is selected from TAT-derived peptides, antennal leader peptides, antennal peptides, and polyarginine peptides. The first tag sequence and the second tag sequence may be the same or different, and are selected from His tag, Myc tag, HA tag, and Flag tag.

10. The fusion protein as described in claim 9, characterized in that, The cell-penetrating peptide is selected from YGRKKRRQRRR, GRKKRRQRRR, KKWKMRRNQFWVKVQRG, RQIKIWFQNRRMKWKK, or a polyarginine peptide composed of 6-9 arginine residues.

11. The fusion protein as described in claim 9 or 10, characterized in that, The first tag sequence is different from the second tag sequence, which are the His tag and the Flag tag, respectively.

12. The fusion protein as described in claim 11, characterized in that, The first tag sequence is HHHHHH, and the second tag sequence is DYKDHDGDYKDHDIDYKDDDDK, or the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK, and the second tag sequence is HHHHHH.

13. The fusion protein as described in claim 9, characterized in that, The amino acid sequence of the fusion protein is MGQVQLVESGGGSVQAGGSLRLSCTASGGSEYSYSTFSLGWFRQAPGQEREAVAAIASMGGLTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAALSGPSWDWECLSAIVASGELNWGQGTQVTVSSGSGDYKDHDGDYKDHDIDYKDDDDKGSGSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINVYGRKKRRQRRRHHHHHH.

14. A polynucleotide, characterized in that: The polynucleotide is a polynucleotide encoding a single-domain antibody as described in any one of claims 1-8 or a fusion protein as described in any one of claims 9-13.

15. The polynucleotide of claim 14, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO. 12 or SEQ ID NO.

18.

16. An expression carrier, characterized in that, It contains the polynucleotide of claim 14 or 15.

17. A host cell, characterized in that, It includes the expression vector as described in claim 16.

18. A pharmaceutical composition or a reagent kit, characterized in that, The pharmaceutical composition or the kit comprises a single-domain antibody as described in any one of claims 1-8 or a fusion protein as described in any one of claims 9-13.

19. The use of the single-domain antibody of any one of claims 1-8 or the fusion protein of any one of claims 9-13 in the preparation of reagents or kits for detecting NUPR1 in samples.

20. The use of the single-domain antibody of any one of claims 1-8 or the fusion protein of any one of claims 9-13 in the preparation of a medicament for treating cancer, wherein the cancer is breast cancer, pancreatic cancer, lung cancer or colorectal cancer.

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