Anti-HIF-1alpha monoclonal antibody and application thereof

Abstract

The invention discloses an anti-HIF-1alpha monoclonal antibody and application thereof, and belongs to the technical field of biological medicines. The anti-HIF-1alpha monoclonal antibody provided by the invention can be specifically combined with the epitope of the 402nd amino acid and / or the 564th amino acid of the HIF-1alpha protein, and has the advantages of high affinity and strong specificity. Besides, the anti-HIF-1alpha monoclonal antibody not only can be used for quantitatively detecting the content of HIF-1alpha protein, but also can play an intervention role by blocking the structural stability or functional activity of the HIF-1alpha due to the fact that the binding site is clear and is located in a key functional region of the HIF-1alpha; therefore, the probe has potential application prospects in diagnosis and treatment of HIF-1alpha related diseases. Moreover, the antibody provided by the invention has important value in the aspects of scientific research, clinical detection, drug development and the like, and a new technical means is provided for research and application of HIF-1alpha related diseases.

Description

[0001] This application is a divisional application of the following patent application: The original application was filed on October 13, 2025. Application number: 202511456190.2; Invention title: A monoclonal antibody against HIF-1α and its application. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to a monoclonal antibody against HIF-1α and its application. Background Technology

[0003] Hypoxia-inducible factor-1α (HIF-1α) is a core regulator of the hypoxia response pathway. It plays a crucial role in the progression of various diseases by regulating downstream target genes, participating in angiogenesis, energy metabolism reprogramming, inflammatory cytokine release, and the maintenance of cell survival signals. Numerous studies have demonstrated that HIF-1α is persistently highly expressed in ischemic stroke, acute lung injury, inflammatory diseases, and various solid tumors, and is closely related to disease severity and prognosis.

[0004] Existing antibodies against HIF-1α generally suffer from low specificity and an inability to accurately identify key functional regions, hindering their effectiveness in scientific research and clinical applications. In particular, the 402nd and / or 564th amino acid residue region of the HIF-1α protein, located near its oxygen-dependent degradation domain (ODD) and transcriptional regulatory functional regions, is crucial in determining its stability and transcriptional activity. Specific identification of this region could not only enable the detection of HIF-1α expression levels but also potentially interfere with its functional activity, thus possessing significant scientific and practical value.

[0005] Currently, there are no research reports on monoclonal antibodies targeting the 402nd and / or 564th amino acid epitopes of the HIF-1α protein. Summary of the Invention

[0006] The purpose of this invention is to provide a monoclonal antibody against HIF-1α and its applications. This addresses the problems of existing HIF-1α antibodies, such as poor affinity, poor specificity, and inability to accurately identify key functional regions.

[0007] In a first aspect, the present invention provides a monoclonal antibody against HIF-1α or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region as shown in A1) or A2) below; A1) the heavy chain variable region comprises complementarity-determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and the light chain variable region comprises complementarity-determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 6-8; A2) the heavy chain variable region comprises complementarity-determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 11-13, and the light chain variable region comprises complementarity-determining regions CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 16-18.

[0008] The anti-HIF-1α monoclonal antibody or its antigen-binding fragment provided by the present invention includes at least one of Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibody (ScFv), and single-domain antibody (sdAb).

[0009] In some implementations, the anti-HIF-1α monoclonal antibody includes at least one of murine antibodies, chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0010] In some implementations, the monoclonal antibody against HIF-1α is an IgG subtype, preferably IgG1.

[0011] In this invention, the anti-HIF-1α monoclonal antibody or its antigen-binding fragment can specifically bind to the epitopes located at amino acid positions 402 and / or 564 of the HIF-1α protein, exhibiting advantages of high affinity and strong specificity. Furthermore, this anti-HIF-1α monoclonal antibody or its antigen-binding fragment can not only be used for the quantitative detection of HIF-1α protein levels but also block the functional activity of the HIF-1α protein, thereby enabling the diagnosis and treatment of ischemic stroke, acute lung injury, and tumor hypoxia-related diseases. Therefore, it has promising application prospects.

[0012] In some embodiments, in A1), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 9; in A2), the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 14, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 14; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19, or an amino acid sequence having more than 80% sequence identity with the sequence shown in SEQ ID NO: 19.

[0013] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0014] In a second aspect, the present invention provides a biological material selected from any of the following: B1) a nucleic acid molecule encoding any of the above-mentioned monoclonal antibodies against HIF-1α or its antigen-binding fragment; B2) a recombinant vector containing the nucleic acid molecule B1); B3) a recombinant cell containing the nucleic acid molecule B1 or the recombinant vector B2.

[0015] In some embodiments, in B1), the nucleic acid molecule is selected from any of the following: C1) a nucleic acid molecule having the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10; C2) a nucleic acid molecule having the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 20.

[0016] In this invention, the nucleic acid molecule also includes a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 10 or the nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 20. The codon preference can be conventionally designed according to the actual species.

[0017] In a third aspect, the present invention provides the use of any of the above-described anti-HIF-1α monoclonal antibodies or antigen-binding fragments thereof, or any of the above-described biological materials, in the preparation of products for detecting HIF-1α protein.

[0018] In some implementations, the product may include reagents, kits, chips, test strips, test cards, and immunosensors (such as electrochemical immunosensors).

[0019] In this invention, because anti-HIF-1α monoclonal antibodies or their antigen-binding fragments have the advantages of high affinity and strong specificity, rapid and accurate detection of HIF-1α protein content can be achieved.

[0020] In a fourth aspect, the present invention provides a method for detecting HIF-1α protein content, comprising the following steps: contacting the sample to be tested with any of the above-mentioned anti-HIF-1α monoclonal antibodies or their antigen-binding fragments; detecting the level of contact to obtain the HIF-1α protein content in the sample to be tested.

[0021] The method for detecting HIF-1α protein content provided by this invention is simple, and the detection results have the advantages of good accuracy and high sensitivity.

[0022] In some implementation schemes, the sample to be tested includes at least one of tissue samples, cell samples, and blood samples; the detection method includes at least one of ELISA, flow cytometry, immunoblotting, and immunohistochemistry.

[0023] In a fifth aspect, the present invention provides the use of any of the above-described anti-HIF-1α monoclonal antibodies or antigen-binding fragments thereof, or any of the above-described biological materials, in the preparation of medicaments for the prevention and / or treatment of diseases associated with HIF-1α overexpression.

[0024] In some implementations, the disease includes at least one of ischemic stroke, acute lung injury, and tumor hypoxia-related disease.

[0025] In a sixth aspect, the present invention provides a pharmaceutical composition comprising any of the above-described anti-HIF-1α monoclonal antibodies or antigen-binding fragments thereof.

[0026] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0027] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0028] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0029] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0030] In some implementations, solid dosage forms include at least one of tablets, capsules, powders, pills, granules, and suppositories; semi-solid dosage forms include at least one of ointments, creams, and patches; and liquid dosage forms include at least one of injections, oral liquids, syrups, and inhalants.

[0031] The pharmaceutical compositions provided by this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0032] The beneficial effects of this invention are as follows: Unlike existing technologies, the anti-HIF-1α monoclonal antibody or its antigen-binding fragment provided by this invention can specifically bind to the epitopes located at amino acid positions 402 and / or 564 of the HIF-1α protein, exhibiting advantages of high affinity and strong specificity. Furthermore, this anti-HIF-1α monoclonal antibody or its antigen-binding fragment can not only be used for the quantitative detection of HIF-1α protein levels, but also, due to its clearly defined binding site located in a key functional region of HIF-1α, may exert an interventional effect by blocking its structural stability or functional activity, thus showing potential application prospects in the diagnosis and treatment of HIF-1α-related diseases. Moreover, the antibody provided by this invention has significant value in scientific research, clinical testing, and drug development, providing a new technical means for the research and application of HIF-1α-related diseases. Attached Figure Description

[0033] Figure 1 The affinity test results for HIF-1α protein of the monoclonal antibody 19G7E12 prepared in Example 2 of this invention are shown. Figure 2 The affinity test results for HIF-1α protein of the monoclonal antibody 41D9F1 prepared in Example 2 of this invention are shown. Figure 3 The results of Western blot analysis of the expression of HIF-1α protein in different cell lines and tissues by the monoclonal antibody 41D9F1 prepared in Example 2 of this invention. Figure 4The results of Western blot analysis of the expression of HIF-1α protein in different cell lines and tissues by the monoclonal antibody 19G7E12 prepared in Example 2 of this invention are shown. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0036] In this invention, the recombinant HIF-1α protein (commercially available, product code: CSB-MP730731MO) has an expression range covering 380-585 amino acids and a His tag at the C-terminus. Its amino acid sequence is shown below: SEDTSCLFDKLKKEPDALTLLAPAAGDTIISLDFGSDDTETEDQQLEDVPLYNDVMFPSSNEKLNINLAMSPLPSSETPKPLRSSADPALNQEVALKLESSPESLGLSFTMPQIQDQPASPSDGSTRQSSPERLLQENVNTPNFSQPNSPSEYCFDVDSDMVNVFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDF(SEQ ID NO:21).

[0037] Example 1: Preparation of monoclonal antibodies against HIF-1α 1.1 Immunizing mice with recombinant HIF-1α protein Balb / c mice aged 6-8 weeks were selected and immunized subcutaneously with the above-mentioned recombinant HIF-1α protein as antigen, emulsified with Freund's adjuvant, for a total of 3-4 times, with each immunization spaced 2 weeks apart. Peripheral blood was then collected, and serum titer was determined by indirect ELISA (titer >1:64000 was considered acceptable; if unacceptable, the dosage of recombinant HIF-1α protein or the number of immunizations could be increased). Subsequently, spleens of mice with acceptable titers were collected and fused with NS-1 myeloma cells. Hybridoma cells were screened using HAT medium. The obtained hybridoma cells were initially screened by ELISA. The positive wells were further analyzed by flow cytometry with endogenous cells (CP-R137 cells, HeLa cells, and HeLa cells treated with DMOG) to verify their binding activity with HIF-1α protein.

[0038] The indirect ELISA assay was performed as follows: HIF-1α protein was coated onto a 96-well microplate, incubated overnight at 4°C, washed with PBS-T, and blocked with 1% BSA. Diluted serum was then added to the coated plate, incubated at 37°C for 1 hour, washed, and incubated again with HRP-labeled anti-mouse IgG secondary antibody. After washing again, TMB chromogenic solution was added, and the reaction was terminated with stop solution. The absorbance was measured at 450 nm.

[0039] The flow cytometry detection method is as follows: Suspended cells were collected by centrifugation at 1000 rpm; adherent cells were also collected and centrifuged after trypsin digestion. Tissue samples were digested with trypsin or collagenase, and digestion was stopped after single-cell suspension was observed under a microscope. The obtained cells were washed three times with PBS on ice, and the appropriate fixation and permeabilization method was selected according to the location of the protein to be detected: if membrane proteins were to be detected, they could be detected directly; if intracellular proteins were to be detected, they were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 for 5 min. The fixed cells were washed three times with PBS and blocked with 10% normal goat serum to reduce non-specific binding. Then, the primary antibody solution was prepared at the recommended dilution and incubated overnight at 4°C, with an isotype control set up; after washing three times with PBS, the fluorescently labeled secondary antibody solution was prepared at the recommended dilution and incubated at 4°C for 30 min in the dark. After incubation, the cells were washed three times with PBS and resuspended in PBS, kept in the dark, and the fluorescence signal was detected immediately. The results are shown in Tables 1 and 2 below.

[0040] Table 1. Results of flow cytometry analysis of positive wells from the initial screening and CP-R137 cells.

[0041] Table 2. Flow cytometry results of primary screening positive wells, HeLa cells, and DMOG-treated HeLa cells.

[0042] As shown in Tables 1 and 2, the monoclonal antibodies secreted by most positive hybridoma cell lines exhibited extremely high positive binding rates in CP-R137 cells, HeLa cells, and DMOG-treated HeLa cells (M3% Parent was generally above 99%, indicating good specific binding ability to HIF-1α protein). Furthermore, the average fluorescence intensity of FITC (Mean FITC-A) was significantly higher than that of the blank control and isotype control mIgG-FITC on the same plate, further demonstrating the reliability of the detection.

[0043] Furthermore, four short peptides were designed targeting the key amino acid epitope regions at positions 402 and 564 of the recombinant HIF-1α protein, and these peptides were coupled to BSA as rescreening antigens to rescreen the aforementioned positive hybridoma cell lines, thereby obtaining positive hybridoma cell lines that target and bind to positions 402 and 564 of the recombinant HIF-1α protein.

[0044] Specifically, the short peptides that target and bind to site 402 of the recombinant HIF-1α protein include: Polypeptide 1: BSA-Cys-DKLKKEPDALTILLAPAA (SEQ ID NO: 22); Peptide 2: LAPAAGDTIISLDFGSDDTE-Cys-BSA (SEQ ID NO: 23); Short peptides targeting site 564 of the recombinant HIF-1α protein include: Polypeptide 3: BSA-Cys-VEKLFAEDTEAKNPFSTQ (SEQ ID NO: 24); Polypeptide 4: PFSTQDTDLDLEMLAPYI-Cys-BSA (SEQ ID NO: 25).

[0045] Then, using the aforementioned short peptides as antigens, indirect ELISA was performed. The results showed that the positive hybridoma cell line 19G7E12 exhibited the highest OD after binding to peptides 1 and 2. 450nm Values ​​(2.0916 and 4.2905, respectively); the positive hybridoma cell line 41D9F1 showed the highest OD after binding to peptides 3 and 4. 450nm The values ​​are 2.455 and 1.603, respectively.

[0046] Subcloning was performed on the above-mentioned positive hybridoma cell lines 19G7E12 and 41D9F1 to obtain monoclonal cell lines 19G7E12 and 41D9F1, respectively, and the antibodies secreted by them were named monoclonal antibodies 19G7E12 and 41D9F1.

[0047] 1.2 Large-scale preparation and titer determination of monoclonal antibodies Freund's adjuvant was subcutaneously injected into 6-8 week old Balb / c mice. Two weeks later, the monoclonal cell lines 19G7E12 and 41D9F1 obtained in step 1.1 were subcutaneously injected, and ascites fluid was collected from the mice multiple times. The ascites fluid was then purified using Protein G affinity chromatography to obtain highly pure monoclonal antibodies 19G7E12 and 41D9F1.

[0048] The titers of the above monoclonal antibodies 19G7E12 and 41D9F1 were determined using indirect ELISA, and the results are shown in Table 3 below.

[0049] Table 3. Titer assay results of monoclonal antibodies 19G7E12 and 41D9F1

[0050] As shown in Table 3, the titers of monoclonal antibodies 19G7E12 and 41D9F1 are respectively, indicating that both monoclonal antibodies 19G7E12 and 41D9F1 have high titers.

[0051] 1.3 Identification and Sequencing of Monoclonal Antibodies To determine the subtype of the prepared monoclonal antibody, this invention employs an antigen-coated ELISA plate followed by the addition of the antibody to be tested for subtype identification. The specific steps are as follows: First, the recombinant antigen is coated onto the surface of a 96-well ELISA plate. After blocking, the supernatant of the monoclonal antibody to be tested or purified antibody is added. Then, enzyme-labeled secondary antibodies targeting different immunoglobulin subtypes are added. The binding status is determined by the colorimetric reaction; the subtype corresponding to the positive signal is the subtype of the antibody to be tested.

[0052] Upon identification, the monoclonal antibodies 19G7E12 and 41D9F1 in this invention are both IgG1 subtypes.

[0053] Further, during the gene sequencing of the monoclonal antibody, total RNA was first extracted from hybridoma cells. After chloroform layering, isopropanol precipitation, and washing with 75% ethanol, the RNA was dissolved and its integrity was detected by agarose gel electrophoresis. Subsequently, cDNA was obtained by reverse transcription using Oligo dT primers and a reverse transcriptase system at 37°C. Using this cDNA as a template, the antibody light and heavy chain genes were amplified separately using specific primers. The PCR products were separated by agarose gel electrophoresis, gel-cleaved, purified, and then digested with restriction endonucleases. The resulting recombinant plasmids were ligated into vectors overnight at 16°C using T4 DNA ligase. The purified recombinant plasmids were transformed into competent cells. The strains were heat-shocked and inoculated into LB solid medium containing ampicillin, cultured overnight, and single colonies were picked for amplification and plasmid DNA extraction. Finally, the samples were sent to a sequencing company for light and heavy chain gene sequencing to obtain the amino acid sequence information of the monoclonal antibody. Details are as follows: The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 19G7E12 is shown below: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPIYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTMIKESYWGQGTLVTVSA (SEQ ID NO: 4).

[0054] The complementarity-determining region of the heavy chain variable region of monoclonal antibody 19G7E12 is shown below: CDR1: DYSMH (SEQ ID NO: 1); CDR2: WINTETGEPIYVDDFKG (SEQ ID NO: 2); CDR3: IKESY (SEQ ID NO: 3).

[0055] The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 19G7E12 is shown below: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATAACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCC AATATATGTAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGCACTATGATTAAGGAGTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO: 5).

[0056] The amino acid sequence of the light chain variable region of monoclonal antibody 19G7E12 is shown below: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK (SEQ ID NO: 9).

[0057] The complementarity-determining region of the light chain variable region of monoclonal antibody 19G7E12 is shown below: CDR1:KSSQSLLYSNGKTYLN(SEQ ID NO: 6); CDR2:LVSKLDS(SEQ ID NO: 7); CDR3: VQGTHFPQT (SEQ ID NO: 8).

[0058] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 19G7E12 is shown below: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTC TAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTAGAAATCAAA(SEQ ID NO: 10).

[0059] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 41D9F1 is shown below: EVQLVESGGGLVKPGGSLKLSCAASGFNFSNYAMSWVRQTPARRLEWVTTISYTGVTTYYLDTVKGRITISRDNAKNTLYLQMTSLRSEDTAMYYCARHRRTLVTTYFDYWGQGTTLTVSS (SEQ ID NO: 14).

[0060] The complementarity-determining region of the heavy chain variable region of monoclonal antibody 41D9F1 is shown below: CDR1: NYAMS (SEQ ID NO: 11); CDR2: TISYTGVTTYYLDTVKG (SEQ ID NO: 12); CDR3:HRRTLVTTYFDY (SEQ ID NO: 13).

[0061] The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 41D9F1 is shown below: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCAATTTTAGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCGGCGAGGAGGCTGGAGTGGGTCACAACCATTAGTTATACTGGTGTTACCACCTACTATTTA GACACTGTGAAGGGCCGAATCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGACCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATAGGAGGACTCTAGTGACTACGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ IDNO: 15).

[0062] The amino acid sequence of the light chain variable region of monoclonal antibody 41D9F1 is shown below: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSRNPWTFGGGTKLEIK (SEQ ID NO: 19).

[0063] The complementarity-determining region of the light chain variable region of monoclonal antibody 41D9F1 is shown below: CDR1: SASSSVSYIH (SEQ ID NO: 16); CDR2:DTSKLAS(SEQ ID NO: 17); CDR3: QQWSRNPWT (SEQ ID NO: 18).

[0064] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 41D9F1 is shown below: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACGATGACCTGCAGTGCCAGTTCAAGTGTAAGTTACATACACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTACGACACTTCCAAACTGGC TTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGAAACCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATTAAA(SEQ ID NO: 20).

[0065] Example 2: Evaluation of the binding activity of monoclonal antibodies The affinity of the monoclonal antibodies 19G7E12 and 41D9F1 prepared in Example 1 for HIF-1α protein was determined using indirect ELISA. The ELISA reaction plate was coated with HIF-1α antigen at a concentration of 2 μg / mL. The detection results are shown below. Figure 1 and 2 As shown.

[0066] from Figure 1 and 2 As can be seen, both monoclonal antibodies 19G7E12 and 41D9F1 exhibited typical dose-dependent binding curves, with absorbance gradually increasing and then plateauing as antibody concentration increased. Specifically, monoclonal antibody 41D9F1 showed significant binding activity in the range of 0-2 μg / mL, with an absorbance of approximately 3.0; monoclonal antibody 19G7E12 also showed a stable binding signal within the corresponding concentration range, with a maximum absorbance close to 3.5. These results indicate that both antibodies possess good binding ability and high sensitivity, making them suitable for subsequent HIF-1α detection.

[0067] Example 3: Expression analysis of HIF-1α protein in various cell lines and tissues To assess the expression of HIF-1α protein in different cell lines and tissues, Western blot analysis was performed using two monoclonal antibodies (41D9F1 and 19G7E12). The target band size was approximately 92 kDa. The results are as follows: Figure 3 and 4 As shown.

[0068] The Western blot detection method is as follows: Cell samples were lysed with pre-cooled RIPA lysis buffer and centrifuged. The supernatant was used for protein quantification. Equal amounts of protein were separated on an SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 1 h, incubated with HIF-1α primary antibody overnight at 4°C, and then incubated with HRP-labeled secondary antibody for 1 h the next day. The results were then analyzed by ECL chemiluminescence staining and imaging.

[0069] from Figure 3 and 4 As can be seen, the 41D9F1 antibody showed a significant HIF-1α band in DMOG-treated U87 cells, while the signal was weaker in HeLa and SY5Y cells; no significant bands were observed in other cell lines (HepG2, JK, MCF-7, R137) and mouse and rat skeletal muscle. The 19G7E12 antibody exhibited a different recognition spectrum; specifically, strong signals were detected in DMOG-treated HeLa and JK cells, and some expression was also observed in DMOG-treated HepG2 cells; in addition, a weak band was observed in mouse kidney tissue, while almost no band was detected in mouse lung and intestinal tissue. The results indicate that HIF-1α expression is induced by DMOG and is cell type-dependent, and the binding of monoclonal antibodies 41D9F1, 19G7E12, and HIF-1α is specific.

[0070] In summary, the anti-HIF-1α monoclonal antibody or its antigen-binding fragment provided by the present invention can specifically bind to the epitope containing amino acids 402 and / or 564 of the HIF-1α protein, and has the advantages of high affinity and strong specificity.

[0071] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0072] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A monoclonal antibody against HIF-1α or its antigen-binding fragment, characterized in that, Includes the heavy chain variable region and the light chain variable region shown in A2) below; A2) The heavy chain variable region includes complementarity-determining regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 11-13, and the light chain variable region includes complementarity-determining regions CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 16-18, respectively.

2. The anti-HIF-1α monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, In A2), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

19.

3. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: B1) A nucleic acid molecule encoding a monoclonal antibody against HIF-1α or an antigen-binding fragment thereof as described in any one of claims 1-2; B2) A recombinant vector containing the nucleic acid molecules described in B1); B3) Recombinant cells containing the nucleic acid molecule described in B1) or the recombinant vector described in B2).

4. The biomaterial according to claim 3, characterized in that, In B1), the nucleic acid molecule is selected from any of the following: C2) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 15 and SEQ ID NO:

20.

5. The use of the anti-HIF-1α monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2, or the biological material as described in any one of claims 3-4, in the preparation of products for detecting HIF-1α protein.

6. A method for detecting HIF-1α protein content for non-diagnostic purposes, characterized in that, Includes the following steps: The sample to be tested is contacted with the anti-HIF-1α monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2; The level of contact was detected to obtain the content of HIF-1α protein in the sample to be tested.

7. The method according to claim 6, characterized in that, The sample to be tested includes at least one of tissue samples, cell samples, and blood samples; The detection methods include at least one of ELISA, flow cytometry, immunoblotting, and immunohistochemistry.

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