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

Abstract

The application relates to the field of biotechnology, in particular to a nanobody targeting annexin A1 and a preparation method and application thereof, discloses a nanobody targeting annexin A1, the nanobody comprises a nanobody Nb6, wherein the amino acid sequence of the nanobody Nb6 is shown in Seq.ID NO.1, the nanobody Nb4 provided has higher affinity with annexin A1, the nanobody can be used for targeted action on annexin A1, has high-strength combination capacity with annexin A1, is favorable for development of a nanobody related medicine of annexin A1, realizes targeted treatment and imaging of tumors, and has very great clinical application value. Meanwhile, a molecular probe can be prepared based on the nanobody, the molecular probe is specifically combined with a tumor blood vessel surface annexin antigen, is applied to specific imaging and diagnosis of tumors, and is used for delivery of related medicines, and is widely applied.

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] The effectiveness of a drug depends on its ability to accumulate and exert its effect at the target site. The poor therapeutic efficacy and toxic side effects of most chemotherapy drugs for tumors are mainly due to their weak targeting. Designing targets against proteins that are highly expressed specifically in different tumors can allow the drug to accumulate rapidly at the target site, reducing its toxic side effects.

[0003] Annexin A1 (ANXA1) is a calcium-regulated phospholipid-binding protein belonging to the calcium ion group. 2+ ANXA1 is a protein that depends on tumor cells. Human ANXA1 consists of 346 amino acids (37 kDa). ANXA1 is highly expressed in some tumor cells and participates in tumor cell survival, proliferation, apoptosis, differentiation, and migration; it is associated with tumor initiation, proliferation, and metastasis. ANXA1 has three subcellular localizations: one in the cytoplasm, the second in the nucleus, and the third loosely or tightly attached to the cell membrane. ANXA1 participates in tumorigenesis in different ways depending on its location. The formylpeptide receptor (FPR) is the only known exocrine ANXA1 receptor. Many studies have found that the ANXA1 / FPR complex is involved in inflammation, the neuroendocrine system, regulation of skeletal muscle differentiation, and cancer progression. FPR can be activated by ANXA1 or by synthetic peptides from its N-terminal region. Because these receptors can be activated or silenced by various synthetic ligands, they represent a very attractive family of pharmacological targets.

[0004] Secondly, pits form in the vascular endothelial cell membranes of human tumors. These pits are a special type of lipid raft, and in many cell types, especially endothelial cells, they are small invaginations (50-100 nm) of the plasma membrane. They are often linked to pressure fibers to ensure molecular transport into and, more importantly, across the cell. Results indicate that target proteins expressed in endothelial cell pits can specifically pump intravenously injected antibodies into the lungs. ANXA1 is expressed in pits of human tumors (breast, kidney, liver, lung, brain, and prostate), on the luminal surface of tumor microcirculation, but not in normal blood vessels. Some researchers have used full-length ANXA1 antibodies conjugated with Alexa Fluor488 or with radioactive 125I for tumor vascular imaging and therapy. These potential findings suggest that ANXA1 holds promise as a target for human tumor imaging, drug delivery, and internal radiotherapy.

[0005] However, there are currently no nanobodies that bind strongly to the ANXA1 protein, which prevents the beneficial properties of the ANXA1 protein target from being widely applied. Summary of the Invention

[0006] 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.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] In a first aspect, this application provides a nanobody targeting annexin A1, the nanobody comprising nanobody Nb4, wherein the amino acid sequence of the nanobody Nb4 is shown in Seq. ID NO.1.

[0009] Secondly, this application provides a method for preparing a nanobody targeting annexin A1, comprising the following steps:

[0010] Annexin A1 was expressed and purified to obtain the target protein, annexin A1.

[0011] The target protein, annexin A1, was coated onto an immunotube for enrichment and screening to obtain a phage library.

[0012] 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.

[0013] 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.

[0014] Thirdly, this application provides the application of nanobodies targeting annexin A1 in the preparation of molecular probes targeting annexin A1.

[0015] Fourthly, this application provides the application of nanobodies targeting annexin A1 in the preparation of drugs for targeted tumor therapy.

[0016] Fifthly, this application provides the application of nanobodies targeting annexin A1 in immunotherapy and drug delivery modified with nanomaterials.

[0017] The first aspect of this application provides a nanobody targeting annexin A1, comprising nanobody Nb4, wherein the amino acid sequence of nanobody Nb4 is shown in Seq. ID NO.1. The provided nanobody Nb4 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, molecular probes can be prepared based on this nanobody, specifically binding to annexin antigens on the surface of tumor blood vessels for tumor-specific imaging and diagnosis; and it can also be used for drug delivery, with a wide range of applications.

[0018] 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 Nb4, which exhibits high binding affinity to both human and mouse annexin A1. Therefore, nanobody Nb4 has good application value. This preparation method is rapid and simple, facilitating large-scale screening and improving screening efficiency.

[0019] The application of the nanobody targeting annexin A1 provided in the third aspect of this application in the preparation of molecular probes targeting annexin A1; since the obtained nanobody targeting annexin A1 includes nanobody Nb4, which has a high binding capacity to annexin A1, it can facilitate the development of molecular probes related to nanobodies of annexin A1, and can be combined with radionuclides, near-infrared dyes, ultrasonic microbubbles, etc., and can be used for specific imaging and diagnosis of tumors by specifically binding to annexin antigens on the surface of tumor blood vessels.

[0020] The application of the nanobody targeting annexin A1 provided in the fourth aspect of this application in the preparation of drugs for targeted tumor therapy is beneficial because the obtained nanobody targeting annexin A1 includes nanobody Nb4, which has a high binding capacity to annexin A1. Therefore, it is advantageous to develop the application of nanobody targeting annexin A1 in the preparation of drugs for targeted tumor therapy. Furthermore, because nanobodies have a small molecular weight, they can cross the blood-brain barrier and bind to corresponding antigens on the surface of tumor blood vessels, thereby achieving targeted tumor therapy.

[0021] The fifth aspect of this application provides the application of nanobodies targeting annexin A1 in immunotherapy and drug delivery modified with nanomaterials. Since the obtained nanobodies targeting annexin A1 include nanobodies Nb4, which have a high binding capacity to annexin A1, it is beneficial to develop nanobodies related to annexin A1 and prepare various therapeutic methods designed for annexin A1, including but not limited to NDC (nanobody-drug conjugate), bispecific antibody-drug conjugates, and immunotherapies such as CAR-T and CAR-NK, as well as drug delivery modified with related nanomaterials; thus facilitating wide application. Attached Figure Description

[0022] 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.

[0023] in:

[0024] Figure 1 The example image shows the screening analysis of ANXA1 nanobodies;

[0025] Figure 2 The example image shows an analysis of positive clones of ANXA1-bound nanobodies.

[0026] Figure 3 The example shows the SDS-PAGE electrophoresis results of the nanobody Nb4 and the control nanobody;

[0027] Figure 4 The example image shows the identification and analysis of the nanobody Nb4 and the control nanobody HA.

[0028] Figure 5 The example image shows the identification and analysis of the nanobody Nb4 and the control nanobody His antibody;

[0029] Figure 6 The above is an ELISA binding verification analysis diagram of the ANXA1 Nb4 nanobody in the example.

[0030] Figure 7 The example image shows the affinity analysis of Nb4 nanobody with human and mouse ANXA1. Detailed Implementation

[0031] 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.

[0032] The first aspect of this application provides a nanobody targeting annexin A1, the nanobody comprising nanobody Nb4, wherein the amino acid sequence of the nanobody Nb4 is shown in Seq. ID NO.1.

[0033] The first aspect of this application provides a nanobody targeting annexin A1, comprising nanobody Nb4, wherein the amino acid sequence of nanobody Nb4 is shown in Seq. ID NO.1. The provided nanobody Nb4 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, molecular probes can be prepared based on this nanobody, specifically binding to annexin antigens on the surface of tumor blood vessels for tumor-specific imaging and diagnosis; and it can also be used for drug delivery, with wide-ranging applications.

[0034] In some embodiments, the amino acid sequence of the nanobody Nb4 is shown in Seq. ID NO.1, which is as follows:

[0035] MAVQLVESGGGLVQAGGSLRLSCAASGRTFSRYDMVAWFRQAPGKEREFVAAISWGSGNIYYADSVKGRFTISSRDNAKNTVYLQMNSLKPEDTAVYYCAAKSLQLRLTREPDVHDYWGQGTQVTVSS.

[0036] In some embodiments, the nanobody includes four framework regions FR1, FR2, FR3, FR4 and three complementarity-determining regions CDR1, CDR2, CDR3.

[0037] In some embodiments, the amino acid sequence of FR1 in the nanobody Nb4 is shown in SEQ ID NO.2, which is as follows: MAVQLVESGGGLVQAGGSLRLSCAASGRTFS.

[0038] In some embodiments, the amino acid sequence of FR2 in the nanobody Nb4 is as shown in SEQ ID NO.3, which is specifically as follows: WFRQAPGKEREFVA.

[0039] In some embodiments, the amino acid sequence of FR3 in the nanobody Nb4 is shown in SEQ ID NO.4, which is as follows: RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA.

[0040] In some embodiments, the amino acid sequence of FR4 in the nanobody Nb4 is as shown in SEQ ID NO.5, specifically SEQ ID NO.5: WGQGTQVTVSS.

[0041] In some embodiments, the amino acid sequence of CDR1 in the nanobody Nb4 is shown in SEQ ID NO.6, which is specifically RYDMVA.

[0042] In some embodiments, the amino acid sequence of CDR2 in the nanobody Nb4 is shown in SEQ ID NO.7, which is as follows: AISWGSGNIYYADSVKG.

[0043] In some embodiments, the amino acid sequence of CDR3 in the nanobody Nb4 is shown in SEQ ID NO.8, specifically as follows: KSLQLRLTREPDVHDY.

[0044] In some embodiments, the base sequence of the nanobody Nb4 is shown in Seq. ID NO.9, which is as follows:

[0045] ATGGCCGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGGCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCAGGACCTTCAGCAGGTACGACATGGTGGCCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCGTGGCCGCCATCAGCTGGGGCAGCGGCAACATCTACTACGCCGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAAGAGCCTGCAGCTGAGGCTGACCAGGGAGCCCGACGTGCACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC.

[0046] The second aspect of this application provides a method for preparing a nanobody targeting annexin A1, comprising the following steps:

[0047] S01. Express and purify annexin A1 to obtain the target protein annexin A1;

[0048] S02. The target protein, annexin A1, is coated onto an immunotube for enrichment and screening to obtain a phage library;

[0049] 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.

[0050] 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.

[0051] 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 Nb4, which exhibits high binding affinity to both human and mouse annexin A1. Therefore, nanobody Nb4 has good application value. This preparation method is rapid and simple, facilitating large-scale screening and improving screening efficiency.

[0052] 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.

[0053] In step S02, the target protein annexin A1 is coated onto an immunotube for enrichment and screening to obtain a phage library.

[0054] 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.

[0055] 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.

[0056] In some embodiments, the resulting phage library has a display library size of 2 x 10^6. 9 .

[0057] 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.

[0058] 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 10⁻⁶. 9 The screening steps are as follows: a) The target protein was coated onto an immunotube at a concentration of 25 ug / ml and three rounds of enrichment screening were performed; b) PCR amplification was performed using the third round of phage elution buffer; d) 192 phage nanobody clones were randomly selected and phage ELISA was performed to identify potential positive clones.

[0059] 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.

[0060] In some embodiments, the expression vector is selected from the PET-14B vector.

[0061] 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.

[0062] In a specific embodiment, the nanobody gene sequence was cloned into the PET-14B vector, and a hemagglutinin HA tag was fused for subsequent detection. The expression and purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16°C using 0.2 mM IPTG; 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 60 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.

[0063] Furthermore, the obtained nanobody targeting annexin A1 needs to be analyzed using ELISA and surface plasmon resonance experiments.

[0064] The specific procedure for the ELISA experiment of nanobodies includes: fusing the HA tag into the gene coding sequence of the nanobody, expressing the HA-tagged nanobody, coating the ELISA plate with mouse and human ANXA1 antigen proteins respectively, incubating overnight at 4°C, blocking with bovine serum albumin at room temperature for 2 hours, then adding nanobody of gradient concentrations 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 a control of irrelevant nanobody and a blank control of irrelevant protein antigen.

[0065] 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.

[0066] The third aspect of this application provides the application of nanobodies targeting annexin A1 in the preparation of molecular probes targeting annexin A1.

[0067] The application of the nanobody targeting annexin A1 provided in the third aspect of this application in the preparation of molecular probes targeting annexin A1; since the obtained nanobody targeting annexin A1 includes nanobody Nb4, which has a high binding capacity to annexin A1, it is beneficial to develop molecular probes related to nanobodies of annexin A1, and can be combined with radionuclides, near-infrared dyes, ultrasonic microbubbles, etc., and can be used for specific imaging and diagnosis of tumors by specifically binding to annexin antigens on the surface of tumor blood vessels.

[0068] The fourth aspect of this application provides the use of nanobodies targeting annexin A1 in the preparation of medicaments for targeted tumor therapy.

[0069] The application of the nanobody targeting annexin A1 provided in the fourth aspect of this application in the preparation of drugs for targeted tumor therapy is beneficial because the obtained nanobody targeting annexin A1 includes nanobody Nb4, which has a high binding capacity to annexin A1. Therefore, it is advantageous to develop the application of nanobody targeting annexin A1 in the preparation of drugs for targeted tumor therapy. Furthermore, because nanobodies have a small molecular weight, they can cross the blood-brain barrier and bind to corresponding antigens on the surface of tumor blood vessels, thereby achieving targeted tumor therapy.

[0070] The fifth aspect of this application provides the application of nanobodies targeting annexin A1 in immunotherapy and drug delivery modified with nanomaterials.

[0071] The fifth aspect of this application provides the application of nanobodies targeting annexin A1 in immunotherapy and drug delivery modified with nanomaterials. Since the obtained nanobodies targeting annexin A1 include nanobodies Nb4, which have a high binding capacity to annexin A1, it is beneficial to develop nanobodies related to annexin A1 and prepare various treatment methods designed for annexin A1, including but not limited to NDC (nanobody-drug conjugate), bispecific antibody-drug conjugates, and immunotherapies such as CAR-T and CAR-NK, as well as drug delivery modified with related nanomaterials; thus, it is beneficial for wide application.

[0072] The following description is based on specific embodiments.

[0073] Example 1

[0074] (I) Preparation method of nanobodies targeting annexin A1

[0075] 1. Screening of ANXA1 nanobodies

[0076] An immunotube method was used to screen natural alpaca-derived phage-displaying nanobody libraries. The selected phage display libraries had a capacity of 2 x 10⁻⁶. 9 The screening steps are as follows: a) The target protein was coated onto immunotubes at a concentration of 25 μg / ml, and three rounds of enrichment screening were performed; b) PCR amplification was performed using the third round of phage elution buffer; d) 192 phage nanobody clones were randomly selected and subjected to phage ELISA to identify potential positive clones.

[0077] 2. Expression and purification of ANXA1 nanobodies

[0078] The nanobody gene sequence was cloned into the PET-14B vector, and a hemagglutinin (HA) tag was fused for subsequent detection. The expression and purification steps were as follows: a) To prevent inclusion body formation and protein degradation, induction was performed using 0.2 mM IPTG 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 at 4°C for 60 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, collecting 1 mL at a time, and analyzing to obtain the nanobody Nb4 targeting annexin A1.

[0079] (II) ELISA assay for nanobodies

[0080] The HA tag was fused into the gene coding sequence of the nanobody to express the HA-tagged nanobody. ELISA plates were coated with mouse and human ANXA1 antigen proteins and incubated overnight at 4°C. Bovine serum albumin was then used as a blocking agent for 2 hours at room temperature. After that, nanobody of varying concentrations was 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 nanobody and a blank control of irrelevant protein antigen were performed.

[0081] (III) Surface plasmon resonance (SPR) experiment

[0082] 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.

[0083] Results Analysis

[0084] 1. Purification of mouse ANXA1 / human ANXA1 domains and screening of nanobodies

[0085] The results are as follows Figure 1 As shown in A, the molecular weights of both mouse ANXA1 and human ANXA1 are approximately 37 kDa. Figure 1 A) After three rounds of screening of natural alpaca nanobody libraries targeting ANXA1, the phage titer results after the three rounds of screening 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. Figure 1 B)

[0086] 2. ELISA verification of positive clones

[0087] 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.

[0088] 3. Purification and identification of Nb4 nanobodies

[0089] Nb4 nanobodies screened from phage libraries were expressed and purified using E. coli prokaryotic expression and purification methods, and identified by Western blot using HA-tag and His-tag antibodies. Figure 3 As shown, SDS-PAGE indicates that the Nb4 nanobody is approximately 15 kDa in size. The purified Nb4 nanobody underwent HA-tag (…). Figure 4 ) and His-tag Figure 5 Immunoblot analysis confirmed that the nanobody was expressed correctly.

[0090] 4. Validation of Nb4 nanobody binding to ELISA

[0091] Verification was performed using ELISA testing. Figure 6 As shown in Figure A, the Nb4 nanobody exhibits high binding activity to both mouse ANXA1 and human ANXA1, such as... Figure 6 As shown in B, the control nanobody did not show binding activity with either mouse ANXA1 or human ANXA1.

[0092] 5. Affinity determination of Nb4 nanobody with human and mouse ANXA1

[0093] like Figure 7 A and Figure 7 As shown in B, the Nb4 nanobody was further analyzed by SPR to determine its binding affinity to human and mouse-derived ANXA1. The results showed that, as Figure 7 As shown in Figure A, the binding affinity constant between Nb4 and human ANXA1 is 1.599 nM; Figure 7 As shown in Figure B, the binding affinity constant between Nb4 and mouse ANXA1 is 2.069 nM.

[0094] In summary, the nanobody targeting annexin A1 provided in this application, comprising nanobody Nb4, wherein the amino acid sequence of nanobody Nb4 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 drugs for targeted tumor therapy and imaging, thus possessing significant clinical application value. Furthermore, molecular probes can be prepared based on this nanobody, specifically binding to annexin antigens on tumor vascular surfaces for tumor-specific imaging and diagnosis; and it can also be used for drug delivery, with a wide range of applications.

[0095] 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 composed of nanobody Nb4, wherein the amino acid sequence of the nanobody Nb4 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 Nb4, 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 Nb4 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 molecular probes targeting annexin A1.

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