Anti-CD39 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an anti-CD39 nano antibody and application thereof. The anti-CD39 nano antibody has an amino acid sequence as shown in SEQ ID NO.1, 5, 9, 13, 17, 21 or 25. The anti-CD39 nano antibody of some examples has good antigen specificity, can be effectively combined with a target antigen CD39, and can effectively block or inhibit the hydrolysis of ATP (adenosine triphosphate) by the CD39, so that the depletion of T cells by a tumor microenvironment is effectively inhibited, and the curative effect of tumor immunotherapy is expected to be improved. The anti-CD39 nano antibody of some examples of the invention can further improve the curative effect of T cell immunotherapy related to CAR-T, TCR-T, TIL, TAL, NK, CIK and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-CD39 nanobody and its application. Background Technology

[0002] CD39, also known as exonucleoside triphosphate diphosphate hydrolase 1 (ENTPDase 1), is widely expressed in various tissues and organs, such as the bladder, brain, breast, colon, uterus, stomach, and prostate, and is mainly expressed on endothelial cells and immune cells. CD39 is highly expressed in various human tumors, including lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer.

[0003] In addition, CD39 plays a crucial role in the tumor microenvironment (TME). Extracellular ATP (eATP) is a pro-inflammatory metabolite found in the intercellular space. It can bind to receptors on T cells, promoting T cell activation and proliferation. Therefore, the hydrolysis of eATP inhibits T cell function. Furthermore, AMP can be further hydrolyzed into adenosine by CD73. Adenosine binds to the A2A receptor on the CD8+ TIL membrane, leading to increased expression levels of various inhibitory receptors, reduced secretion of cytokines, decreased proliferation and effector capacity, ultimately inhibiting the response of T cells and NK cells, and suppressing the anti-tumor response. CD39 can hydrolyze eATP into adenosine, thereby inhibiting the function of CD8+ TILs, leading to T cell exhaustion.

[0004] Given the crucial role of CD39 in the tumor microenvironment, it has become an emerging target for researchers developing tumor immunotherapy. Targeting CD39 in the tumor microenvironment offers dual anti-tumor immunomodulatory effects: ① by inhibiting ATP hydrolysis, it increases ATP levels in the TME, which have pro-inflammatory and pro-cell proliferation effects; ② by inhibiting the accumulation of downstream product ADO, it reverses the immunosuppressive effects directly mediated by ADO receptors and the long-established immunosuppressive tumor microenvironment. CD73 antagonists or A2ARi only focus on reversing the immunosuppressive effects of adenosine (ADO), underestimating the role of eATP; CD39-targeted therapies, however, have the potential to regulate tumor immune responses from multiple perspectives. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and provide an anti-CD39 nanobody and its application.

[0006] The technical solution adopted in this invention is: A first aspect of the present invention provides: an anti-CD39 nanobody, the anti-CD39 nanobody comprising a complementarity-determining region (CDR), the CDR comprising complementarity-determining region CDR1, complementarity-determining region CDR2, and complementarity-determining region CDR3. The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 6 to SEQ ID NO. 8, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.14 to SEQ ID NO.16, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.18 to SEQ ID NO.20, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.12 to SEQ ID NO.24, respectively; or The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.18, SEQ ID NO.26 and SEQ ID NO.27, respectively.

[0007] In some instances, the amino acid sequences of the CD39 nanobody are as shown in SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21 or SEQ ID NO.25, or amino acid sequences thereof modified by substitution, deletion or addition of one or more amino acids and having the same or similar function.

[0008] A second aspect of the present invention provides: a fusion protein comprising a first domain and a second domain, wherein the first domain is the anti-CD39 nanobody described in the first aspect of the present invention.

[0009] In some instances, the second domain is used to extend the half-life of the first domain in vivo.

[0010] In some instances, the second domain is a secretion signal peptide. Antibodies expressed in this way can be more effectively excreted.

[0011] In some instances, the second domain includes, but is not limited to, serum albumin (such as human HSA) or fragments thereof, a domain that binds to serum albumin (such as anti-serum albumin antibodies, including nanobodies), polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof; and / or Molecules that have an affinity for T cell surface molecules and / or are able to bind to surface molecules (such as CD3) present on T cells.

[0012] In some instances, the human immunoglobulin Fc region includes mutations that alter Fc-mediated effector functions, including one or more combinations of CDC activity, ADCC activity, and ADCP activity.

[0013] In some instances, the immunoglobulin is one or more combinations selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0014] In some instances, the IgG is selected from one or more of the IgG1, IgG2, IgG3, or IgG4 subtypes.

[0015] In some instances, the amino acid sequence of the Fc region of the immunoglobulin is as shown in SEQ ID NO.28; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO.28 after substitution, deletion, or addition of one or more amino acids.

[0016] In some instances, the first and second domains are directly or interconnected via connectors.

[0017] In some instances, the linker is a flexible linker; preferably, the flexible linker is selected from the hinge region of the antibody, YAPVDV, GSAS, or (GGCAGCGCCAGC). n (GGCGGCGGCAGC) n (GGCGGCGGCGGCAGC) n (GGGS) n (GGSG) n (GGGGS) n (G) n At least one of the following, where n is an integer not less than 1, preferably, n is an integer between 1 and 10, or between 1 and 5.

[0018] In some instances, the amino acid sequence of the hinge region is as shown in SEQ ID NO.29, or an amino acid sequence thereof that is functionally identical or similar after substitution, deletion or addition of one or more amino acids.

[0019] In some instances, the amino acid sequence of the secretion signal peptide is shown in SEQ ID NO.30.

[0020] The above features can be combined arbitrarily as long as they do not conflict with each other.

[0021] A third aspect of the invention provides: a biomaterial selected from any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-CD39 nanobody of the first aspect of the present invention or the fusion protein of the second aspect of the present invention; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecule described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

[0022] In some instances, the transgenic animal cell lines do not contain reproductive material.

[0023] In some instances, the 5' transcriptional control region contains a promoter (which may be a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeper" promoter), a transcription start site, an enhancer, and / or a silencing element.

[0024] In some instances, the 3'UTR may encode AU-rich elements, which, via the 3'-5' exosome pathway, are common regulators of mRNA stability and are typically located in the 3'UTR. AU-rich elements may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements with the sequence AUAUAU.

[0025] In some instances, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0026] In some instances, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0027] In some instances, the viral vector includes at least one of lentiviral vectors, adenovirus vectors, baculovirus vectors, retroviral vectors, poxvirus vectors, Sendai virus vectors, and herpes simplex virus vectors.

[0028] In some instances, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0029] In some instances, the vector is a plasmid vector, phage particle, viral vector, cell vector, bacteriophage, sclerotium, F sclerotium, or artificial chromosome.

[0030] In some instances, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0031] In some instances, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0032] In some instances, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal species.

[0033] In some instances, the prokaryotic cells include bacteria well-known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which can be used to express the target protein.

[0034] In some instances, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0035] In some instances, the cells include engineered cell lines such as CHO, CHO-K1, and CHO-GS, and lymphocyte lines such as T cells, NK cells, and CIK cells.

[0036] A fourth aspect of the present invention provides: a biological agent comprising: an anti-CD39 nanobody or conjugate thereof according to the first aspect of the present invention, a fusion protein or conjugate thereof according to the second aspect of the present invention, and a biological material according to the third aspect of the present invention.

[0037] In some instances, the conjugates include, but are not limited to, cytotoxins, detectable markers, bioactive proteins, molecules that target tumor surface markers, molecules that inhibit tumors, molecules that target immune cell surface markers, extracellular hinge regions, transmembrane regions, and intracellular signaling regions based on chimeric antigen receptor technology.

[0038] In some instances, the bioactive proteins include molecules that target tumor surface markers, molecules that inhibit tumors, molecules that target surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, cytokines, antibodies, antibody Fc fragments, and antibody scFv fragments.

[0039] In some instances, the molecules that target tumor surface markers are antibodies or ligands that bind to tumor surface markers.

[0040] In some instances, the tumor-inhibiting molecules are anti-tumor cytokines or anti-tumor toxins. In some instances, the detectable markers include, but are not limited to, radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, and nanoparticles.

[0041] In some instances, the detectable markers include, but are not limited to, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents or enzymes capable of producing detectable products.

[0042] In some instances, the nanoparticles include, but are not limited to, gold nanoparticles / nanorods, magnetic nanoparticles, liposomes, viral particles, and pseudovirus particles.

[0043] In some instances, the biological agent further includes acceptable excipients. Preferably, the excipients include, but are not limited to, fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, colorants, fragrances, and solvents.

[0044] In some instances, the biological agent also includes combination drugs, including but not limited to immune effector molecules, cells, cytotoxic substances, and multi-kinase inhibitors.

[0045] These features can be combined arbitrarily as long as they do not conflict with each other.

[0046] A fifth aspect of the present invention provides the use of the anti-CD39 nanobody described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the biomaterial described in the third aspect of the present invention, or the biological agent described in the fourth aspect of the present invention in the preparation of a formulation, kit, or drug for the diagnosis, treatment, or prevention of cancer.

[0047] In some instances, the cancers include lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer.

[0048] The beneficial effects of this invention are: The anti-CD39 nanobodies of some examples of the present invention have good antigen specificity, can effectively bind to the target antigen CD39, can effectively block or inhibit CD39 hydrolysis of ATP, and thus effectively inhibit the depletion of T cells by the tumor microenvironment, which is expected to improve the efficacy of tumor immunotherapy.

[0049] Some examples of the anti-CD39 nanobody of the present invention can further improve the efficacy of T-cell immunotherapy involving CAR-T, TCR-T, TIL, TAL, NK, CIK, etc. Attached Figure Description

[0050] Figure 1 Anti-CD39 fusion antibody structure.

[0051] Figure 2 Flow cytometry report on the construction of CD39 target antigen cell lines.

[0052] Figures 3-9 Flow cytometry report of specific detection of anti-CD39 supernatant in jurkaat target cells.

[0053] Figure 10 :jurkat target cells: Specific detection results of Anti-CD39 supernatant.

[0054] Figures 11-18 CD39 antibody blocking ability flow cytometry detection report.

[0055] Figure 19 Anti-CD39 blocks the hydrolysis of ATP. Detailed Implementation

[0056] The present invention will be further described in detail below through specific embodiments.

[0057] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments. Example 1: Plasmid construction (including mutation of the target fragment)

[0060] Multiple candidate nanobody sequences were obtained by immunizing alpacas with the human CD39 target antigen (amino acid sequence shown in SEQ ID NO. 31). Several high-titer anti-CD39 nanobodies were screened and identified as: FP858, FP859, FP860, FP861, FP862, FP863, and FP864. Analysis revealed: The amino acid sequence of FP858 is shown in SEQ ID NO.1, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively. The amino acid sequence of FP859 is shown in SEQ ID NO.5, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.6 to SEQ ID NO.8, respectively. The amino acid sequence of FP860 is shown in SEQ ID NO.9, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively. The amino acid sequence of FP861 is shown in SEQ ID NO.13, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.14 to SEQ ID NO.16, respectively. The amino acid sequence of FP862 is shown in SEQ ID NO.17, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.18 to SEQ ID NO.20, respectively. The amino acid sequence of FP863 is shown in SEQ ID NO.21, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.12 to SEQ ID NO.24, respectively. The amino acid sequence of FP864 is shown in SEQ ID NO.25, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.18, SEQ ID NO.26 and SEQ ID NO.27, respectively.

[0061] The VHH region nucleic acid sequence of the nanobody was ligated with the human IgG Fc nucleic acid sequence using multi-fragment homologous recombination technology. The linker region was the hinge. After homologous recombination, the plasmid was identified by enzyme digestion and then sequenced to analyze the fusion antibody sequence structure. Figure 1 As shown.

[0062] The sequence of the constructed fusion antibody is as follows: (1) Preparation and procedure of single-fragment amplification reaction system Prepare the single-fragment amplification reaction system as shown in Table 1, and carry out the reaction according to the reaction procedure shown in Table 2. Primers were designed using existing methods.

[0063] Table 1. Single-fragment amplification reaction system

[0064] Table 2. Reaction Procedure

[0065] (2) Multi-fragment homologous recombination preparation system and procedure Prepare the multi-fragment homologous recombination reaction system as shown in Table 3. Perform the multi-fragment recombination reaction at 50°C for 15 min; then cool to 4°C or immediately place on ice to cool.

[0066] Table 3. Multi-fragment homologous recombination reaction system

[0067] The above-mentioned carriers involve nanobodies with multiple clone numbers of the human CD39 target antigen linked to IgG1 Fc (SEQ ID NO. 28) via hinge (SEQ ID NO. 29), and their fusion antibody structures are as follows. Figure 1 . Example 2: Plasmid Extraction

[0068] (1) Transform the vector with the correct sequence alignment into a plasmid at 37°C for 14 h. (2) Pick single colonies, culture a small amount of bacterial solution, culture volume 4 mL Amp+ LB (100 mg / L Amp+), culture at 37℃, 220 rpm for 10 h; (3) Large-scale culture of bacterial culture: The next day, the seed culture was inoculated into 250mL Amp+ LB (100mg / L Amp+) and cultured at 37 ℃ and 220rpm for 14 h. (4) Plasmid extraction was performed using the Tiangen plasmid large-scale extraction kit: S1) Add the bacterial culture to a collection bottle (500 mL), centrifuge at 4000 rpm for 30 min, and remove as much supernatant as possible; S2) Column equilibration step: Add 2.5 mL of equilibration solution BL to the adsorption column CP6 (the adsorption column is placed in a 50 mL collection tube); S3) Add 10 mL of solution P1 to the bacterial precipitate to resuspend the bacterial cells; add 10 mL of solution P2 to the bacterial suspension, and gently invert 6-8 times to allow the bacterial cells to fully lyse. S4) Add 10 mL of solution P4 to the centrifuge tube, gently invert 6 to 8 times to mix thoroughly until a white, dispersed flocculent precipitate appears in the solution; S5) Centrifuge the lysate at 3580 × g for 30 min; S6) Carefully pour the supernatant lysis buffer into filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50 mL tube; S7) Add 3 mL of red endotoxin-free solution ER to each solution, mix by inverting, and the solution will turn into a uniform transparent yellow. S8) Add 0.3 times the volume of the above filtrate in isopropanol, mix by inverting the container, and then transfer it to the adsorption column CP6. S9) Centrifuge at 8228×g for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube; S10) Add 10 mL of buffer ED to the adsorption column CP6, centrifuge at 8228×g for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; repeat this step. S11) Add 10 mL of washing buffer PW to the adsorption column CP6, centrifuge at 8228×g for 2 min, discard the waste liquid in the collection tube, put the adsorption column back into the collection tube, and repeat the operation steps. S12) Place the adsorption column CP6 back into the collection tube, centrifuge at 8228×g for 5 min, then open the cap of the adsorption column CP6 and place it at room temperature for several minutes to thoroughly dry the residual rinsing solution in the adsorption material. S13) Place the adsorption column CP6 in a clean 50 mL collection tube, add 1-2 mL of elution buffer TB to the middle of the adsorption membrane, incubate at room temperature for 5 min, and then centrifuge at 8228×g for 5 min at room temperature. S14) Take 1 μL of plasmid DNA to determine the concentration (zero with Buffer TE), aliquot the plasmid DNA into 1.5 mL centrifuge tubes, and label the name, concentration, batch number and volume with a marker. Store at -20℃. Example 3: Antibody Production

[0069] (1) Observe the 293T cells used for packaging under a microscope and screen out cells with a cell density of 80% to 95%; (2) Prepare 10% packaged culture medium. Open a new bottle of 500 mL DMEM culture medium, add 55 mL FBS, then add 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mix well. (3) Take the screened cells out of the incubator, pour the old culture medium in the culture flask into the waste liquid container, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt; (4) Calculate the amount of plasmid to be added (30 μg plasmid per bottle) and 0.125 mol / L calcium chloride solution according to the number of culture bottles to be transfected, and prepare a DNA-CaCl2 mixture; then take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl2 mixture while vortexing the DNA-CaCl2 mixture; after the addition is complete, let it stand at room temperature for 20 min. (5) Remove the cells obtained in step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, put them back into the incubator, and change the medium with 5% packaged culture medium for 4-6 h. To prepare 5% packaged culture medium, open a new 500 mL bottle of DMEM medium, add 27.5 mL of FBS and 27.5 mL of gibco KnockOut™ (catalog number 10828028) serum substitute, then add 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mix well. (6) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25 mL of 5% packaged culture medium to each bottle, and then put them back into the incubator to continue culturing. After culturing for 72 h, harvest the supernatant to obtain the fusion antibody. Example 4: Preparation of Lentiviral Virus Overexpressing Target Antigen

[0070] (1) Observe the 293T cells used for packaging under a microscope and screen out cells with a cell density of 80% to 95%; (2) Prepare 10% packaged culture medium. Open a new 500ml bottle of DMEM culture medium, add 55ml of FBS, then add 550μl of 1mol / L sodium pyruvate solution and 550μl of 25mmol / L chloroquine phosphate solution, and mix well. (3) Take the screened cells out of the incubator, pour the old culture medium in the culture flask into the waste liquid container, add 17ml of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt; (4) Calculate the added expression plasmid, packaging helper plasmid, coating plasmid and 0.125 mol / L calcium chloride solution, and prepare DNA-CaCl2 mixture; then take an equal volume of 2×HBS solution and add it dropwise to DNA-CaCl2 mixture while vortexing the DNA-CaCl2 mixture; after the addition is complete, let it stand at room temperature for 20 min until a white precipitate forms; (5) Remove the cells obtained in (3) from the incubator, add 8 ml of calcium phosphate-DNA precipitation complex to each bottle, and mark the virus name and operation date on the bottle. Put it back into the incubator and change the medium with 5% packaged culture medium for 4-6 hours. (6) Prepare 5% packaged culture medium. Take a 500ml bottle of DMEM culture medium, add 27.5ml FBS and 27.5ml serum substitute, add 550μl 1 mol / L sodium pyruvate solution and 550μl 25mmol / L chloroquine phosphate solution, and mix well; (7) Take out (5) 293T cells to be replaced from the incubator, discard the old culture medium, add 25ml of 5% packaged culture medium to each bottle, and then put them back into the incubator to continue culturing. Harvest the virus after culturing for 42-48 hours. Example 5: Construction of target antigen overexpression cell lines

[0071] Resuscitating the Jurakat cell line: Remove the Jurakat cells to be resuscitated from the liquid nitrogen tank, thaw them rapidly in a 37°C constant temperature water bath, and then use a 10ml pipette to transfer the cell suspension to RM1640 medium containing 10ml of 10% FBS. Centrifuge at 1000rpm for 5min, discard the supernatant after centrifugation, resuspend the cell pellet in 10ml of fresh RM1640 medium containing 10% FBS, transfer it to T25, and incubate in a 37°C CO2 incubator. Passage the cells every two days at a ratio of 1:3. Cells that had been stably cultured for three generations from the start of resuscitation were seeded into 24-well plates at 4E5 cells / well. The harvested lentiviral supernatant was transduced at doses of 10 μL, 20 μL, and 40 μL. EGFP expression was detected by flow cytometry after 72 hours; the positive rate was greater than 85%. The positive rate of the ENTPD1 (CD39) target antigen overexpressing cells involved in this invention was 95.90%. Figure 2 As shown, the cell line was successfully constructed and can be used for subsequent antibody supernatant affinity property detection. Example 6: Antibody supernatant specificity test

[0072] (1) Collect 1 mL of the antibody supernatant obtained 72 h after transfection (Example 3) into a 1.5 mL centrifuge tube; (2) Centrifuge at 12,000 rpm for 5 min to remove cell debris, and then clarify using a 0.22 μm filter; (3) Cell staining: Take CD39-jurkat cells and jurkat cells to be stained by flow cytometry, wash with sodium chloride / PBS, add 50 μL of antibody supernatant from step (2), 200 μL, incubate at 4℃ for 30 min, and protect from light; (4) Wash the stained cells with sodium chloride, centrifuge, discard the supernatant, add anti-hunan-IG1-FC-APC flow cytometry antibody, incubate at 4℃ for 60 min, and protect from light; (5) Wash the cells to be tested with sodium chloride, centrifuge, add flow cytometry buffer, and then test.

[0073] Flow cytometry results showed that FP858, FP859, FP860, FP861, FP862, FP863, and FP864 did not bind to Jurakat cells. Figures 3 to 10 Similarly, after co-incubation with jurkat-CD39 cells, flow cytometry analysis was performed after 1 h of incubation with mouse anti-human anti-hunan-IG1-FC-APC secondary antibody. As shown in Table 4, the anti-CD39 nanobodies FP858, FP859, FP860, FP861, FP862, FP863, and FP864 all bound to jurkat-CD39.

[0074] The above conclusions indicate that the anti-CD39 nanobodies FP858, FP859, FP860, FP861, FP862, FP863, and FP864 specifically bind to jurkat-CD39.

[0075] Table 4. Binding test results of jurakat-CD39 target antigen cells and anti-CD39 fusion antibody. Example 7: ELISA Quantitative Detection of Antibody Supernatant

[0076] S1) Remove the ELISA plate from the aluminum foil bag after equilibration to room temperature for 20 minutes; S2) Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well; S3) Add 50 μL of the sample to be tested (gradient dilution, 1:100 / 1:1000 / 1:10000) to the sample wells, and do not add any sample to the blank wells; S4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min. S5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). S6) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min. S7) Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0077] The final quantitative results are shown in Table 5. Among the fusion antibodies, FP858, FP862, and FP861 showed the strongest expression.

[0078] Table 5. Quantitative results of anti-CD39 fusion antibody ELISA Example 8: Detection of binding ability of anti-CD39 fusion antibody after quantification

[0079] S1) Cell staining: CD39-jurkat cells and jurkat cells to be stained by flow cytometry were taken, washed with sodium chloride / PBS, and fusion antibody was added at 100ng, 25ng, 6.25ng, 0.391ng, and 0.156ng respectively. The cells were incubated at 4℃ for 30 min in the dark. S2) Wash the stained cells with sodium chloride, centrifuge, discard the supernatant, add anti-hunan-IG1-FC-APC flow cytometry antibody, incubate at 4°C for 60 min, and protect from light; S3) Wash the cells to be tested with sodium chloride, centrifuge, add flow cytometry buffer, and then perform the test.

[0080] The test results are shown in Tables 6 and 7. Figures 11-18 As shown, with the increase of antibody amount, the binding ability of anti-CD39 to the target antigen cell line gradually increases. Among them, FP859, FP860, FP861 and FP858 are more likely to bind to the target cells when the antibody amount is low, indicating that the affinity is better.

[0081] The final results showed that FP858, FP859, FP860, and FP861 were superior in terms of Anti-CD39 antibody affinity screening.

[0082] Table 6

[0083] Table 7 Example 9: Blocking ability test of anti-CD39 fusion antibody

[0084] After adjusting the density of Mino (CD39 positive) cells to 4E6 cells / mL, 50 μL of each cell was transferred to a 96-well U-bottom cell culture plate. Antibody was prepared with medium at 20 μg / mL as the starting material. Ten 3-fold dilutions were made, and 100 mL of antibody was added to each well of the cells. After mixing, the cells were incubated at 37°C for 60 min. ATP was prepared with medium at 200 mM and added to each well at 50 μL. After mixing, the cells were incubated at 37°C for 45 min. The cells were centrifuged at 350 G for 5 min, and 80 μL of the supernatant was transferred to a white plate. An equal volume of CellTiter-Glo® 2.0 Assay (promega G9242) was added, and the plates were incubated at room temperature in the dark for 10 min before analysis.

[0085] like Figure 19 The results showed that Anti-CD39 nanobodies FP858, FP859, FP860, and FP861 can effectively block the hydrolysis of ATP substrates by CD39 on Mino cells.

[0086] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An anti-CD39 nanobody, wherein the anti-CD39 nanobody comprises a complementarity-determining region (CDR), the CDR comprising complementarity-determining region CDR1, complementarity-determining region CDR2, and complementarity-determining region CDR3, characterized in that, The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 6 to SEQ ID NO. 8, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.14 to SEQ ID NO.16, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.18 to SEQ ID NO.20, respectively; or The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO.12 to SEQ ID NO.24, respectively; or The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.18, SEQ ID NO.26 and SEQ ID NO.27, respectively.

2. The CD39 nanobody according to claim 1, characterized in that, The amino acid sequences of the CD39 nanobody are shown in SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21 or SEQ ID NO.25, respectively, or amino acid sequences that are functionally identical or similar after substitution, deletion or addition of one or more amino acids.

3. A fusion protein comprising a first domain and a second domain, characterized in that, The first domain is the anti-CD39 nanobody as described in claim 1 or 2; preferably, the second domain is used to prolong the half-life of the first domain in vivo, or the second domain is a secretory signal peptide.

4. The fusion protein according to claim 3, characterized in that, The second domain is selected from at least one of the following: immunoglobulin Fc region, serum albumin or a fragment thereof, a domain that binds to serum albumin, polyethylene glycol, and polyethylene glycol-liposome complex; Preferably, the amino acid sequence of the Fc region of the immunoglobulin is as shown in SEQ ID NO.28; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO.28 after substitution, deletion or addition of one or more amino acids.

5. The fusion protein according to claim 3, characterized in that, The first and second structural domains are directly or interconnected through connectors.

6. The fusion protein according to claim 5, characterized in that, The connector is a flexible connector. Preferably, the flexible linker is selected from the hinge region of the antibody, YAPVDV, GSAS, and (GGCAGCGCCAGC). n (GGCGGCGGCAGC) n (GGCGGCGGCGGCAGC) n (GGGS) n (GGSG) n (GGGGS) n (G) n At least one of them, where n is an integer not less than 1, preferably, n is an integer between 1 and 10, or between 1 and 5; Preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO.29, or an amino acid sequence thereof that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar function.

7. A biomaterial, characterized in that, The biomaterial is selected from any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-CD39 nanobody as described in claim 1 or 2 or the fusion protein as described in any one of claims 3 to 6; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

8. A biological agent, characterized in that, include: One of the anti-CD39 nanobody or its conjugate as described in claim 1 or 2, the fusion protein or its conjugate as described in any one of claims 3 to 6, and the biomaterial as described in claim 7; Preferably, the conjugate includes at least one of cytotoxin, detectable marker, and bioactive protein; Preferably, the cytotoxic agent includes a prodrug-activating enzyme or a chemotherapeutic agent; Preferably, the bioactive protein includes molecules that target tumor surface markers, molecules that inhibit tumors, molecules that target immune cell surface markers, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, cytokines, antibodies, antibody Fc fragments, and antibody scFv fragments. Preferably, the detectable markers include radioactive isotopes, fluorescent substances, chemiluminescent markers, colored substances, MRI or CT contrast agents, enzymes capable of producing detectable products, viral particles, liposomes, and nanoparticles.

9. The use of the anti-CD39 nanobody of claim 1 or 2, the fusion protein of any one of claims 3 to 6, the biomaterial of claim 7, or the biological agent of claim 8 in the preparation of a formulation, kit, or drug for the diagnosis, treatment, or prevention of cancer.

10. The application according to claim 9, characterized in that, The cancers mentioned include lymphoma, sarcoma, lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, thyroid cancer, and testicular cancer.

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