Monoclonal nanobodies against cd31 and uses thereof

Abstract

The application discloses an anti-CD31 monoclonal nanobody and application thereof, and relates to the technical field of phage display. The anti-CD31 monoclonal nanobody provided by the application is CD31-1040 and CD31-1042; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the monoclonal nanobody CD31-1040 are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the monoclonal nanobody CD31-1042 are respectively shown as SEQ ID NO. 6-8. The two camel-derived monoclonal nanobody sequences with high affinity and specific recognition of human CD31 protein can be used for the expression of CD31 on tissues or cells, and further for the assessment of the conditions of various hemangiomas.

Description

Technical Field

[0001] The present invention relates to the technical field of phage display, and in particular to an anti-CD31 monoclonal nanobody and applications thereof. Background Art

[0002] Phage display technology involves fusing exogenous proteins or peptides with specific phage capsid proteins and displaying them on the phage surface. This technology can mimic the process of natural selection, enabling high-throughput screening of target-specific receptors. It also seamlessly integrates the genotype and phenotype of screened proteins, making it a common technique for screening monoclonal antibodies against different target proteins.

[0003] Nanobodies are a type of heavy-chain, single-domain antibody. Unlike other antibodies, they naturally lack light chains and are only one-tenth the size of traditional IgG antibodies. They offer advantages such as high penetrability, excellent stability, and simplified humanization. They are widely used in the development of antibody drugs and in clinical disease diagnosis. Common nanobodies are primarily derived from camels and certain cartilaginous fish.

[0004] CD31 (platelet-endothelial cell adhesion molecule) is a member of the immunoglobulin superfamily with a molecular weight of approximately 130 kDa. It is a transmembrane protein composed of 574 amino acids in its extracellular domain, 19 hydrophobic amino acids in its transmembrane domain, and 118 amino acids in its intracellular domain. CD31 protein is primarily found on the surfaces of platelets, neutrophils, monocytes, and certain T cell types, as well as at tight junctions between endothelial cells. Due to its high expression on vascular endothelial cells, CD31 is used as an endothelial cell marker to assess the development of vascular tumors. CD31 protein is detected in most types of vascular tumors, such as hemangioendotheliomas, angiofibromas, hemangiomas, and angiosarcomas. Furthermore, due to its crucial role in tumor growth and metastasis, overexpression of CD31 protein is often used as a key indicator for the diagnosis of benign or malignant hemangiomas.

[0005] Therefore, if antibodies with high affinity and specific recognition for CD31 protein can be developed, immunohistochemistry experiments can be used to detect the expression of CD31 on tissues or cells, thereby achieving the purpose of evaluating various types of hemangiomas. Summary of the Invention

[0006] The present invention provides an anti-CD31 monoclonal nanobody and its applications. This monoclonal nanobody has high affinity and specific recognition for human CD31 and can be used for detection and identification of CD31 protein and related applications. The technical solution of the present invention is specifically implemented through the following technologies.

[0007] A monoclonal nanoantibody against CD31, characterized in that it is a monoclonal nanoantibody CD31-1040 or CD31-1042; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody CD31-1040 are shown as SEQ ID NOs. 1-3, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanoantibody CD31-1042 are shown as SEQ ID NOs. 6-8, respectively.

[0008] Furthermore, the amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1040 is shown as SEQ ID NO.4; the amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1042 is shown as SEQ ID NO.9.

[0009] Furthermore, the gene sequence encoding the heavy chain of the monoclonal nanobody CD31-1040 is shown as SEQ ID NO.5, and the gene sequence encoding the heavy chain of the monoclonal nanobody CD31-1042 is shown as SEQ ID NO.10.

[0010] The present invention also provides a substance, which comprises any one of the following aspects:

[0011] (1) A nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal Nanobody described in any one of the above items;

[0012] (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the monoclonal nanobody described in any one of the above items;

[0013] (3) An antibody derivative, comprising any of the monoclonal nanobodies described above;

[0014] (4) A pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned monoclonal nanobodies, or the antibody derivatives;

[0015] (5) A detection product, comprising the monoclonal nanobody described in any one of the above, or the nucleic acid molecule, or the vector, or the antibody derivative.

[0016] The present invention also provides an application of the above-mentioned anti-CD31 monoclonal nanobody, wherein the application includes any one of the following aspects:

[0017] (1) Used for the purpose of detecting CD31 expression, detecting vascular tumors, or evaluating the efficacy of drugs for treating vascular tumors, but not for the purpose of disease diagnosis and treatment;

[0018] (2) Products for detecting CD31 expression, diagnosing vascular tumors, or evaluating the efficacy of drugs for treating vascular tumors;

[0019] (3) Used for the preparation of drugs for treating vascular tumors.

[0020] The monoclonal Nanobodies provided by the present invention are not limited to detecting CD31 expression or diagnosing vascular tumors. In fact, due to the high affinity of the monoclonal Nanobodies of the present invention for CD31, they can be used in any other environment and scenario requiring qualitative or quantitative detection of CD31 expression.

[0021] It should also be noted that the aforementioned use of the monoclonal Nanobodies provided by the present invention, when stated as "not for the purpose of disease diagnosis and treatment," refers to the use of the monoclonal Nanobodies of the present invention for qualitative / quantitative detection of CD31 or vascular tumor detection under non-clinical conditions, such as routine scientific research or laboratory settings. In fact, the monoclonal Nanobodies provided by the present invention can also be used in clinical settings to assist in the detection of CD31 or vascular tumor diseases.

[0022] Furthermore, the product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

[0023] The present invention also provides a camel-derived nanoantibody library for screening any of the above-mentioned monoclonal nanoantibodies; the camel-derived nanoantibody library is obtained by extracting total RNA from natural camel-derived PBMCs cells, performing reverse transcription and PCR amplification to obtain the gene sequence of the antibody variable region VHH, connecting the gene sequence of the antibody variable region VHH to a phage expression vector, and transforming it into competent cells.

[0024] Compared with the existing technology, the benefits of the present invention are as follows: the present invention extracts total RNA from alpaca lymphocytes after immunization with human CD31 protein, obtains camel-derived nanoantibody variable region gene fragments through reverse transcription and two rounds of PCR, and finally successfully constructs a phage-displayed camel-derived nanoantibody library; using this library through enrichment panning and high-throughput ELISA screening, two high-affinity camel-derived monoclonal nanoantibody sequences that specifically recognize human CD31 protein are obtained, which can be used to express CD31 on tissues or cells, and then evaluate the condition of various hemangiomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of total RNA electrophoresis detection of PBMCs cells in Example 1.

[0026] Figure 2 1% agarose gel electrophoresis results of the first-round PCR products of Example 1.

[0027] Figure 3 1% agarose gel electrophoresis results of the second round PCR products of Example 1.

[0028] Figure 4 These are the expression and purification results of the monoclonal nanoantibodies CD31-1040 and CD31-1042 of Example 3.

[0029] Figure 5 and 6 These are the results of Western Blot in Example 4. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] In the embodiments of the present invention, the term "nucleic acid (molecule)" may include coding nucleic acids or nucleic acids that also include additional coding and / or non-coding sequences. DNA forms of the nucleic acids of the present invention include, but are not limited to, cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. Nucleic acids encoding the high-affinity rabbit monoclonal antibodies or antigen-binding fragments thereof described herein include, but are not limited to, coding sequences encoding only the structure of the mature high-affinity rabbit monoclonal antibody or antigen-binding fragment thereof; coding sequences encoding the mature high-affinity rabbit monoclonal antibody or antigen-binding fragment thereof and various additional coding sequences; coding sequences encoding the structure of the mature high-affinity rabbit monoclonal antibody or antigen-binding fragment thereof (and optional additional coding sequences) and non-coding sequences, etc.

[0032] In some embodiments of the present invention, the full-length nucleic acid sequence of the high-affinity rabbit monoclonal antibody or fragments thereof can generally be obtained by PCR amplification, recombination, or synthetic methods. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Generally, the relevant sequence is cloned into a vector, then transferred into cells, and then isolated from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, high-affinity rabbit monoclonal antibodies, and antigen-binding fragments thereof, etc.) referred to in the present invention include biomolecules in isolated form.

[0033] The vectors include viral vectors (such as adenoviral vectors, retroviral vectors, adeno-associated viral vectors) and non-viral vectors (plasmids, transposon vectors).

[0034] In some preferred embodiments, the vector is an expression vector.

[0035] The terms "vector," "cloning vector," and "expression vector" refer to vehicles that can introduce DNA or RNA sequences into a host cell in a manner that transforms the host and promotes expression (eg, transcription and translation) of the introduced sequence.

[0036] Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrating plasmids.

[0037] The vector can be a recombinant expression vector or a cloning vector. The vector provided by the present invention (e.g., an expression vector) comprises a nucleic acid sequence provided by the present invention encoding an antibody, at least one promoter operably linked to the nucleic acid sequence, and / or at least one selective marker.

[0038] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses, lambda phage, M13 phage, plasmids, and the like.

[0039] The "modified cell" provided by the present invention refers to the introduction of a "foreign" (i.e., external or extracellular) gene or DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a substance of interest, usually a protein encoded by the gene or introduced sequence. The host cell that receives and expresses the introduced DNA or RNA has been "modified".

[0040] In a specific embodiment of the present invention, the host cells include but are not limited to mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells, etc.

[0041] The present invention provides an antibody derivative, which comprises the high-affinity rabbit monoclonal antibody provided by the present invention, directly or indirectly connected to a connectable substance to form a complex.

[0042] The attachable species include a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0043] In a specific embodiment of the present invention, the connectable substance is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL), chemotherapeutic agents (such as cisplatin)) or any form of nanoparticles, etc.

[0044] The term "detectable label" refers to any moiety that generates a measurable signal by a change in an optical, electrical, or other physical indicator of the state of a molecule to which the moiety is coupled. Such physical indicators include spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, and chemical means, such as, but not limited to, fluorescence, chemiluminescence, and chemiluminescence. When used with respect to a labeled detection agent, a "direct label" is a detectable label that is attached to the detection agent by any means. When used with respect to a labeled detection agent, an "indirect label" is a detectable label that specifically binds to the detection agent. Thus, indirect labels include the following moieties: a specific binding partner of the detection agent. Biotin and avidin are examples of such moieties that are employed, for example, by contacting a biotinylated antibody with a labeled avidin to produce an indirectly labeled antibody.

[0045] The pharmaceutical compositions provided herein include pharmaceutically acceptable carriers or excipients. In some embodiments, excipients include pharmaceutically acceptable solvents, dispersants, additives, plasticizers, and the like. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions can be administered via conventional routes, including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

[0046] In other embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition (e.g., an aqueous solution), or the high affinity rabbit monoclonal antibody is provided with salt in water without a buffer, or contains an aqueous buffer or other type of solvent (e.g., an organic solvent).

[0047] The term "effective amount" as used herein refers to an amount sufficient to achieve a beneficial or desired result. A "therapeutically effective amount" is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount required to prevent the onset of a disease or disease symptoms. An effective amount may be administered, applied, or dosed once or more. The "therapeutically effective amount" (i.e., effective dose) of a therapeutic compound depends on the therapeutic compound selected. For example, the composition may be administered from one or more times daily to one or more times weekly, to one or more times monthly, to one or more times annually. Certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the subject's general health and / or age, and the presence of other diseases.

[0048] Example 1: Construction of a camel-derived nanobody library specific for CD31, and in vitro enrichment and screening of monoclonal nanobodies

[0049] (1) Animal immunization: A 2.5-year-old female alpaca was purchased (provided by Qingdao Kangda Aibo Biotechnology Co., Ltd.) and immunized with Recombinant Human CD31 / PECAM1 and C-His protein (AntibodySystem, catalog number EHD16401).

[0050] (2) 500 μg of monkeypox virus core protein A29L was emulsified with Freund's incomplete adjuvant and injected subcutaneously into the alpaca at multiple points. Immunization was performed every two weeks, and venous blood was collected from the alpaca seven days after each immunization.

[0051] Serum titers were determined using an indirect ELISA method. After five rounds of immunization, 80 ml of venous blood was extracted and PBMCs were isolated from the camel blood using the Tianjin Haoyang Company's Camel Peripheral Blood Mononuclear Cell Separation Kit (Cat. No. LDS1078).

[0052] PBMCs were lysed in 1 ml of trizol solution (Invitvogen, Catalog No. 15596018), and total RNA was extracted using Thermo Fisher Scientific's PureLink™ RNA Extraction Kit (Catalog No. 12183020). The total RNA was then analyzed by 1% agarose gel electrophoresis. Figure 1 shown.

[0053] (3) Total RNA from natural alpaca PBMCs was used as a template and reverse transcribed to obtain cDNA using Vazyme's HiScript® III1st Strand cDNA Synthesis Kit (+gDNA wiper) (Cat. No. R312-02);

[0054] Using cDNA as a template, two rounds of PCR amplification were performed using Vazyme's Phanta® Max Super-Fidelity DNA Polymerase (Cat. No. P505-d3) to obtain the gene sequence of the antibody variable region VHH. The reaction systems for the first and second rounds of PCR are shown in Tables 1 and 2 below, and the PCR reaction procedures are shown in Table 3. The results of 1% agarose gel electrophoresis of the products of the first round of PCR are shown in Figure 2 As shown, a DNA fragment of 630-740 bp was recovered.

[0055] The results of 1% agarose gel electrophoresis of the second round of PCR products are as follows Figure 3 As shown, a DNA fragment of about 450 bp was recovered.

[0056] Table 1 First round PCR reaction system

[0057]

[0058] Table 2 Second round PCR reaction system

[0059]

[0060] Table 3 First and second round PCR reaction procedures

[0061]

[0062] (4) The target fragment obtained by PCR and the vector pCANTAB5E (Amersham biosciences, phage display system) were digested with SfiI double-site enzymes at 50°C for 5-6 hours;

[0063] The enzyme digestion system of vector and target DNA fragment is shown in Table 4 below.

[0064] Table 4 Enzyme digestion system for vector and target DNA fragment

[0065]

[0066] Ligate the target fragment to the pCANTAB 5E vector using T4 ligase (NEB) at 16°C overnight. The specific ligation conditions are: 16°C for 15 hours, followed by inactivation at 65°C for 10 minutes. The enzyme ligation system is shown in Table 5.

[0067] Table 5 Enzyme ligation system

[0068]

[0069] (5) The ligation product was transformed into TG1 electrocompetent cells (Lucigen) by electroporation, plated on 2YT-Amp-Glucose solid culture medium (same recipe as below), and cultured at 37°C overnight.

[0070] The formula of the 2YT solid culture medium is: 1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) NaCl, dissolved in 1 L of distilled water.

[0071] The formula of the 2YT-Amp-Glucose culture medium is as follows: 100 μg / ml ampicillin and 2% glucose are added to the 2YT solid culture medium.

[0072] (6) Scrape the colonies on the culture medium, mix them evenly, add glycerol to a final concentration of 15%, and store at -80°C; inoculate the bacterial solution into 2YT-Amp-Glucose culture medium and culture at 37°C for 1-2 hours until the OD 600 =0.4-0.6.

[0073] Add helper phage and culture at 37°C for 45 min-1 h; centrifuge the bacterial solution at 3000-5000 rpm and discard the supernatant;

[0074] Resuspend the cells with an equal volume of 2YT-Amp-Kan medium and culture at 30°C overnight;

[0075] The next day, centrifuge and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5× PEG / NaCl solution. Mix thoroughly and place on ice or at 4°C for 1-2 hours. Centrifuge and discard the supernatant. Resuspend the pellet in PBS. The resulting monoclonal nanobody library is stored at 4°C or -20°C.

[0076] The formula of the 2YT-Amp-Kan culture medium is as follows: 100 μg / ml of ampicillin and 50 μg / ml of kanamycin are added to the 2YT culture medium.

[0077] Example 2: In vitro enrichment of camelid-derived nanobody (VHH) sequences specific for human CD31 protein and high-throughput monoclonal nanobody screening

[0078] The reagents and materials used in this example are shown in Table 6 below.

[0079] Table 6 Experimental reagents and materials

[0080]

[0081] 1. Immunotube solid phase panning

[0082] (1) Coating of immunotubes: 50 μg / ml antigen Recombinant Human CD31 / PECAM1, C-His protein, 1 ml / tube, coating overnight at 4°C; wash the immunotubes with 5 ml PBST for a total of 3 washes.

[0083] (2) Blocking: Take 5 ml of 5% skim milk / PBST and block at 30℃ for 1 hour; take 5 ml of PBS and wash twice.

[0084] (3) Incubation: Add 1 ml to the immunotube, with a total volume of (1-2) × 10 12 Incubate the pfu of nanolibrary phage at 30°C for 1.5 h; then wash 6-8 times with 5 ml of PBST.

[0085] (4) Elution: Add 1 ml of Gly-HCl (pH = 2.2) to the immunotube to elute the phage, and incubate at room temperature with shaking for about 6-8 minutes; add Tris-HCl (pH = 9.6) to neutralize the solution to pH = 7.0-8.0.

[0086] 2. Determine the titer of phage after elution

[0087] (1) Cultivate E. coli TG1 until OD 600 =0.4-0.6; mix 10 μL of diluted eluted phage and 190 μL of E. coli TG1.

[0088] (2) The resulting mixture was incubated at 37°C for 30 min, then poured onto 2×YT-A (Amp 100 μg / ml) solid culture medium and cultured at 37°C overnight.

[0089] 3. Amplification of eluted phage

[0090] (1) Directly aspirate 800 μl of the eluted phage solution and add it to 10 ml of the logarithmic phase TG1 bacterial solution. Incubate at 37°C for 30 min; incubate at 220 rpm at 37°C for 30 min.

[0091] (2) Add the bacterial solution to 30 ml of 2YT-Amp-Glucose medium and culture at 37°C and 220 rpm until the OD 600 = about 0.4-0.6. Add 3×10 11 pfu helper phage, incubate at 37°C for 30 min; culture at 220 rpm for 45 min-1 h.

[0092] (3) Centrifuge the bacteria at 3000-5000 rpm, discard the supernatant, and resuspend the bacteria in the same volume of 2YT-Amp-Kan medium. Incubate at 30°C, 220 rpm, overnight.

[0093] (4) The next day, centrifuge the bacteria at 8000 rpm, 4°C, 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5x PEG / NaCl solution, mix well, and place on ice or at 4°C for 1-2 h. Centrifuge to discard the supernatant, resuspend the precipitate with about 1 ml PBS, centrifuge to remove impurities, and transfer the supernatant to a new centrifuge tube.

[0094] 4. Test the titer of the amplified phage

[0095] The test method is the same as step 2.

[0096] 5. Repeat steps 1 to 4 to perform the 2nd to 4th rounds of enrichment panning

[0097] The enrichment panning method is shown in Table 7 below.

[0098] Table 7 Enrichment panning method

[0099]

[0100] The results of in vitro enrichment panning are shown in Table 8 below.

[0101] Table 8 Results of in vitro enrichment panning

[0102]

[0103] 6. Polyclonal phage ELISA test

[0104] (1) Coat the immunoplate with 4 μg / ml of the antigen Recombinant Human CD31 / PECAM1, C-His protein, 100 μl, 4°C overnight, and coat the control wells with 100 μl PBS. Wash 3 times with 300 μl PBST.

[0105] (2) Block: Take 300 μl of 5% skimmed milk / PBST, and block at 30°C for 1 h. Wash 2-3 times with 300 μl PBST.

[0106] (3) Incubate: Dilute the amplified phage from each round with PBS, with a 3-fold increase in dilution factor. The initial concentration is 10 12 pfu / ml; add 100 μl of the diluted amplified phage to each well. Incubate at 30°C for 1 h.

[0107] (4) Wash: Wash 3 times with 300 μl PBST.

[0108] (5) Secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.

[0109] (6) Washing: Wash three times with 300 μl PBST.

[0110] (7) Color development: Add 100 μl of TMB colorimetric solution and color for 3-8 min in the dark. (8) Stop the reaction and read the plate: Add 100 μl of 2 M HCl to stop the reaction and read the plate on a microplate reader (450 nm-620 nm). The ELISA test results are shown in Table 9 below.

[0111] Table 9 Polyclonal phage ELISA test results

[0112]

[0113] 7. Monoclonal phage ELISA screening

[0114] (1) Select an appropriate round number (R4), dilute the eluted phage to an appropriate concentration, infect TG1 in the logarithmic phase, and plate it.

[0115] (2) The next day, 96 single clones were picked from the plate and inoculated into a 96-deep-well plate (600 μl of 2YT-Amp-Glucose medium was added to each well) and cultured at 37°C, 250 rpm for 2 h until the bacterial solution OD 600 =0.4-0.6.

[0116] Pipette 100 μl of bacterial solution into each well of the cell culture plate, add sterile glycerol (final concentration 20%-25%) to each well, mix well and store at -20°C.

[0117] (3) Add helper phage to the remaining 96-well plate culture medium. The amount of helper phage added = 0.6 × liquid volume × 5 × 10 8 ×30 pfu; incubate at 37°C for 30 min, shake at 250 rpm, and incubate at 37°C for 45 min-1 h.

[0118] (4) Centrifuge the 96-well plate at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial solution in 600 μl of 2YT-Amp-Kan medium per well and incubate at 30°C, 250 rpm, with shaking overnight.

[0119] (5) The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 min and collect the supernatant for ELISA experiment.

[0120] (6) Coating: Coat the immunoplate with 4 μg / ml antigen Recombinant Human CD31 / PECAM1, C-His protein, 100 μl, overnight at 4°C. Control wells were coated with 100 μl PBS; washed three times with 300 μl PBST.

[0121] (7) Blocking: Take 300 μl of 5% skim milk / PBST and block at 30°C for 1 h; wash 2-3 times with 300 μl of PBST.

[0122] (8) Incubation: Add 100 μl of supernatant phage to each well and incubate at 30°C for 1 h.

[0123] (9) Washing: Wash three times with 300 μl PBST.

[0124] (10) Secondary antibody: Add 100 μl of secondary antibody diluent (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.

[0125] (11) Washing: Wash three times with 300 μl PBST.

[0126] (12) Color development: Add 100 μl of TMB color development solution and color for 3-8 min in the dark. Add 100 μl of 2M HCl to terminate the reaction and read the result with a microplate reader (450 nm-620 nm).

[0127] The results of the ELISA test for monoclonal phage No. 1-48 are shown in Table 10 below, and the results of the ELISA test for monoclonal phage No. 49-96 are shown in Table 11 below.

[0128] Table 10 ELISA test results of monoclonal phage No. 1-48

[0129]

[0130] Table 11 ELISA test results of monoclonal phage No. 49-96

[0131]

[0132] According to the monitoring results in Tables 10 and 11 above, (OD 600 (Antigen group)-OD 600 High-affinity positive clones were defined as those with a p-value greater than 2.5 (control group). These positive clones were sequenced to eliminate erroneous and duplicated antibody sequences. Ultimately, two high-affinity antibody sequences were identified: high-affinity nanobodies that specifically recognize the CD31 protein, designated CD31-1040 and CD31-1042.

[0133] After sequencing, the amino acid sequence of the complementary determining region CDR1 of the heavy chain of the monoclonal nanoantibody CD31-1040 is: GRAFSSLS, as shown in SEQ ID NO.1; the amino acid sequence of CDR2 is: IAVAGGST, as shown in SEQ ID NO.2; the amino acid sequence of CDR3 is ASSLNPGTVRTPTGYSY, as shown in SEQ ID NO.3.

[0134] The amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1040 is:

[0135] QVQLVESGGGLVQAGGSLRLSCAASGRAFSSLSMGWIRQAPGKEREFVASIAVAGGSTYYTDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCASSLNPGTVRTPTGYSYWGQGTQVTVSS, as shown in SEQ ID NO.4.

[0136] The DNA sequence (nucleotide sequence) encoding the heavy chain of the monoclonal nanobody CD31-1040 is:

[0137] caggtgcagctcgtggagtcggggggagggttggtgcaggctgggggttctctgagactctcctgtgcagcctctggacgcgccttcagtagcttgtccatgggctggatccgccaggctccagggaaggagcgtgagtttgtagcgtccattgcggtggcaggtggtagcacatactatacagactc cgtgaagggccgattcaccatctccagagacaacgccaagaacatggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattattgtgcatcttcgttgaaccccggcacagtgcgtacacctacggggtattcatactggggccaggggacccaggtcaccgtctcctca, as in SEQ Shown as ID NO.5.

[0138] The amino acid sequence of the complementary determining region CDR1 of the heavy chain of the monoclonal nanoantibody CD31-1042 is: GRTFSSLS, as shown in SEQ ID NO.6; the amino acid sequence of CDR2 is: IAVAGGST, as shown in SEQ ID NO.7; the amino acid sequence of CDR3 is ASSVNSGVTRSPSGYHY, as shown in SEQ ID NO.8.

[0139] The amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1042 is:

[0140] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSLSMGWIRQAPGKEREFVASIAVAGGSTYYADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCASSVNSGVTRSPSGYHYWGQGTQVTVSS, as shown in SEQ ID NO.9.

[0141] The DNA sequence (nucleotide sequence) encoding the heavy chain of the monoclonal nanobody CD31-1042 is:

[0142] caggtgcagctcgtggagtcggggggagggttggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcaccttcagtagct tgtccatgggctggatccgccaggctccagggaaggagcgtgagtttgtcgcgtccattgcggtggcaggtggtagcacctactatgcagactc cgtgaagggccgattcaccatctccagagacaacgccaagaacatggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcatcttcagtgaactccggcgtaacgcgttcaccgtcggggtatcactactggggccaggggacccaggtcaccgtctcctca, as in SEQ Shown as ID NO.10.

[0143] Example 3: Recombinant expression of monoclonal nanobodies CD31-1040 and CD31-1042

[0144] The gene sequences of the two nanobodies screened in Example 2 were subcloned into the pATX1 vector (ATAGENIX), with restriction sites EcoR1 / Not1 and mouse IgG Fc added to the C-terminus. Xten CHO cells were transfected. 72 hours after transfection, the cells were removed by centrifugation, and the culture supernatant was purified using Protein A resin (12% non-reduced SDS-PAGE). Figure 4 shown.

[0145] Example 4: Identification of monoclonal nanobodies CD31-1040 and CD31-1042

[0146] 1. ELISA detection of monoclonal nanoantibodies CD31-1040 and CD31-1042

[0147] (1) Coating the immunoplate: 5 μg / ml antigen Recombinant Human CD31 / PECAM1, C-His protein, 100 μl, overnight at 4°C; control wells are coated with 100 μl PBS. Wash three times with 300 μl PBST.

[0148] (2) Blocking: Take 300 μl 3% BSA / PBS and block at 37°C for 1.5 h; wash 2-3 times with 300 μl PBST.

[0149] (3) Incubation: Dilute the monoclonal nanobody with PBS in 5-fold increments; the initial concentration is 10 μg / ml. Add 100 μl of diluted nanobody to each well and incubate at 37°C for 1 h. Wash three times with 300 μl of PBST.

[0150] (4) Add secondary antibody: Add 100 μl of secondary antibody diluent Goat anti Mouse (H+L)-HRP, 1:5000 (AntibodySystem, Cat. No. PMB96431), incubate at 37°C for 30 min; wash three times with 300 μl of PBST.

[0151] (5) Color development: Add 100 μl of TMB colorimetric solution and color for 8 min in the dark. Add 100 μl of 2 M HCl to terminate the reaction. Read the result with a microplate reader (450 nm-620 nm). The results are shown in Table 12.

[0152] Table 12 ELISA test results

[0153]

[0154] 2. Western Blot Detection of Monoclonal Nanobodies CD31-1040 and CD31-1042

[0155] (1) SDS-PAGE electrophoresis: 100 ng of CD31 protein was subjected to SDS-PAGE gel electrophoresis using a 10% polyacrylamide precast gel (Thermo, catalog number XP00102BOX).

[0156] (2) Membrane transfer: Transfer the electrophoretically separated CD31 protein to a nitrocellulose membrane.

[0157] (3) Blocking: Remove the electrotransferred nitrocellulose membrane, wash it once with TBST, and block it with 5% milk / TBST on a shaker at room temperature for 1 h; rinse it with TBST for 2 min.

[0158] (4) Binding of primary antibody and target protein: Monoclonal nanoantibodies CD31-1040 and CD31-1042 were diluted with 1% BSA / PBST, incubated with nitrocellulose membrane, and stored in a refrigerator at 4°C overnight; washed with PBST three times, 10 min each time.

[0159] (5) Incubation of secondary and primary antibodies: Place the treated nitrocellulose membrane in a secondary antibody diluent (1:5000) Goat anti Mouse (H+L)-HRP diluted in 5% milk / PBST and incubate on a shaker at room temperature for 1 h; wash three times with PBST, 10 min each time.

[0160] (6) Color development: Place the treated nitrocellulose membrane with the front side facing down in contact with the luminescent reagent. After color development for 1.5-2.0 minutes, use a gel imaging system to observe the results. The results of Western Blot are as follows: Figure 5 and 6 shown.

[0161] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. An anti-CD31 monoclonal nanobody, characterized in that It is a monoclonal nanobody CD31-1040 or CD31-1042; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanobody CD31-1040 are shown in SEQ ID NOs. 1-3, respectively; the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the monoclonal nanobody CD31-1042 are shown in SEQ ID NOs. 6-8, respectively.

2. The anti-CD31 monoclonal nanobody according to claim 1, characterized in that The amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1040 is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain of the monoclonal nanobody CD31-1042 is shown in SEQ ID NO.

9.

3. The anti-CD31 monoclonal nanobody according to claim 2, characterized in that The gene sequence encoding the heavy chain of the monoclonal nanobody CD31-1040 is shown in SEQ ID NO.5, and the gene sequence encoding the heavy chain of the monoclonal nanobody CD31-1042 is shown in SEQ ID NO.

10.

4. A substance, characterized in that The substance includes any one of the following aspects: (1) A nucleic acid molecule or a vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the monoclonal nanobody according to any one of claims 1 to 3; (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain is used to express the monoclonal nanobody according to any one of claims 1 to 3; (3) an antibody derivative comprising the monoclonal nanobody according to any one of claims 1 to 3, directly or indirectly linked to a detectable marker, drug or toxin; (4) A detection product comprising the monoclonal nanobody according to any one of claims 1 to 3, or the nucleic acid molecule, or the vector, or the antibody derivative.

5. A use of the anti-CD31 monoclonal nanobody according to any one of claims 1 to 3, characterized in that: The application includes any one of the following aspects: (1) Used for the detection of CD31 expression for purposes other than disease diagnosis and treatment, or for in vitro evaluation of the efficacy of drugs for the treatment of vascular tumors; (2) Products used to detect CD31 expression or evaluate the efficacy of drugs for treating vascular tumors.

6. The use according to claim 5, characterized in that The product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

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