CD47 nano antibody as well as preparation method, application and product thereof

Abstract

The invention provides a CD47 nano antibody as well as a preparation method, application and a product thereof, and belongs to the technical field of nano antibodies. The CD47 nano antibody comprises a complementarity determining region (CDR), and the complementarity determining region is composed of CDR1, CDR2 and CDR3 with specific sequences. The CD47 nano antibody can be combined with CD47 with high specificity, and has high affinity to CD47. The preparation method of the CD47 nano antibody is simple and easy to operate, and a large number of high-purity CD47 nano antibodies can be obtained. The invention provides a nano antibody capable of specifically binding CD47, which can be used for detecting CD47 and treating related diseases, and provides a drug candidate for diagnosis and treatment of CD47 related diseases.

Description

Technical Field

[0001] This invention belongs to the field of nanobody technology, and particularly relates to a CD47 nanobody and its preparation method, application and product. Background Technology

[0002] Integrin-associated protein (CD47) is a member of the immunoglobulin superfamily with a five-fold transmembrane structure, widely expressed on the surface of normal cells. It transmits an inhibitory signal by binding to the SIRPα receptor on the surface of macrophages, inhibiting the phagocytosis of normal cells by macrophages. Studies have shown that this protein is universally overexpressed on the surface of tumor cells, and the inhibitory signal generated when it binds to SIRPα prompts tumor cells to escape phagocytosis by macrophages, thereby inducing tumor development and progression. It is one of the key molecules in the tumor microenvironment that mediates tumor cell escape from immune surveillance.

[0003] By preparing a specific antibody that binds to CD47 on the surface of tumor cells, the inhibitory signaling pathway transmitted by the binding of CD47 to SIRPα on the tumor cell surface can be blocked, promoting the phagocytosis of tumor cells by macrophages, thus providing a new theory and method for tumor immunotherapy. Hu5F9-G4 is a humanized antibody targeting human CD47 designed and screened by Forty Seven Biotech, Inc. Because CD47 is expressed on erythrocytes, Hu5F9 has a certain toxic effect on erythrocytes, which may lead to a series of adverse reactions in the hematologic system, posing certain safety risks to its clinical application. These risks undoubtedly limit the development and application of Hu5F9.

[0004] Nanobodies (Nb) are single-domain antibody fragments derived from the variable region (VHH) of naturally occurring heavy-chain antibodies (HCAbs) in camel-like animals, obtained through cloning and expression. Nanobodies are gradually replacing other small antibodies and becoming a hot topic in the development of novel antibody drugs. Nb typically has a size of only about 15 kDa, approximately one-tenth the size of traditional antibodies. It contains disulfide bonds internally and has numerous hydrophilic residues on its surface, exhibiting strong resistance to heat and pH. The absence of an Fc fragment and light chain in Nb allows it to recognize cryptic or small epitopes that traditional antibodies cannot, while avoiding complement reactions. Furthermore, nanobodies possess numerous advantages, including high stability, low toxicity, high solubility, ease of target screening, and direct expression in prokaryotic microorganisms, as well as good cost-effectiveness. Currently developed CD47 antibodies not only suffer from significant side effects but also exhibit low affinity; therefore, further exploration and development of new nanobodies targeting human CD47 are needed to provide options for subsequent clinical research and applications. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a CD47 nanobody, its preparation method, application and product, wherein the CD47 nanobody can bind to CD47.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a CD47 nanobody comprising a complementarity-determining region, wherein the complementarity-determining region is composed of CDR1, CDR2 and CDR3; CDR1 as shown in SEQ ID NO.17, CDR2 as shown in SEQ ID NO.18, and CDR3 as shown in SEQ ID NO.19; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19; CDR1 as shown in SEQ ID NO.20, CDR2 as shown in SEQ ID NO.21, and CDR3 as shown in SEQ ID NO.22; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22; CDR1 as shown in SEQ ID NO.20, CDR2 as shown in SEQ ID NO.23, and CDR3 as shown in SEQ ID NO.24; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.20, SEQ ID NO.23, and SEQ ID NO.24; CDR1 as shown in SEQ ID NO.25, CDR2 as shown in SEQ ID NO.26, and CDR3 as shown in SEQ ID NO.27; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27; CDR1 as shown in SEQ ID NO.28, CDR2 as shown in SEQ ID NO.23, and CDR3 as shown in SEQ ID NO.29; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.28, SEQ ID NO.23, and SEQ ID NO.29.

[0007] Preferably, the amino acid sequence of the CD47 nanobody is shown in one or more of SEQ ID NO. 8 to SEQ ID NO. 16; Alternatively, the amino acid sequence of the CD47 nanobody may be any one or more amino acid sequences that have at least 85% sequence similarity to those in SEQ ID NO. 8 to SEQ ID NO. 16.

[0008] The present invention provides a nucleotide encoding the above-mentioned CD47 nanobody.

[0009] The present invention provides a biomaterial comprising an expression vector or a host cell; the expression vector contains the aforementioned nucleotides; and the host cell contains the aforementioned nucleotides or the expression vector.

[0010] This invention provides a method for preparing CD47 nanobodies, comprising the following steps: The above expression vector was transformed into host cells and cultured, and then induced to express to obtain a culture containing CD47 nanobodies. The CD47 nanobodies were then isolated and purified from the culture containing CD47 nanobodies.

[0011] The present invention provides an antibody conjugate comprising the above-mentioned CD47 nanobody and conjugating agent; The conjugates include immunoglobulin Fc domains, His tags, GST tags, MBP tags, FLAG tags, SUMO tags, Strep-tag II tags, Twin-Strep-tag tags, MyC tags, HA tags, AviTag tags, HaloTag tags, EGFP tags, SNAP tags, V5 tags, thioredoxin tags, GFP tags, mCherry tags, protein C tags, BCCP tags, Spot-tag tags, Isopeptag, SpyTag, serum albumin, albumin-binding peptides, prealbumin, C-terminal peptides, elastin-like peptides, cytokines, single-chain antibodies, virus-like particles, ligases, kinases, prodrug activating enzymes, chemotherapeutic agents, gold nanoparticles, or liposomes.

[0012] The present invention provides a medicament or pharmaceutical composition for the prevention and / or treatment of tumors, comprising the above-described CD47 nanobody or antibody-drug conjugate.

[0013] The present invention provides a kit for detecting CD47, the kit comprising the above-mentioned CD47 nanobody or antibody conjugate.

[0014] The present invention provides a drug or drug composition for immune regulation or binding to CD47 targeting, wherein the drug or drug composition comprises an active ingredient that interacts with CD47; the active ingredient includes the aforementioned CD47 nanobody or antibody-drug conjugate.

[0015] This invention provides an application of the above-mentioned CD47 nanobody, the CD47 nanobody prepared by the preparation method, or the antibody conjugate in the preparation of at least one of the following (1) to (6): (1) Diagnostic reagents or kits for detecting CD47; (2) Molecular probes or detection reagents that bind to CD47; (3) Drugs that enhance the phagocytic activity of macrophages; (4) Antitumor drugs or drug compositions; (5) Drugs or drug compositions used for immune regulation; (6) Targeted drugs or drug compositions that bind to CD47.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a CD47 nanobody, its preparation method, applications, and products. This CD47 nanobody can bind to CD47 with high specificity and high affinity. The preparation method of the CD47 nanobody of this invention is simple and easy to operate, and can obtain large quantities of high-purity CD47 nanobodies. This invention provides potential new nanobody drugs for CD47 detection and disease treatment. Attached Figure Description

[0017] Figure 1 Flow cytometry results of CD47 nanobodies HBM-C12~HBM-C20 specifically binding to CD47 on different cell surfaces; Figure 2 The results show the competitive binding efficiency of CD47 nanobodies HBM-C12~HBM-C20 with SIRPα ligands for CD47; Figure 3 The results show the affinity of different concentrations of CD47 antibody to CD47 as determined by flow cytometry. Figure 4 The results show the affinity of different concentrations of CD47 nanobodies HBM-C12~HBM-C15 with CD47. Figure 5 The results show the affinity of different concentrations of CD47 nanobodies HBM-C16~HBM-C20 with CD47. Figure 6 The IC50 values ​​of CD47 nanoantibodies HBM-C12~HBM-C15 at different concentrations are shown. Figure 7 The IC50 values ​​of CD47 nanoantibodies HBM-C16~HBM-C19 at different concentrations are shown. Figure 8The IC50 values ​​of CD47 nanoantibodies HBM-C20, Ligufalimab, and Evorpacept at different concentrations were measured. Detailed Implementation

[0018] This invention provides a CD47 nanobody comprising a complementarity-determining region, wherein the complementarity-determining region is composed of CDR1, CDR2 and CDR3; CDR1 as shown in SEQ ID NO.17, CDR2 as shown in SEQ ID NO.18, and CDR3 as shown in SEQ ID NO.19; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.17, SEQ ID NO.18, and SEQ ID NO.19; CDR1 as shown in SEQ ID NO.20, CDR2 as shown in SEQ ID NO.21, and CDR3 as shown in SEQ ID NO.22; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22; CDR1 as shown in SEQ ID NO.20, CDR2 as shown in SEQ ID NO.23, and CDR3 as shown in SEQ ID NO.24; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.20, SEQ ID NO.23, and SEQ ID NO.24; CDR1 as shown in SEQ ID NO.25, CDR2 as shown in SEQ ID NO.26, and CDR3 as shown in SEQ ID NO.27; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27; CDR1 as shown in SEQ ID NO.28, CDR2 as shown in SEQ ID NO.23, and CDR3 as shown in SEQ ID NO.29; or a CDR region having at least 85% sequence similarity to any one of the sequences in SEQ ID NO.28, SEQ ID NO.23, and SEQ ID NO.29.

[0019] In this invention, the amino acid sequence of the CD47 nanobody is shown in one or more of SEQ ID NO. 8 to SEQ ID NO. 16; Alternatively, the amino acid sequence of the CD47 nanobody may be any one or more amino acid sequences that have at least 85% sequence similarity to those in SEQ ID NO. 8 to SEQ ID NO. 16.

[0020] In this invention, the CD47 nanobody shown in any one of SEQ ID NO. 8 to SEQ ID NO. 16 binds to CD47 with high affinity and strong targeting specificity. In this invention, the CD47 nanobody shown in SEQ ID NO. 8 to SEQ ID NO. 16 is named HBM-C12 to HBM-C20, respectively. The EC50 values ​​of HBM-C12 to HBM-C20 binding to CD47 are 14.8 ng / mL, 23.5 ng / mL, 25.4 ng / mL, 48.9 ng / mL, 56.5 ng / mL, 81.9 ng / mL, 99 ng / mL, 99.8 ng / mL, and 204 ng / mL, respectively. Among them, HBM-C12~HBM-C17, HBM-C19 and HBM-C20 can competitively bind to CD47 with SIRPα ligands and can block the interaction between CD47 and SIRPα. In particular, HBM-C12, HBM-C13, HBM-C14, HBM-C15, HBM-C17, HBM-C19 and HBM-C20 have high efficiency in competitively binding to CD47 with SIRPα ligands.

[0021] The present invention provides a biomaterial comprising an expression vector or a host cell; the expression vector contains the aforementioned nucleotides; and the host cell contains the aforementioned nucleotides or the expression vector.

[0022] In this invention, the expression vector comprises the pComb3x plasmid. The expression vector is obtained by introducing nucleotides encoding the aforementioned CD47 nanobody into the pComb3x plasmid.

[0023] The present invention provides a host cell containing the aforementioned nucleotides or expression vectors.

[0024] In this invention, the host cell comprises bacteria or fungi, wherein the bacteria are *Escherichia coli*, *E. coli* ER2738 competent cells, or *E. coli* WK6 competent cells. The host cell is obtained by transfecting the expression vector into *E. coli*.

[0025] This invention provides a method for preparing CD47 nanobodies, comprising the following steps: The above expression vector was transformed into host cells and cultured, and then induced to express to obtain a culture containing CD47 nanobodies. The CD47 nanobodies were then isolated and purified from the culture containing CD47 nanobodies.

[0026] In this invention, the above-mentioned expression vector is transformed into host cells and cultured, followed by induced expression to obtain a culture containing CD47 nanobodies. The host cells are preferably WK6 competent cells. This invention does not specifically limit the culture method; any culture method known in the art can be used. The induced expression can be performed overnight using IPTG, with the IPTG concentration being 0.5–1.5 mM, such as 1 mM. After induction, the precipitate is collected by centrifugation to obtain a culture containing CD47 nanobodies. After obtaining the culture containing CD47 nanobodies, the CD47 nanobodies are separated and purified. The separation involves incubating pre-cooled TES buffer at 4°C with shaking overnight, then adding TES / 4, incubating at 4°C with shaking for 4 hours, and centrifuging to collect the supernatant. The TES buffer consists of the following components: 200 mM Tris-HCl, pH 8.0, 500 mM sucrose, and 1 mM EDTA. The overnight incubation time is 10–14 h, such as 12 h. The TES / 4 solution consists of 250 mL TES buffer and 750 mL ddH2O. Centrifugation is performed at 10000 g for 30 minutes at 4°C. Purification is achieved by subjecting the supernatant to nickel column purification using a Ni-NTA column purchased from Thermo, 88223.

[0027] The method for preparing CD47 nanobodies of the present invention can produce high-purity CD47 nanobodies. In particular, the purity of HBM-C16 and HBM-C18 is 100%.

[0028] The present invention provides an antibody conjugate comprising the above-mentioned CD47 nanobody and conjugating agent; The conjugates include immunoglobulin Fc domains, His tags, GST tags, MBP tags, FLAG tags, SUMO tags, Strep-tag II tags, Twin-Strep-tag tags, MyC tags, HA tags, AviTag tags, HaloTag tags, EGFP tags, SNAP tags, V5 tags, thioredoxin tags, GFP tags, mCherry tags, protein C tags, BCCP tags, Spot-tag tags, Isopeptag, SpyTag, serum albumin, albumin-binding peptides, prealbumin, C-terminal peptides, elastin-like peptides, cytokines, single-chain antibodies, virus-like particles, ligases, kinases, prodrug activating enzymes, chemotherapeutic agents, gold nanoparticles, or liposomes.

[0029] The present invention provides a medicament or pharmaceutical composition for the prevention and / or treatment of tumors, comprising the above-described CD47 nanobody or antibody-drug conjugate.

[0030] The present invention provides a kit for detecting CD47, the kit comprising the above-mentioned CD47 nanobody or antibody conjugate.

[0031] The present invention provides a drug or drug composition for immune regulation or binding to CD47 targeting, wherein the drug or drug composition comprises an active ingredient that interacts with CD47; the active ingredient includes the aforementioned CD47 nanobody or antibody-drug conjugate.

[0032] This invention provides an application of the above-mentioned CD47 nanobody, the CD47 nanobody prepared by the preparation method, or the antibody conjugate in the preparation of at least one of the following (1) to (6): (1) Diagnostic reagents or kits for detecting CD47; (2) Molecular probes or detection reagents that bind to CD47; (3) Drugs that enhance the phagocytic activity of macrophages; (4) Antitumor drugs or drug compositions; (5) Drugs or drug compositions used for immune regulation; (6) Targeted drugs or drug compositions that bind to CD47.

[0033] In this invention, the product may include reagents or kits.

[0034] In the application of this invention in the preparation of drugs or antitumor drugs or pharmaceutical compositions that enhance macrophage phagocytosis, the CD47 nanobody is preferably one of HBM-C12, HBM-C13, HBM-C14, HBM-C15, HBM-C16, HBM-C17, HBM-C19, or HBM-C20, i.e., any one of SEQ ID NO. 8~SEQ ID NO. 13, SEQ ID NO. 15, and SEQ ID NO. 16. This invention, through the competitive binding of the aforementioned CD47 nanobody to SIRPα, blocks the interaction between CD47 and SIRPα on the surface of tumor cells, thereby promoting the phagocytosis of tumor cells by macrophages. Therefore, it has the effects of activating macrophages and antitumor activity.

[0035] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] In the following examples, the human ovarian cancer cells CAOV4 were purchased from Orex, ORC1509; the plasmid pLCRISPR-CMV was purchased from Addgene, 102609.

[0038] Example 1 Screening of nanobodies 1.1 Animal Immunization The human CD47 protein antigen (His Tag) was prepared by Genscript Biotech (order number C938UDWTG0-4), using pcDNA3.4 as the expression plasmid and CHO-S as the cell line.

[0039] The nucleotide sequence of the human CD47 protein is as follows: CAGCTTGCTGTTCAACAAGACCAAGTCCGTGGAGTTCACCTTCTGCAACGACACCGTGGTGATCCCTTGCTTCGTGACCAACATGGAAGCTCAGAACACCACCGAGGTGTACGTGAAGTGGAAGTTCAAGGGACGGGACATCTACACCTTTGATGGCGCTCTGAATAAATCCACCGTGCCCACCGACTTCTCC TCCGCCAAGATCGAGGTGTCCCAGCTGCTCAAAGGCGACGCCTCTCTGAAGATGGACAAGAGCGATGCCGTGTCTCACCACCGGCAACTACACCTGTGAGGTCACAGAACTGACCAGAGAGGGCGAGACAATCATCGAGCTGAAGTACAGAGTGGTCTCTTGGTTTAGCCCTCACCACCATCATCACCAC (SEQ ID NO.1).

[0040] The human CD47 protein antigen was entrusted to Shenzhen Kangti Biomedical Technology Co., Ltd. for immunization of camels. The specific immunization plan is as follows: immunize with Human CD47 Protein, His Tag once every two weeks for a total of 5 immunizations. In the first two immunizations, the antigen immunization dose is 0.4 mg each time. From the third to the fifth immunization, the antigen immunization dose is halved each time, i.e., 0.2 mg.

[0041] (1) Emulsify the antigen according to its properties; (2) Immunize camels with emulsified antigens once every two weeks for a total of 5 times; (3) One week after the first to fifth immunizations, 5 mL of serum was drawn each time, and 200 ng of human CD47 protein antigen was used as the coating antigen for ELISA titer detection. (4) One week after the fourth and fifth immunizations, 50 mL of peripheral blood was drawn from each of them, and serum and PBMCs were separated.

[0042] The results of the potency test are shown in Table 1.

[0043] Table 1. Valence Test Results

[0044] The results in Table 1 show that the serum titers after the fourth and fifth immunizations reached 10. 3 Finally, the five-immune serum was selected for subsequent experiments.

[0045] 1.2 Construction of Phage-Displayed Nanobody Library 1.2.1 Total RNA extraction and reverse transcription The serum of the five immune patients was used to isolate B lymphocytes and extract and purify total RNA using the LeukoLOCK Total RNA Isolation System (AM1923) from Thermo. Then, cDNA was obtained by reverse transcription using the PrimeScript™ RT-PCR Kit (RR014A) from Takara.

[0046] 1.2.2 Amplification and purification of nanobody genes Nested PCR was performed using cDNA as a template to amplify the nanobody gene fragment. The specific steps are as follows: (1) First round of PCR reaction The reaction system for the first round is shown in Table 2, the primer sequences are shown in Table 3, and the reaction program is 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 5 min.

[0047] Table 2 First-round reaction system

[0048] Table 3 Primer sequences

[0049] In Table 3, W represents a degenerate base, which is either A or T.

[0050] (2) Second round of PCR reaction The reaction system is shown in Table 4.

[0051] Table 4 Second Round Reaction System

[0052] In Table 4, R3 / R4 / R5 (10 μM) refers to three PCR amplification reactions performed with F1 / R3, F1 / R4, or F1 / R5 as the upper and lower primers, respectively, with a downstream primer concentration of 10 μM, resulting in nanobody fragments 1-3.

[0053] The second round of reaction was performed at 94℃ for 3 min; 94℃ for 30 s, 55℃ for 30 s, 68℃ for 1 min, for a total of 30 cycles; and then at 68℃ for 5 min.

[0054] 1.2.3 Enzyme digestion and ligation of nanobody fragments and vectors (1) The nanobody fragments 1-3 and the pComb3x plasmid (Addgene, catalog number 63890) were digested with the restriction endonuclease SfiI (Catalog No. R0123L) from NEB and reacted overnight at 50°C to obtain the digested nanobody fragments 1-3 and the digested plasmid vector.

[0055] (2) The nanobody fragments 1-3 after enzyme digestion were purified using Qiagen's QIAquick PCR Purification Kit (catalog number 28106). The 3400 bp bands after plasmid digestion were recovered by gel extraction using Qiagen's QIAquick Gel Extraction Kit (catalog number 28706), and the fragment and vector concentrations were determined to obtain the recovered nanobody fragments 1-3.

[0056] (3) The digested plasmid vector and the recovered nanobody fragments 1-3 were ligated together using NEB's T4 ligase (catalog number M0202L) and reacted overnight at 16°C to obtain the ligation product.

[0057] (4) The enzyme-linked product was electroporated into ER2738 competent cells and rescued using helper phages (NEB, catalog number N0315S) to construct a phage-displaying nanobody library.

[0058] The library has a capacity of 5.3 × 10⁻⁶. 8 The CFU reservoir, after rescue by helper phages, has a capacity of 2 × 10⁻⁶. 13 The library has good insertion rate and diversity, with a pfu / mL concentration.

[0059] 1.2.4 Screening of CD47 nanobodies Human CD47 protein antigen was coated in wells 3 and 4 of a 96-well microplate at a concentration of 100 μg / mL and incubated overnight at 4°C. The next day, the coating solution was poured out, and the plates were washed three times with PBST. Wells 1 and 2 were blocked with 300 μL of 1% gelatin, and wells 3-8 were blocked with 300 μL of 1% BSA. The plates were incubated at room temperature for 1 h. The blocking solution was poured out, and the plates were washed three times with PBST. 100 μL of the phage nanobody library obtained in step (4) above was mixed with 110 μL of 3% BSA (prepared with PBS) and premixed at 37°C and 220 rpm for 1 h. The mixture was added to wells 1 and 2 and reverse-selected for 1 h. The liquid from wells 1 and 2 was transferred to wells 3 and 4 and bound for 1.5 h. The liquid was poured out, patted dry on clean absorbent paper, and washed five times with PBST. 100 μL of 0.1M Glycine-HCl buffer (pH 100- ... 2.2) Add the solution to wells 3 and 4, vortex and elute for 10 min, then add 10 μL of 1M Tris-HCl buffer (pH 9.1) and mix well to neutralize. Aspirate the liquid from wells 3 and 4 and transfer it to wells 5, 6, 7, and 8 (50 μL / well), and incubate for 1 h. Collect the liquid from wells 5, 6, 7, and 8, using 10 μL for titer determination, and add 3 mL of *E. coli* ER2738 culture solution shaken to the logarithmic phase. Incubate at 37°C for 1 h. Add 1.5 μL of 100 mg / mL carbenicillin and 3 mL of 2×YT medium, and incubate at 37°C and 220 rpm for 1 h. Add 1.5 μL of 100 mg / mL carbenicillin, and incubate at 37°C and 220 rpm for 1 h. Add helper phage (NEB, catalog number N0315S), 22 μL of 100 mg / mL carbenicillin, and 44 μL of... Incubate 2×YT medium at 37℃ and 220 rpm for 1 h; add 75 μL of 50 mg / mL kanamycin and incubate overnight; the next day, centrifuge and collect the supernatant, add PEG-NaCl solution to precipitate and purify the phage, and obtain the amplification product. Perform the next round of screening on the amplification product, ensuring the same amount of antigen is added in each round, decreasing the antigen coating concentration by a factor of 2, calculating the titers of eluted phage and amplification product in each round, selecting single clones for amplification and ELISA identification, and obtaining positive single-clone plasmids.

[0060] In this embodiment, 22 anti-CD47 nanoantibodies with different sequences were finally selected from the obtained positive monoclonal plasmids C01~C22.

[0061] Example 2 Expression and purification of CD47 nanobodies Positive monoclonal plasmids C12-C20 prepared in Example 1 were extracted and transformed into WK6 competent cells (Honorgene, catalog number HG-VDA1597). After recovery, the cells were plated on solid medium (carbenicillin resistant) and cultured overnight. The next day, single clones were picked and cultured in TB-carbenicillin medium, and IPTG was added to a final concentration of 1 mM to induce expression overnight. The following day, the bacteria were harvested by centrifugation at 9000 g for 15 minutes at room temperature. The bacterial pellet was carefully resuspended in 5 mL of pre-chilled TES buffer (200 mM Tris-HCl, pH 8.0, 500 mM sucrose, 1 mM EDTA) and incubated overnight at 4°C with shaking. The next day, 10 mL of TES / 4 (diluted with 750 mL ddH2O in 250 mL TES buffer) was added to the resuspended cell pellet and incubated at 4°C with shaking for 4 hours. Centrifuge at 10000 g for 30 minutes at 4℃, collect the supernatant and purify it with nickel column to obtain the purified anti-CD47 nanoantibodies, namely HBM-C12~HBM-C20.

[0062] The nickel column purification steps are as follows: (1) Take out the Ni-NTA-filled gravity column (2 mL column volume) at 4℃, add 4 mL ddH2O to wash the column each time, and repeat for a total of 5 times; (2) Column equilibration: Each time, 4 mL of binding buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH 7.4) was passed through the column, and the process was repeated 5 times in total. (3) Mix 15 mL of the above supernatant with 2 mL of packing material, transfer to a 50 mL centrifuge tube, and shake on a shaker at 4°C for 1 h to allow the protein to fully bind with the resin and obtain a supernatant-resin mixture. (4) Refill the tube with the supernatant-resin mixture and let the liquid drip down slowly.

[0063] (5) Add 10 mL of binding buffer (20 mM sodium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH 7.4) to wash the nickel column and remove non-specifically bound proteins.

[0064] (6) Add 10 mL of washing buffer (20 mM sodium phosphate, 500 mM sodium chloride, 40 mM imidazole, pH 7.4) to wash the nickel column twice.

[0065] (7) Elution: Add 8 mL of elution buffer (20 mM sodium phosphate, 50 mM sodium chloride, 300 mM imidazole, pH 7.4) to elute the target protein and collect all the elution buffer.

[0066] The sequence information of the anti-CD47 nanobody corresponding to HBM-C12~HBM-C20 is as follows: HBM-C12 (SEQ ID NO.8): QVQLVESGGGSAQAGGSLRLSCAASGYTFSSERIGWFRQAPGKEREGVAIIVPSNGVTYYTDSVKGRFTISQDNAKNTLFLQMNSLKPEDTATYYCAAGWRVGAPALLPKSYRYWGQGTQVTVSS.

[0067] Among them, in the amino acid sequence such as SEQ ID NO.8, CDR1 of HBM-C12 is GYTFSSER (SEQ ID NO.17); CDR2 is IVPSNGVT (SEQ ID NO.18); and CDR3 is AAGURVGAPALLPKSYRY (SEQ ID NO.19).

[0068] HBM-C13 (SEQ ID NO.9): QLQLVESGGGSVQAGGSLRLSCAASGDSSWNLCMGWFRQTPGKEREGVAGIYTGGPNTYYADSVKGRFTISQDNAKNTVYLQMDGLKPEDTAMYYCAARVCLSGDWLSPKLKYTYWAQGTQVTVSS.

[0069] Among them, in the amino acid sequence such as SEQ ID NO.9, CDR1 of HBM-C13 is GDSSWNLC (SEQ ID NO.20); CDR2 is IYTGGPNT (SEQ ID NO.21); and CDR3 is AARVCLSGDWLSPKLKYTY (SEQ ID NO.22).

[0070] HBM-C14 (SEQ ID NO.10): QVQLVESGGGSVQAGGSLRLSCAASGDSSWNLCMGWFRQAPGKEREGVAGIYTGGRNTYYADSVKGRFAISQDNAKNTVHLQMDGLKPEDTAMYYCAARVCLSGSWLSPDLKYTYWAQGTQVTVSS.

[0071] Among them, in the amino acid sequence such as SEQ ID NO.10, CDR1 of HBM-C14 is GDSSWNLC (SEQ ID NO.20); CDR2 is IYTGGRNT (SEQ ID NO.23); and CDR3 is AARVCLSGSWLSPDLKYTY (SEQ ID NO.24).

[0072] HBM-C15 (SEQ ID NO.11): QLQLVESGGGLVQPGGSLRLSCAVPGYTFSSERIGWFRQAPGKEREGVAIIVPSNGVTYYTDSVKGRFTISQDNAKNTLFLQMNSLKPEDTATYYCAAGWRVGAPALLPKSYRYWGQGTQVTVSS.

[0073] Among them, in the amino acid sequence such as SEQ ID NO.11, CDR1 of HBM-C15 is GYTFSSER (SEQ ID NO.17); CDR2 is IVPSNGVT (SEQ ID NO.18); and CDR3 is AAGURVGAPALLPKSYRY (SEQ ID NO.19).

[0074] HBM-C16 (SEQ ID NO.12): QVQLVDSGGGSAQAGGSLRLSCAASGYTFSSERIGWFRQAPGKEREGVAIIVPSNGVTYYTDSVKGRFTISQDNAKNTLFLQMNSLKPEDTATYYCAAGWRVGAPALLPKSYRYWGQGTQVTVSS.

[0075] Among them, in the amino acid sequence such as SEQ ID NO.12, CDR1 of HBM-C16 is GYTFSSER (SEQ ID NO.17); CDR2 is IVPSNGVT (SEQ ID NO.18); and CDR3 is AAGRVGAPALLPKSYRY (SEQ ID NO.19).

[0076] HBM-C17 (SEQ ID NO.13): QLQLVESGGGSVQAGGSLRLSCAASGITVSRRYMTWFRQAPGKEREGVASIYIGDGHTDYADSVKGRFTISQDNAKNTLYLRMNSLKPEDTAMYYCAAKLRSGSNWGAWYWPHEYIYWGQGTQVTVSS.

[0077] Among them, in the amino acid sequence such as SEQ ID NO.13, CDR1 of HBM-C17 is GITVSRRY (SEQ ID NO.25); CDR2 is IYIGDGHT (SEQ ID NO.26); and CDR3 is AAKLRSGSNWGAWYWPHEYIY (SEQ ID NO.27).

[0078] HBM-C18 (SEQ ID NO.14): QVQLVESGGGSVQAGGSLRLSCAASGITVSRRYMTWFRQAPGKEREGVASIYIGDGHTDYADSVKGRFTTSQDNAKNTLYLRMNSLKPEDTAMYYCAAKLRSGSNWGAWYWPHEYIYWGQGTQVTVSS.

[0079] Among them, in the amino acid sequence such as SEQ ID NO.14, CDR1 of HBM-C18 is GITVSRRY (SEQ ID NO.25); CDR2 is IYIGDGHT (SEQ ID NO.26); and CDR3 is AAKLRSGSNWGAWYWPHEYIY (SEQ ID NO.27).

[0080] HBM-C19 (SEQ ID NO.15): QLQLVESGGGSVQAGGSLLRLSCTASTSSSPNLCMGWFRQAPGKEREGVAGIYTGGRNTYYADSVKGRFTISQDNVKNTVYLQMDGLKPEDTAMYYCAARVCLSGSWLSAELKYTYWGQGTQVTVSS.

[0081] Among them, in the amino acid sequence such as SEQ ID NO.15, the CDR1 of HBM-C19 is TSSSPNLC (SEQ ID NO.28); the CDR2 is IYTGGRNT (SEQ ID NO.23); and the CDR3 is AARVCLSGSWLSAELKYTY (SEQ ID NO.29).

[0082] HBM-C20 (SEQ ID NO.16): QVQLVESGGGSVQAGGSLRLSCAASGITVSRRYMTWFRQAPGKEREGVASIYIGDGHTDYADSVKGRFTISQDNAKNTLYLRMNSLKPEDTAMYYCAAKLRSGSNWGAWYWPHEYIYWGQGTQVTVSS.

[0083] Among them, in the amino acid sequence such as SEQ ID NO.16, CDR1 of HBM-C20 is GITVSRRY (SEQ ID NO.25); CDR2 is IYIGDGHT (SEQ ID NO.26); and CDR3 is AAKLRSGSNWGAWYWPHEYIY (SEQ ID NO.27).

[0084] After performing 12.5% ​​SDS-PAGE gel electrophoresis on the above eluent, Coomassie Brilliant Blue staining was performed, and the purity of the obtained HBM-C12~HBM-C20 was detected using a gel imaging system (Tanon 1600). The results are shown in Table 5.

[0085] Table 5 Purity results of different nanobodies

[0086] The results in Table 5 show that the purified anti-CD47 nanobodies prepared in this invention have relatively high purity. Except for HBM-C17, the purity of the other numbered anti-CD47 nanobodies after purification is above 60%, especially HBM-C16 and HBM-C18, which have a purity of 100%.

[0087] Example 3 PL-CRISPR-CAOV4 and KO-CD47 cells were digested and resuspended in culture medium. 100 μL / well was seeded into 96-well U-shaped plates, and 1 μg / mL of the CD47 nanoantibody (HBM-C12~HBM-C20) prepared in Example 2 was added to each well. After mixing, the plates were incubated at 37°C in a 5% CO2 incubator for 15 min. After incubation, the 96-well U-shaped plates were centrifuged at 1200 rpm for 5 min, the plates were shaken, the supernatant was discarded, and 100 μL of FACS was added to wash twice. After centrifugation at 1200 rpm for 5 min, the plates were shaken, and the supernatant was discarded. 50 μL of 1 μg / mL anti-HA (Biolegend, 901518, B390917, 0.2 mg / mL) was added to each well, and the plates were gently mixed. The plates were stained at 4°C for 15 min. After staining, the plates were centrifuged at 1200 rpm for 5 min, the plates were shaken, and 100 μL of FACS buffer was added to each well to resuspend the cells. After centrifugation at 1200 rpm for 5 min, the plates were shaken, and 100 μL of FACS buffer was added to each well to resuspend the cells. Cells were resuspended in μL of FACS buffer for flow cytometry and data analysis.

[0088] Among them, PL-CRISPR-CAOV4 cells are CD47-unknockout CAOV4 cells, serving as control cells; KO-CA47 cells are CD47 knockout cells.

[0089] Construction of PL-CRISPR-CAOV4 cells: pLCRISPR-CMV plasmid (purchased from Addgene, 102609) was mixed with psPAX2 helper plasmid (Miaoling Biotechnology, P0261) and pMD2.G helper plasmid (Miaoling Biotechnology, P0262) in a specific ratio and analyzed by Lipofectamine. TM Lentiviral fluid was obtained by transfecting 293T cells with lentivirus 3000 (Thermo Fisher Scientific, L3000015). PL-CRISPR-CAOV4 cells were constructed by infecting CAOV4 cells with the lentivirus.

[0090] Construction of KO-CD47 cells: gRNA was amplified using Human CD47-Fw 5'-CACCGTAAATATAGATCCGGTGGTA-3' (SEQ ID NO.30) and CD47-Rw 5'-AAACTACCACCGGATCTATATTTAC-3' (SEQ ID NO.31). The gRNA was then ligated with pLCRISPR-CMV after enzyme digestion to obtain the pLCRISPR-CMV-gRNA plasmid. The pLCRISPR-CMV-gRNA plasmid was mixed with psPAX2 helper plasmid (Miaoling Biotechnology, P0261) and pMD2.G helper plasmid (Miaoling Biotechnology, P0262) in a specific ratio and analyzed using Lipofectamine.TM Lentiviral fluid was obtained by transfecting 293T cells with lentivirus 3000 (Thermo Fisher Scientific, L3000015). KO-CD47 cells were constructed by infecting CAOV4 cells with the lentivirus.

[0091] Figure 1 The results showed that the 10 sequences of the CD47 nanobody HBM-C12 to HBM-C20 could specifically bind to CD47 on the surface of PL-CRISPR-CAOV4 cells.

[0092] Example 4 Ligand competition experiment CD47 OE cells were digested and resuspended in culture medium. 50 μL / well was seeded into 96-well U-shaped plates, and 10 μL of 0.1 mg / mL SIRPα (Bepsys, SIA-H5251, MW 63.9 kDa) and 0.3 μg of the CD47 nanobodies (HBM-C12~HBM-C20) prepared in Example 2 were added at a molar ratio of 1:1. After mixing, incubate at 37°C in a 5% CO2 incubator for 20 min. After incubation, centrifuge the 96-well U-shaped plate at 1200 rpm for 5 min, discard the supernatant, and wash once with 100 μL PBS. Centrifuge at 1200 rpm for 5 min, discard the supernatant, and add 50 μL of flow cytometry antibody dilution buffer (anti-HA (Biolegend, 901518, 0.2 mg / mL) and anti-human IgG Fc (Biolegend, 410711) to each well, mix gently, and stain at 4°C for 15 min. After staining, centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the cells in 100 μL of FACS buffer in each well. Centrifuge at 1200 rpm for 5 min, discard the supernatant, and resuspend the cells in 100 μL of FACS buffer in each well for flow cytometry and data analysis.

[0093] CD47 OE cells are cells that overexpress CD47. The construction of CD47 OE cells includes: using the target plasmid pLV3-CMV-CD47(human)-3×FLAG-CopGFP-Puro (purchased from Miaoling Biotechnology, P60560) to infect 293T cells (Pronosai, CL-0005) with lentivirus to construct PCDH-CD47 OE-293T cells, which are CD47 OE cells.

[0094] The nanobody competition rate is calculated as follows: (Median fluorescence intensity of CD47 OE cells APCs with only ligand added - Median fluorescence intensity of CD47 OE cells APCs with both ligand and CD47 nanobody added) ÷ Median fluorescence intensity of CD47 OE cells APCs with only ligand added × 100%. The ligand is SIRPα.

[0095] Table 6. Ligand competition rates of different CD47 nanobodies

[0096] Figure 2 The results in Table 6 show that CD47 nanobodies HBM-C12~HBM-C17 and HBM-C19~HBM-C20 all have ligand competition ability, and HBM-C12~HBM-C15, HBM-C17 and HBM-C19~HBM-C20 have high ligand competition rates, which meet the requirements of ligand competition.

[0097] Example 5 (1) Affinity test Remove CD47 antigen (0.565 mg / mL, 1:1 glycerol) from -20℃, thaw at room temperature, vortex for more than 30 seconds, add an appropriate amount of PBS, vortex to mix and dilute to the final concentration: 0.5 μg / mL; add 100 μL of 0.5 μg / mL CD47 antigen to each well in rows A to F of the ELISA plate, seal with sealing film, and incubate overnight at 4℃. The next day, the liquid in the wells of the ELISA plate was removed and the plate was cleaned. The plate was washed three times with PBST for 1 minute each time, and the liquid was cleaned after the last wash. Each well of the plate was then blocked with 200 μL of 3% BSA solution (prepared with CB solution) and incubated at room temperature for 1 hour. After blocking, the liquid in the wells was removed and the plate was cleaned. The plate was washed three times with PBST for 1 minute each time, and the liquid was cleaned after the last wash. The CD47 nanobodies (HBM-C12~HBM-C20) and CD47 flow cytometry antibody (Biolegend, Cat#323124) prepared in Example 2 were diluted 3-fold with PBS to obtain 11 concentrations. In rows AC + G (excluding column 12), 100 μL of nanobodies 1 (one of HBM-C12~HBM-C20) was added sequentially from high to low concentration. In rows DF + H (excluding column 12), 100 μL of nanobodies 1 was added sequentially from high to low concentration. Add 100 μL of PBS to column 12 of the microplate containing 2 μL nanobody antibodies (one of HBM-C12~HBM-C20) and incubate at room temperature with shaking for 1 h. After incubation, remove the liquid from the wells and blot dry. Wash three times with PBST for 1 min each time, and blot dry after the last wash. Secondary antibody incubation: Add 100 μL of anti-HA-Peroxidase (Roche, 120138190001, diluted 1:600 ​​with PBS) and incubate at room temperature with shaking for 1 h. After incubation, remove the liquid from the wells and blot dry. Wash three times with PBST for 1 min each time, and blot dry after the last wash. Prepare the colorimetric solution: A solution:B solution = 1:1, 100 μL / well, and develop color at room temperature for 10 min. Add 50 μL of 2 mol / L H2SO4 / well to stop the colorimetric reaction and read the value at 450 nm using a microplate reader.

[0098] (2) Detection of dissociation equilibrium constant (KD) The binding constant (Ka), dissociation constant (Kdis), and dissociation equilibrium constant (KD) of the CD47 nanobodies (HBM-C12~HBM-C20) prepared in Example 2 were determined using a molecular interaction analyzer (Sartorius, Octet R8).

[0099] The CD47 nanobodies (HBM-C12~HBM-C20) and CD47 flow cytometry antibody (Biolegend, Cat#323124) prepared in Example 2 were diluted with PBST to 1.5 μg / mL (100 nM).

[0100] Immobilize biotinylated human CD47 antigen (final concentration 5 μg / mL). Set the program as follows: buffer is 200 μL of the same buffer as the sample (PBST); sample wells contain 200 μL; regeneration wells contain 200 μL of 10 mM Gly-HCl (pH 2.45); baseline 60 s, binding 120 s, dissociation 60 s; regeneration conditions are default. Run the program for kinetic analysis. After the program finishes running, open Octet Analysis Studio 13.1 data analysis software and obtain the KD value according to the kinetic analysis program.

[0101] Figures 3-5 The results in Table 7 show that the EC50 value of the CD47 flow cytometry antibody was 28.3 ng / mL, and the EC50 values ​​of HBM-C12~HBM-C14 were 14.8 ng / mL, 23.5 ng / mL, and 25.4 ng / mL, respectively. This indicates that the CD47 nanobodies HBM-C12~HBM-C14 provided by this invention have superior binding activity and meet the requirements of high affinity. Furthermore, other CD47 nanobodies of this invention can also bind to CD47.

[0102] Table 7. Results of EC50, ka, kdis, and KD values ​​for different CD47 nanobodies.

[0103] Example 6 Half-inhibition rate (IC50) detection The IC50 of the CD47 nanobodies (HBM-C12~HBM-C20) prepared in Example 2 was determined using a molecular interaction analyzer (Sartorius, Octet R8).

[0104] The CD47 nanobodies (HBM-C12~HBM-C20) prepared in Example 2 were diluted with PBST to 9 μg / mL (final concentration 4.5 μg / mL, 300 nM), and then diluted 2-fold to obtain a total of 8 concentrations (including 0 μg / mL); the commercial monoclonal antibodies Ligufalimab (MCE, HY-P99706, MW 144.58 kDa) and Evorpacept (MCE, HY-P99950, MW 76.61 kDa) were diluted with PBST to 600 nM (final concentration 300 nM), and then diluted 2-fold to obtain a total of 8 concentrations (including 0 μg / mL); the CD47 antigen (1.13 mg / mL, MW 14 kDa) was diluted with PBST to 2.8 μg / mL (final concentration 1.4 μg / mL, 100 nM).

[0105] Immobilize biotinylated protein SIRPα-Fc, Avitag (Bepsys, CDA-H82F2) (final concentration 5 μg / mL). Set the program as follows: buffer 200 μL (same buffer as the sample), 200 μL per well, regeneration well 200 μL 10 mM Gly-HCl (pH 2.0), baseline 60 s, binding 120 s, dissociation 60 s, regeneration conditions default, and run the program for kinetic analysis. After the program finishes running, open Octet Analysis Studio 13.1 data analysis software and obtain the IC50 value based on the concentration response program.

[0106] Table 8 IC50 values ​​of different CD47 nanobodies

[0107] Figures 6-8 The results in Table 8 show that the IC50 value of Ligufalimab is 21.7610 nM, the IC50 value of Evorpacept is 16.5553 nM, and the IC50 values ​​of CD47 nanobodies HBM-C12~HBM-C17 and HBM-C19~HBM-C20 are 84.9486~572.4965 nM, indicating that CD47 nanobodies HBM-C12~HBM-C17 and HBM-C19~HBM-C20 can block the binding of CD47 antigen to SIRPα.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A CD47 Nanobody, characterized in that, complementarity determining regions consisting of CDR1, CDR2 and CDR3; CDR1 as shown in SEQ ID NO. 17, CDR2 as shown in SEQ ID NO. 18, and CDR3 as shown in SEQ ID NO. 19; or CDR regions having at least 85% sequence similarity to any one of SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19; CDR1 as shown in SEQ ID NO. 20, CDR2 as shown in SEQ ID NO. 21, and CDR3 as shown in SEQ ID NO. 22; or CDR regions having at least 85% sequence similarity to any one of SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22; CDR1 as shown in SEQ ID NO. 20, CDR2 as shown in SEQ ID NO. 23, and CDR3 as shown in SEQ ID NO. 24; or CDR regions having at least 85% sequence similarity to any one of SEQ ID NO. 20, SEQ ID NO. 23 and SEQ ID NO. 24; CDR1 as shown in SEQ ID NO. 25, CDR2 as shown in SEQ ID NO. 26, and CDR3 as shown in SEQ ID NO. 27; or CDR regions having at least 85% sequence similarity to any one of SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27; CDR1 as shown in SEQ ID NO. 28, CDR2 as shown in SEQ ID NO. 23, and CDR3 as shown in SEQ ID NO. 29; or CDR regions having at least 85% sequence similarity to any one of SEQ ID NO. 28, SEQ ID NO. 23 and SEQ ID NO.

29.

2. The CD47 Nanobody according to claim 1, characterized in that, The amino acid sequence of the CD47 nanobody is shown in one or more of SEQ ID NO. 8~SEQ ID NO. 16; Or the amino acid sequence of the CD47 nanobody is any one or more of the amino acid sequences having at least 85% sequence similarity to SEQ ID NO. 8~SEQ ID NO.

16.

3. Nucleotide encoding the CD47 nanobody of claim 1 or 2.

4. A biomaterial, characterized by, The biological material comprises an expression vector or a host cell; the expression vector comprises the nucleotide of claim 3; the host cell contains the nucleotide or expression vector of claim 3.

5. A method of preparing a CD47 Nanobody, characterized in that, Comprising the following steps: The expression vector of claim 4 is transformed into a host cell for culture, then induced for expression, and the culture containing the CD47 nanobody is obtained, then separated and purified from the culture containing the CD47 nanobody to obtain the CD47 nanobody.

6. An antibody conjugate, characterized in that, The CD47 nanobody of claim 1 or 2 and a conjugating agent; The conjugating agent comprises an immunoglobulin Fc domain, a His tag, a GST tag, a MBP tag, a FLAG tag, a SUMO tag, a Strep-tag II tag, a Twin-Strep-tag tag, a MyC tag, a HA tag, an AviTag tag, a HaloTag tag, an EGFP tag, a SNAP tag, a V5 tag, a thioredoxin tag, a GFP tag, a mCherry tag, a protein C tag, a BCCP tag, a Spot-tag tag, an Isopeptag, a SpyTag, a serum albumin, an albumin-binding polypeptide, a prealbumin, a carboxy-terminal peptide, an elastin-like polypeptide, a cytokine, a single-chain antibody, a virus-like particle, a ligase, a kinase, a prodrug-activating enzyme, a chemotherapeutic agent, a gold nanoparticle, or a liposome.

7. A medicament or pharmaceutical composition for preventing and / or treating a tumor, characterized by, The CD47 nanobody of claim 1 or 2 or the antibody conjugate of claim 6.

8. A kit for detecting CD47, characterized by, The kit comprises the CD47 nanobody of claim 1 or 2 or the antibody conjugate of claim 6.

9. A medicament or pharmaceutical composition for immunomodulation or targeting of CD47 binding, characterized in that, The pharmaceutical or pharmaceutical composition comprises an effective ingredient that interacts with CD47; the effective ingredient comprises the CD47 nanobody of claim 1 or 2 or the antibody conjugate of claim 6.

10. Use of the CD47 nanobody of claim 1 or 2, the CD47 nanobody prepared by the method of claim 5, or the antibody conjugate of claim 6 in the preparation of at least one of the following (1)~(6): (1) a diagnostic reagent or kit for detecting CD47; (2) a molecular probe or detection reagent that binds to CD47; (3) a drug that enhances macrophage phagocytosis; (4) an anti-tumor drug or pharmaceutical composition; (5) a drug or pharmaceutical composition for immune regulation; (6) a targeted drug or pharmaceutical composition that binds to CD47.

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