Nanobodies targeting annexin a1 and methods of making and using the same

Abstract

This invention relates to the field of biotechnology, and more particularly to a nanobody targeting annexin A1, its preparation method, and its applications. The invention discloses a nanobody targeting annexin A1, comprising nanobody Nb6, wherein the amino acid sequence of nanobody Nb6 is shown in Seq. ID NO.1. The provided nanobody Nb6 exhibits a high affinity for annexin A1, allowing it to target annexin A1 with a strong binding affinity. This facilitates the development of annexin A1-related nanobody drugs, enabling targeted therapy and imaging of tumors, and possesses significant clinical application value. Furthermore, due to their small molecular weight, nanobodies can cross the blood-brain barrier. Assembling drugs or nanomaterials with nanobodies holds promise for imaging and treating intracranial tumors, with a wide range of applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nanobody targeting annexin A1, its preparation method, and its application. Background Technology

[0002] Annexin A1 (ANXA1), also known as lipocorton-1, is a member of the annexin family. It possesses a unique structure and function. ANXA1 is a widely distributed intracellular and extracellular calcium- and phospholipid-binding protein, belonging to the Ca2+-dependent protein family. The ANXA1 gene is located on human chromosome 9q12-q21.2, consisting of 13 exons and 12 introns, with a relative molecular mass of 37,000 Da. Structurally, ANXA1 shares similarities with its family members: a highly conserved central domain and an N-terminal sequence responsible for their respective unique functions. ANXA1 participates in various cellular physiological processes: it regulates glucocorticoid-related anti-inflammatory responses in the host's defense system, and also regulates various inflammatory responses, cell proliferation, cell death-related signaling, cell differentiation, apoptosis, and cellular endocytosis and secretion. In vitro and in vivo studies have shown that ANXA1 can inhibit cell growth, inhibit proliferation, regulate differentiation, and promote apoptosis. The anti-proliferative effects of ANXA1 are mainly manifested in the following ways: By binding to different growth factor proteins, it activates the ERK1 / 2MAPK signaling cascade by influencing the formation and activity of protein complexes upstream of the mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling pathway, thereby inhibiting cyclin D1 expression and reducing cell growth. As a substrate of EGFR tyrosine kinase, it can be phosphorylated and participate in corresponding signal transduction, inhibiting EGF-mediated proliferation. ANXA1 can also promote apoptosis by affecting the activation of caspase-3 and the release of Ca2+, thereby activating p38 and JNK signal transduction. Exogenous ANXA1 can induce apoptosis by increasing intracellular Ca2+ concentration and dephosphorylating Bcl-2 / Bcl-xL-2-associated death promoter (BAD). Furthermore, ANXA1 can also promote hydrogen peroxide-induced apoptosis by regulating PLA2 activity and inhibiting the production of prostaglandin-like factors (such as PGE2) with anti-apoptotic effects.In addition, many studies have shown that ANXA1 is highly expressed in many tumors, such as malignant melanoma, gastric cancer, glioma, breast cancer, prostate cancer, and lung cancer. In particular, ANXA1 is highly expressed in the vascular endothelial cells of the above-mentioned tumors. These findings suggest that ANXA1 may be an important target for the treatment or improvement of certain inflammatory and tumor-related diseases. Using this target, a series of tumor imaging and tumor treatment-related drugs can be developed, thereby enabling precision diagnosis and treatment of tumors.

[0003] However, there are currently no nanobodies that bind strongly to the ANXA1 protein, which prevents the beneficial properties of the ANXA1 protein from being widely applied. Summary of the Invention

[0004] The purpose of this application is to provide a nanobody targeting annexin A1, its preparation method and application, in order to solve the problem that there is a lack of nanobodies that can target annexin A1 in the prior art, which makes it impossible to better utilize the annexin A1 target for the preparation of related drugs.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, this application provides a nanobody targeting annexin A1, the nanobody comprising nanobody Nb6, wherein the amino acid sequence of the nanobody Nb6 is shown in Seq. ID NO.1.

[0007] Secondly, this application provides a method for preparing a nanobody targeting annexin A1, comprising the following steps:

[0008] Annexin A1 was expressed and purified to obtain the target protein, annexin A1.

[0009] The target protein, annexin A1, was coated onto an immunotube for enrichment and screening to obtain a phage library.

[0010] The elution buffer of the phage library was amplified by PCR and verified by ELISA, followed by next-generation sequencing, and the gene sequence of the nanobody was synthesized based on the sequencing results.

[0011] The gene sequence of the nanobody was cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid was then transformed into a host cell to induce expression and was purified to obtain a nanobody targeting annexin A1.

[0012] Thirdly, this application provides the use of nanobodies targeting annexin A1 in the preparation of drugs for the treatment of inflammation or immunotherapy related to annexin A1.

[0013] Fourthly, this application provides the application of nanobodies targeting annexin A1 in the preparation of drugs for tumor-targeted therapy and imaging.

[0014] The first aspect of this application provides a nanobody targeting annexin A1, comprising nanobody Nb6, wherein the amino acid sequence of nanobody Nb6 is shown in Seq. ID NO.1. The provided nanobody Nb6 exhibits a high affinity for annexin A1, allowing it to target annexin A1 with a strong binding affinity. This facilitates the development of nanobody-related drugs targeting annexin A1, enabling targeted therapy and imaging of tumors, and possesses significant clinical application value. Furthermore, due to their small molecular weight, nanobodies can cross the blood-brain barrier. Assembling drugs or nanomaterials with nanobodies holds promise for imaging and treating intracranial tumors, with a wide range of applications.

[0015] The second aspect of this application provides a method for preparing a nanobody targeting annexin A1. This method is based on a phage-based natural nanobody library and completes three rounds of screening for annexin A1 proteins. ELISA verification revealed a nanobody Nb6, which exhibits high binding affinity to both human and mouse annexin A1. Therefore, nanobody Nb6 has good application value. This preparation method is rapid and simple, facilitating large-scale screening and improving screening efficiency.

[0016] The application of the nanobody targeting annexin A1 provided in the third aspect of this application in the preparation of drugs for the treatment of annexin A1-related inflammation or immunotherapy, since the obtained nanobody targeting annexin A1 includes nanobody Nb6, which has a high binding capacity to annexin A1, it can facilitate the development of nanobody-related drugs for annexin A1, enabling the prevention and treatment of related inflammatory diseases such as osteoarthritis, or the preparation of various treatment methods designed for annexin A1, including CAR-T, CAR-NK, bispecific antibodies, nanobody-conjugated drugs, nanomaterials, and other related drugs; thus facilitating its wide application.

[0017] The application of the nanobody targeting annexin A1 provided in the fourth aspect of this application in the preparation of drugs for tumor-targeted therapy and imaging is beneficial because the obtained nanobody targeting annexin A1 includes nanobody Nb6, which has a high binding capacity to annexin A1. Therefore, it is beneficial to develop the application of nanobody targeting annexin A1 in the preparation of drugs for tumor-targeted therapy and imaging. Furthermore, since nanobodies have a small molecular weight and can cross the blood-brain barrier, assembling drugs or nanomaterials with nanobodies is beneficial for imaging and treating intracranial tumors. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] in:

[0020] Figure 1 The example image shows the screening analysis of ANXA1 nanobodies;

[0021] Figure 2 The example image shows an analysis of positive clones of ANXA1-bound nanobodies.

[0022] Figure 3 The following is a diagram illustrating the purification and identification analysis of the ANXA1 nanobody in the example.

[0023] Figure 4 This is an ELISA binding verification analysis diagram of the ANXA1 Nb6 nanobody used in the example.

[0024] Figure 5 The above is an analysis of the affinity of the Nb6 nanobody to human and mouse ANXA1 in the example. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The first aspect of this application provides a nanobody targeting annexin A1, the nanobody comprising nanobody Nb6, wherein the amino acid sequence of the nanobody Nb6 is shown in Seq. ID NO.1.

[0027] The first aspect of this application provides a nanobody targeting annexin A1, comprising nanobody Nb6, wherein the amino acid sequence of nanobody Nb6 is shown in Seq. ID NO.1. The provided nanobody Nb6 exhibits a high affinity for annexin A1, allowing it to target annexin A1. Its strong binding ability to annexin A1 facilitates the development of nanobody-related drugs for annexin A1, enabling targeted therapy and imaging of tumors, and possesses significant clinical application value. Furthermore, due to their small molecular weight, nanobodies can cross the blood-brain barrier. Assembling drugs or nanomaterials with nanobodies holds promise for imaging and treating intracranial tumors, making its applications very broad.

[0028] In some embodiments, the amino acid sequence of the nanobody Nb6 is shown in Seq. ID NO.1, which is as follows:

[0029] MAVQLVESGGGLVQTGDSLRLSCTPSGNSKSNYHMGWFRQAPGKEREWI TYIDPSGLITWHAEAVKGRFAVSRDNAKKTVYLEMNNLQPEDTAVYYCRALR VGDKIEITATWGQGTQVTVSS.

[0030] In some embodiments, the nanobody includes four framework regions FR1, FR2, FR3, FR4 and three complementarity-determining regions CDR1, CDR2, CDR3.

[0031] In some embodiments, the amino acid sequence of FR1 in the nanobody Nb6 is as shown in SEQ ID NO.2, which is as follows: MAVQLVESGGGLVQTGDSLRLSCTPSGNSKS.

[0032] In some embodiments, the amino acid sequence of FR2 in the nanobody Nb6 is as shown in SEQ ID NO.3, which is specifically as follows: WFRQAPGKEREWIT.

[0033] In some embodiments, the amino acid sequence of FR3 in the nanobody Nb6 is shown in SEQ ID NO.4, which is as follows: RFAVSRDNAKKTVYLEMNNLQPEDTAVYYCRA.

[0034] In some embodiments, the amino acid sequence of FR4 in the nanobody Nb6 is as shown in SEQ ID NO.5, specifically SEQ ID NO.5: WGQGTQVTVSS.

[0035] In some embodiments, the amino acid sequence of CDR1 in the nanobody Nb6 is as shown in SEQ ID NO.6, specifically SEQ ID NO.6: NYHMG.

[0036] In some embodiments, the amino acid sequence of CDR2 in the nanobody Nb6 is shown in SEQ ID NO.7, specifically: YIDPSGLITWHAEAVKG.

[0037] In some embodiments, the amino acid sequence of CDR3 in the nanobody Nb6 is shown in SEQ ID NO.8, specifically: LRVGDKIEITAT.

[0038] In some embodiments, the base sequence of the nanobody Nb6 is shown in Seq. ID NO.9, which is as follows:

[0039] ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTGCAGACTGGGGACTCTCTGAGACTCTCCTGTACACCCTCTGGCAACAGTAAGAGTAACTATCACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATGGATAACATACATTGACCCCGAGTGGGCTTATCACATGGCAT GCTGAAGCCGTGAAGGGCCGCTTCGCCGTGTCCAGAGACAACGCCAAGAAGACGTGTATTTGGAGATGAACAACCTCCAACCTGAGGATACGGCCGTCTATTATTGTCGTGCACTCCGTGTTGGTGACAAGATCGAAATCACTGCGACCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0040] The second aspect of this application provides a method for preparing a nanobody targeting annexin A1, comprising the following steps:

[0041] S01. Express and purify annexin A1 to obtain the target protein annexin A1;

[0042] S02. The target protein, annexin A1, is coated onto an immunotube for enrichment and screening to obtain a phage library;

[0043] S03. The elution buffer of the phage library is subjected to PCR amplification and ELISA verification, followed by next-generation sequencing, and the gene sequence of the nanobody is synthesized based on the sequencing results.

[0044] S04. The gene sequence of the nanobody is cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is then transferred into a host cell to induce expression and purified to obtain a nanobody targeting annexin A1.

[0045] The second aspect of this application provides a method for preparing a nanobody targeting annexin A1. This method is based on a phage-based natural nanobody library and completes three rounds of screening for annexin A1 proteins. ELISA verification revealed a nanobody Nb6, which exhibits high binding affinity to both human and mouse annexin A1. Therefore, nanobody Nb6 has good application value. This preparation method is rapid and simple, facilitating large-scale screening and improving screening efficiency.

[0046] In step S01, annexin A1 is expressed and purified to obtain the target protein, annexin A1. Conventional methods are used to express and purify annexin A1 to obtain the target protein.

[0047] In step S02, the target protein annexin A1 is coated onto an immunotube for enrichment and screening to obtain a phage library.

[0048] In some embodiments, the annexin A1 target protein is coated onto an immunotube, wherein the concentration of the target protein is 25–27 μg / mL. In some specific embodiments, the concentration of the target protein is 25 μg / mL.

[0049] In some embodiments, the enrichment screening step involves 2 to 3 rounds of enrichment screening. In some specific embodiments, the enrichment screening step involves 3 rounds of enrichment screening.

[0050] In some embodiments, the resulting phage library has a display library size of 2 x 10^6. 9 .

[0051] In step S03, the elution buffer of the phage library is subjected to PCR amplification and ELISA verification, followed by next-generation sequencing, and the gene sequence of the nanobody is synthesized based on the sequencing results.

[0052] In a specific embodiment, the screening of annexin A1 nanobodies was performed using an immunotube method to screen a natural alpaca-derived phage-displayed nanobodies library. The selected phage display library had a capacity of 2 x 10⁹. The screening steps were as follows: a) The target protein was coated onto immunotubes at a concentration of 25 μg / ml and subjected to three rounds of enrichment screening; b) PCR amplification was performed using the third round of phage elution buffer; d) Single clones were randomly selected for ELISA detection, and the results of positive clones were then verified by a second ELISA test; e) A total of 34 clones were selected for sequencing after screening. 33 sequences were normal, and 1 showed no signal. The 33 sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment, resulting in 6 different nanobodies.

[0053] In step S04, the gene sequence of the nanobody is cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is then transferred into a host cell to induce expression and is purified to obtain a nanobody targeting annexin A1.

[0054] In some embodiments, the expression vector is selected from the pColdII vector.

[0055] In some embodiments, the step of cloning the gene sequence of the nanobody into an expression vector to obtain a recombinant plasmid further includes: fusing and expressing a hemagglutinin tag for subsequent detection.

[0056] In a specific embodiment, the nanobody gene sequence was cloned into the pColdII vector, and a hemagglutinin HA tag was fused for subsequent detection. The expression and purification steps are as follows: a) To reduce inclusion body formation and protein degradation, 0.2 mM IPTG was used for induction at 16°C; b) Large-scale induction expression was performed according to the preliminary induction conditions, and autoclaving was performed at 1230 bar; c) Centrifugation was performed at 12000 rpm and 4°C for 40 min, and the supernatant was incubated with Ni packing material at 4°C for 1 hour; g) After purification by Ni column, molecular sieve separation was performed, with AKATA parameters set at a flow rate of 0.5 mL / min, and 1 mL was collected each time.

[0057] Furthermore, the obtained nanobody targeting annexin A1 needs to be analyzed using ELISA and surface plasmon resonance experiments.

[0058] The specific procedure for the ELISA experiment of nanobodies includes: fusing HA and His tags into the coding sequence of the nanobody gene, expressing nanobodies with HA and His tags, coating mouse and human ANXA1 antigens on ELISA plates respectively, incubating overnight at 4°C, blocking with skim milk powder at room temperature for 2 hours the next day, adding gradient concentrations of nanobodies and incubating at room temperature for 1 hour, washing 5 times with PBST, incubating with anti-HA antibody at room temperature for 1 hour, amplifying the signal with horseradish peroxidase-labeled anti-HA antibody, developing color with TMB, and simultaneously setting up controls for irrelevant nanobodies and blank controls for irrelevant protein antigens.

[0059] Surface plasmon resonance (SPR) experiments were primarily used to verify the direct interaction between in vitro expressed and purified nanobodies and in vitro purified antigen proteins, and to calculate their equilibrium constant. The purified antigen protein was immobilized on a chip, and nanobodies of different concentrations were added sequentially to analyze their affinity for the antigen protein. The reaction signal was recorded over 780 seconds, kinetic curves were generated, and relevant parameters were calculated.

[0060] The third aspect of this application provides the use of nanobodies targeting annexin A1 in the preparation of medicaments for the treatment of inflammation or immunotherapy related to annexin A1.

[0061] The application of the nanobody targeting annexin A1 provided in the third aspect of this application in the preparation of drugs for the treatment of annexin A1-related inflammation or immunotherapy is beneficial because the obtained nanobody targeting annexin A1 includes nanobody Nb6, which has a high binding capacity to annexin A1. Therefore, it can facilitate the development of nanobody-related drugs for annexin A1, enabling the prevention and treatment of related inflammatory diseases such as osteoarthritis, or the preparation of various treatment methods designed for annexin A1, including CAR-T, CAR-NK, bispecific antibodies, nanobody-conjugated drugs, nanomaterials, and other related drugs; thus facilitating its widespread application.

[0062] The fourth aspect of this application provides the use of nanobodies targeting annexin A1 in the preparation of medicaments for tumor-targeted therapy and imaging.

[0063] The application of the nanobody targeting annexin A1 provided in the fourth aspect of this application in the preparation of drugs for tumor-targeted therapy and imaging is beneficial because the obtained nanobody targeting annexin A1 includes nanobody Nb6, which has a high binding capacity to annexin A1. Therefore, it is beneficial to develop the application of nanobody targeting annexin A1 in the preparation of drugs for tumor-targeted therapy and imaging. Furthermore, since nanobodies have a small molecular weight and can cross the blood-brain barrier, assembling drugs or nanomaterials with nanobodies is beneficial for imaging and treating intracranial tumors.

[0064] The following description is based on specific embodiments.

[0065] Example 1

[0066] (I) Preparation method of nanobodies targeting annexin A1

[0067] 1. Screening of ANXA1 nanobodies

[0068] An immunotube method was used to screen a natural alpaca-derived phage-displayed nanobody library. The selected phage display library had a capacity of 2 x 10⁹. The screening steps were as follows: a) The target protein was coated onto immunotubes at a concentration of 25 μg / ml and subjected to three rounds of enrichment screening; b) PCR amplification was performed using the third round of phage elution buffer; d) Single clones were randomly selected for ELISA detection, and positive clones were then validated by a second ELISA test; e) A total of 34 clones were selected for sequencing. 33 sequences were normal, and 1 showed no signal. The 33 sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment, yielding 6 different nanobody sequences.

[0069] 2. Expression and purification of ANXA1 nanobodies

[0070] The nanobody gene sequence was cloned into the pColdII vector, and a hemagglutinin (HA) tag was fused for subsequent detection. The expression and purification steps were as follows: a) To reduce inclusion body formation and protein degradation, 0.2 mM IPTG was used for induction at 16°C; b) Large-scale induction expression was performed according to the preliminary induction conditions, and autoclaving was performed at 1230 bar; c) Centrifugation was performed at 12000 rpm and 4°C for 40 min, and the supernatant was incubated with Ni packing material at 4°C for 1 hour; g) After purification by Ni column, molecular sieve separation was performed, with AKATA parameters set at a flow rate of 0.5 mL / min, and 1 mL was collected every time. The nanobody Nb6 targeting annexin A1 was obtained.

[0071] (II) ELISA assay for nanobodies

[0072] The HA and His tags were fused into the coding sequence of the nanobody gene to express nanobodies with HA and His tags. ELISA plates were coated with mouse and human ANXA1 antigens, respectively, and incubated overnight at 4°C. The next day, the plates were blocked with skim milk powder at room temperature for 2 hours. Then, nanobodies of varying concentrations were added and incubated at room temperature for 1 hour. The plates were washed 5 times with PBST and incubated with anti-HA antibody at room temperature for 1 hour. Horseradish peroxidase-labeled anti-HA antibody amplified the signal, and TMB was used for color development. At the same time, a control of irrelevant nanobodies and a blank control of irrelevant protein antigens were performed.

[0073] (III) Surface plasmon resonance (SPR) experiment

[0074] This experiment was used to verify the direct interaction between in vitro expressed and purified nanobodies and in vitro purified antigen proteins, and to calculate their equilibrium constant. The purified antigen protein was immobilized on a chip, and nanobodies of different concentrations were added sequentially to analyze their affinity for the antigen protein. The reaction signal was recorded over 780 seconds, kinetic curves were generated, and relevant parameters were calculated.

[0075] Results Analysis

[0076] 1. Purification of mouse ANXA1 / human ANXA1 domains and screening of nanobodies

[0077] The results are as follows Figure 1 As shown in Figure A, the molecular weights of both mouse ANXA1 and human ANXA1 are approximately 37 kDa. After three rounds of screening using a natural alpaca nanobody library targeting ANXA1, the phage titer results are as follows: Figure 1 As shown in Figure B, the library was enriched more than 400-fold, indicating that nanobodies binding to ANXA1 were amplified.

[0078] 2. ELISA verification of positive clones

[0079] The results are as follows Figure 2 As shown, 192 phage clones were randomly selected for ELISA verification, revealing 33 potentially positive clones (the OD450 value of the target antigen was 3 times greater than the control OD450 value). These 33 clones were sequenced; 32 were normal, and one (Nb4) showed no signal. Based on the nanobody sequencing results, nanobody sequences were obtained. These 32 sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment, yielding a total of 6 different nanobody sequences.

[0080] 3. Purification and identification of Nb6 nanobodies

[0081] Subsequently, gene cloning and protein expression analysis were performed on the Nb6 nanobody sequence, such as... Figure 3 As shown in Figure A, SDS-PAGE analysis of the Nb6 nanobody indicates that the nanobody size is approximately 15 kDa. Figure 3 B is the identification and analysis diagram of the ANXA1 nanobody Nb6 His antibody. Figure 3 C is the identification and analysis diagram of the ANXA1 nanobody Nb6 HA antibody. It can be seen that the purified Nb6 nanobody underwent His-tag (Hydrogen-labeled) assay. Figure 3 B) and HA-tag( Figure 3 C) Immunoblotting analysis showed that the nanobody was expressed correctly.

[0082] 4. Further validation using Nb6 nanobody ELISA

[0083] like Figure 4 A and Figure 4 As shown in B, ELISA detection further confirmed that the Nb6 nanobody had binding activity with both mouse ANXA1 and human ANXA1, while the control nanobody did not show binding activity with either mouse ANXA1 or human ANXA1.

[0084] 5. Affinity determination of Nb6 nanobody with human and mouse ANXA1

[0085] like Figure 5 A and Figure 5 As shown in Figure B, the Nb6 nanobody was further analyzed by SPR to determine its binding affinity to human and mouse ANXA1. The results showed that Nb6 binds well to mouse ANXA1. Figure 5 A) / person ANXA1( Figure 5 The binding affinity constants of B) are 1.19 nM and 0.4888 nM, respectively.

[0086] In summary, the nanobody targeting annexin A1 provided in this application, comprising nanobody Nb6, wherein the amino acid sequence of nanobody Nb6 is shown in Seq. ID NO.1, demonstrates a high affinity for annexin A1. This antibody can target annexin A1 with a strong binding affinity, facilitating the development of annexin A1-related nanobody drugs for targeted tumor therapy and imaging, thus possessing significant clinical application value. Furthermore, due to their small molecular weight, nanobodies can cross the blood-brain barrier. Assembling drugs or nanomaterials with nanobodies holds promise for imaging and treating intracranial tumors, making their applications very broad.

[0087] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A nanobody targeting annexin A1, characterized in that, The nanobody is nanobody Nb6, wherein the amino acid sequence of nanobody Nb6 is shown in Seq.ID NO.

1.

2. The nanobody targeting annexin A1 according to claim 1, characterized in that, The nanobody includes four framework regions FR1, FR2, FR3, and FR4 and three complementarity-determining regions CDR1, CDR2, and CDR3. In the nanobody Nb6, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, the amino acid sequence of FR4 is shown in SEQ ID NO.5, the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

8.

3. The nanobody targeting annexin A1 according to claim 1, characterized in that, The base sequence of the nanobody Nb6 is shown in Seq.ID NO.

9.

4. The use of the nanobody targeting annexin A1 as described in any one of claims 1 to 3 in the preparation of a drug for tumor imaging.

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