An anti-CD27 nanoantibody and its application

Abstract

The present invention discloses an anti-CD27 nano antibody and its application; the nano antibody or its antigen binding fragment comprises a heavy chain variable region containing CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The anti-CD27 nano antibody or its antigen binding fragment has strong specificity and high affinity for CD27 antigen, and can be used for flow cytometry or Western Blot detection of CD27 and lymphocyte grouping, screening of drugs for treating tumors expressing CD27, and diagnosis or treatment of diseases and conditions related to CD27 activity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-CD27 nano antibody and an application thereof. Background Art

[0002] CD27 is a type I transmembrane protein and a member of the tumor necrosis factor receptor superfamily (TNFRSF27). It is mainly expressed as a surface antigen on most T cells, natural killer cells, antibody-secreting plasma cells, and memory B cells (Jacquot 2000). CD70 is the cognate ligand of CD27, also known as tumor necrosis factor ligand superfamily member 7 (TNFSF7) (Goodwin, Alderson et al. 1993, Wajant 2016).

[0003] Related studies have found that CD27 can not only regulate T cell-dependent B cell differentiation, but also induce apoptosis of different cells by binding to CD70 to form a CD27 / CD70 complex (Wajant 2016). In the cell, CD27 interacts with its ligand CD70 and recruits TRAF proteins to the intracellular domain of CD27, activating downstream signals, including canonical and non-canonical NF-κB signaling pathways and JNK signaling pathways (Hendriks, Xiao et al. 2003), and plays an important role in the proliferation, activation and cytotoxicity of CD8+T cells (Soares, Waechter et al. 2007). Agonistic antibodies to CD27 can significantly enhance the killing ability of T cells in mouse tumor models and have anti-tumor activity (van de Ven and Borst 2015). In addition to being expressed on normal cells, CD27 is also highly expressed in B-cell lymphoma and B-cell chronic lymphocytic leukemia cells (Feau, Garcia et al. 2012). Therefore, CD27 is often used as one of the targets for the diagnosis of multiple myeloma or non-small cell lung cancer or monitoring of clinical efficacy (Sakanishi and Yagita 2010, Ahrends, Bąbała et al. 2016). Therefore, developing a CD27 antibody with good biological activity has great research value and application prospects. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide an anti-CD27 nanobody or an antigen-binding fragment thereof.

[0005] The purpose of the second aspect of the present invention is to provide a bivalent anti-CD27 nanobody.

[0006] The third aspect of the present invention aims to provide a recombinant protein.

[0007] The purpose of the fourth aspect of the present invention is to provide a biomaterial related to the anti-CD27 nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, or the recombinant protein of the third aspect of the present invention.

[0008] The fifth aspect of the present invention aims to provide a conjugate.

[0009] The sixth aspect of the present invention aims to provide a solid phase carrier.

[0010] The purpose of the seventh aspect of the present invention is to provide the use of the anti-CD27 nanobody or its antigen-binding fragment of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, or the recombinant protein of the third aspect of the present invention, the biomaterial of the fourth aspect, the conjugate of the fifth aspect, and / or the solid phase carrier of the sixth aspect in the preparation of products.

[0011] The eighth aspect of the present invention aims to provide a product.

[0012] The purpose of the ninth aspect of the present invention is to provide a method for preparing the anti-CD27 nanobody or its antigen-binding fragment of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, or the recombinant protein of the third aspect of the present invention.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides an anti-CD27 nanobody or an antigen-binding fragment thereof, wherein the nanobody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0015] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0016] a1) SEQ ID NO: 11; or

[0017] a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO: 11 and which has the same function as the protein shown in SEQ ID NO: 1; or

[0018] a3) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identical to SEQ ID NO: 11 and has the same function as the protein shown in SEQ ID NO: 11.

[0019] In the present invention, the amino acid sequences of the above-listed CDRs are shown in accordance with the IMGT definition rules. However, it is well known to those skilled in the art that the CDR of an antibody can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to cover complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0020] The term "variable region" refers to the domain of the antibody heavy chain that is involved in antibody binding to antigen. VH consists of four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains 3 CDR regions: CDR1, CDR2 and CDR3. Each VH is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0021] The second aspect of the present invention provides a bivalent anti-CD27 Nanobody, wherein the bivalent Nanobody comprises the Nanobody as described in the present invention.

[0022] Preferably, the bivalent Nanobody further comprises a heavy chain constant region.

[0023] Preferably, the heavy chain constant region is murine IgG Fc.

[0024] Preferably, the amino acid sequence of mouse IgG Fc is as shown in SEQ ID NO:15.

[0025] Preferably, the nucleic acid molecule sequence encoding the amino acid sequence of mouse IgG Fc is shown in SEQ ID NO:16.

[0026] Preferably, the amino acid sequence of the bivalent Nanobody is as shown in SEQ ID NO:13.

[0027] The bivalent nanobody described in the present invention is a new type of antibody molecule consisting only of heavy chains. Although it lacks light chains compared to ordinary antibodies, it still retains the ability to bind to antigens.

[0028] The third aspect of the present invention provides a recombinant protein comprising: the anti-CD27 nanobody or antigen-binding fragment thereof of the first aspect of the present invention or the anti-CD27 bivalent nanobody of the second aspect of the present invention;

[0029] and / or, tag sequences.

[0030] Preferably, the tag sequence includes a tag sequence that assists expression and / or purification.

[0031] Preferably, the tag sequence is selected from at least one of the following groups: a His tag, a GGGS sequence, and a FLAG tag.

[0032] A fourth aspect of the present invention provides a biomaterial related to the anti-CD27 nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, or the recombinant protein of the third aspect of the present invention, wherein the biomaterial comprises at least one of b1)-b8):

[0033] b1) a nucleic acid molecule encoding the anti-CD27 Nanobody or antigen-binding fragment thereof of the first aspect of the invention, the anti-CD27 bivalent Nanobody of the second aspect of the invention, or the recombinant protein of the second aspect;

[0034] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0035] b3) a vector comprising the nucleic acid molecule described in b1);

[0036] b4) a vector comprising the expression cassette described in b2);

[0037] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0038] b6) a transgenic cell line comprising the expression cassette described in b2);

[0039] b7) a transgenic cell line comprising the vector described in b3);

[0040] b8) A transgenic cell line comprising the vector described in b4).

[0041] Preferably, the nucleic acid molecule is DNA.

[0042] Preferably, the transgenic cell line comprises no reproductive material.

[0043] Preferably, the cell comprises at least one of a eukaryotic cell and a prokaryotic cell; further comprises a prokaryotic cell; further is an Escherichia coli cell; further is WK6, BL21 or TG1.

[0044] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the anti-CD27 Nanobody or antigen-binding fragment thereof of the first aspect of the present invention comprises:

[0045] c1) the nucleotide sequence shown in SEQ ID NO: 12; or

[0046] c2) SEQ ID NO: 12 is subjected to one or more nucleotide substitutions and / or deletions and / or additions, and the nucleotide sequence has the same function as the nucleic acid molecule shown in SEQ ID NO: 11; or

[0047] c3) having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70% identity with SEQ ID NO:12, and having a nucleotide sequence with the same function as the nucleic acid molecule shown in SEQ ID NO:12.

[0048] Preferably, the nucleotide sequence encoding the anti-CD27 bivalent Nanobody of the second aspect of the invention comprises:

[0049] c1) the nucleotide sequence shown in SEQ ID NO: 14; or

[0050] c2) SEQ ID NO: 14 is subjected to one or more nucleotide substitutions and / or deletions and / or additions, and has a nucleotide sequence having the same function as the nucleic acid molecule shown in SEQ ID NO: 11; or

[0051] c3) having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70% identity with SEQ ID NO:14, and having a nucleotide sequence with the same function as the nucleic acid molecule shown in SEQ ID NO:14.

[0052] Preferably, the vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0053] Preferably, the vector is a plasmid expression vector.

[0054] The fifth aspect of the present invention provides a conjugate comprising: at least one of the anti-CD27 nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, and the recombinant protein of the third aspect of the present invention;

[0055] and a coupling portion, wherein the coupling portion comprises at least one of a detectable marker, a drug, a toxin, an electron-dense label, biotin, a spin label, an antibody, an antibody Fc fragment, an antibody scFv fragment, a radioactive isotope, an enzyme, a gold nanoparticle, a nanomagnetic particle, and a virus coat protein.

[0056] Preferably, the detectable label is a fluorescent or luminescent label.

[0057] Preferably, the detectable label is selected from any one of acridinium ester, acridinium sulfonamide, luminol, isoluminol, horseradish peroxidase and alkaline phosphatase.

[0058] Preferably, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177 and Re-188.

[0059] Preferably, the drug is other anti-CD27 drug.

[0060] The sixth aspect of the present invention provides a solid phase carrier, the surface of which is coupled with the anti-CD27 nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention and / or the recombinant protein of the third aspect of the present invention.

[0061] The seventh aspect of the present invention provides the use of the anti-CD27 nanobody or its antigen-binding fragment of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect of the present invention, or the recombinant protein of the third aspect of the present invention, the biomaterial of the fourth aspect, the conjugate of the fifth aspect, and / or the solid phase carrier of the sixth aspect in the preparation of products.

[0062] Preferably, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0063] Preferably, the drug has at least one of the functions d1) to d6):

[0064] d1) Induce or enhance T cell proliferation or activation;

[0065] d2) Enhance the killing ability of T cells;

[0066] d3) inhibiting the binding of CD70 and CD27 on cells;

[0067] d4) Treatment of tumors expressing CD27;

[0068] d5) Anti-transplant rejection;

[0069] d6) Treatment and / or prevention of autoimmune diseases.

[0070] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0071] Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, a binder, a wetting agent, a surfactant, a lubricant and a disintegrant.

[0072] Preferably, the tumor includes solid tumors and non-solid tumors; further, the solid tumor includes: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumor includes: leukemia and / or malignant lymphoma; further, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia; the malignant lymphoma includes B cell lymphoma; the lung cancer includes non-small cell lung cancer; further, the chronic leukemia includes B cell chronic lymphocytic leukemia.

[0073] Preferably, the autoimmune diseases include multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune at least one of autoimmune-related infertility, glomerulonephritis, crescentic glomerulonephritis, proliferative glomerulonephritis, bullous pemphigoid, Sjögren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis, Guillain-Barré syndrome, arteriosclerosis, and Alzheimer's disease.

[0074] Preferably, the reagent, detection plate, detection chip or kit has at least one function among e1) to e4):

[0075] e1) Detection of CD27;

[0076] e2) Diagnosis of tumors expressing CD27;

[0077] e3) screening for drugs for treating tumors expressing CD27;

[0078] e4) Lymphocyte clustering. The diagnosis is not intended for the diagnosis and treatment of diseases.

[0079] Preferably, the detecting CD27 comprises flow cytometry or Western Blot detection.

[0080] Preferably, the tumor includes solid tumors and non-solid tumors; further, the solid tumor includes: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumor includes: leukemia and / or malignant lymphoma; further, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia; the malignant lymphoma includes B cell lymphoma; the lung cancer includes non-small cell lung cancer; further, the chronic leukemia includes B cell chronic lymphocytic leukemia.

[0081] The eighth aspect of the present invention provides a product, wherein the product comprises at least one of f1) to f5):

[0082] f1) the anti-CD27 Nanobody or antigen-binding fragment thereof of the first aspect of the invention;

[0083] f2) the anti-CD27 bivalent Nanobody of the second aspect of the invention;

[0084] f3) the recombinant protein according to the third aspect of the present invention;

[0085] f4) the conjugate according to the fifth aspect of the present invention;

[0086] f5) The solid phase carrier according to the sixth aspect of the present invention.

[0087] Preferably, the product comprises at least one of a drug, a reagent, a test plate, a test kit, and a test chip.

[0088] Preferably, the drug has at least one of the functions d1) to d6):

[0089] d1) Induce or enhance T cell proliferation or activation;

[0090] d2) Enhance the killing ability of T cells;

[0091] d3) inhibiting the binding of CD70 and CD27 on cells;

[0092] d4) Treatment of tumors expressing CD27;

[0093] d5) Anti-transplant rejection;

[0094] d6) Treatment and / or prevention of autoimmune diseases.

[0095] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0096] Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, a binder, a wetting agent, a surfactant, a lubricant and a disintegrant.

[0097] Preferably, the tumor includes solid tumors and non-solid tumors; further, the solid tumor includes: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumor includes: leukemia and / or malignant lymphoma; further, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia; the malignant lymphoma includes B cell lymphoma; the lung cancer includes non-small cell lung cancer; further, the chronic leukemia includes B cell chronic lymphocytic leukemia.

[0098] Preferably, the autoimmune diseases include multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune at least one of autoimmune-related infertility, glomerulonephritis, crescentic glomerulonephritis, proliferative glomerulonephritis, bullous pemphigoid, Sjögren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes, autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome, autoimmune uveoretinitis, Guillain-Barré syndrome, arteriosclerosis, and Alzheimer's disease.

[0099] Preferably, the reagent, detection plate, detection chip or kit has at least one function among e1)-e3):

[0100] e1) Detection of CD27;

[0101] e2) Diagnosis of tumors expressing CD27;

[0102] e3) screening for drugs for treating tumors expressing CD27;

[0103] e4) Lymphocyte clustering. The diagnosis is not intended for the diagnosis and treatment of diseases.

[0104] Preferably, the detecting CD27 comprises flow cytometry or Western Blot detection.

[0105] Preferably, the tumor includes solid tumors and non-solid tumors; further, the solid tumor includes: cervical cancer, ovarian cancer, endometrial cancer, vulvar cancer, prostate cancer, parotid cancer, thyroid cancer, salivary gland cancer, lung cancer, fibrosarcoma, skin cancer, nasopharyngeal cancer, tongue cancer, oral cancer, laryngeal cancer, brain tumor, gallbladder cancer, kidney cancer, gum cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumor includes: leukemia and / or malignant lymphoma; further, the leukemia includes acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic myelomonocytic erythroleukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia; the malignant lymphoma includes B cell lymphoma; the lung cancer includes non-small cell lung cancer; further, the chronic leukemia includes B cell chronic lymphocytic leukemia.

[0106] The ninth aspect of the present invention is to provide a method for preparing the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the anti-CD27 bivalent nanobody of the second aspect, or the recombinant protein of the third aspect, which is obtained by culturing the transgenic cell line in the fourth aspect of the present invention.

[0107] The beneficial effects of the present invention are:

[0108] The present invention provides an anti-CD27 nanobody or an antigen-binding fragment thereof, which has strong specificity and high affinity for the CD27 antigen, and can be used for flow cytometry or Western Blot detection of CD27 and lymphocyte grouping, screening of drugs for treating tumors expressing CD27, and diagnosis or treatment of diseases and conditions related to CD27 activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0110] Figure 1 This is a diagram showing the results of animal immune titer testing in Example 1 of the present invention;

[0111] Figure 2 This is a graph showing the enrichment multiples of CD27-specific phages enriched by three rounds of phage panning in Example 2 of the present invention;

[0112] Figure 3 Graphs showing the results of the ELISA screening test of the CD27 nanobody monoclonals in Example 2 of the present invention, wherein A is a graph showing the results of the ELISA screening test of monoclonals 1-25, B is a graph showing the results of the ELISA screening test of monoclonals 26-50, C is a graph showing the results of the ELISA screening test of monoclonals 51-75, and D is a graph showing the results of the ELISA screening test of monoclonals 76-96;

[0113] Figure 4 This is a diagram showing the expression and purification results of CD27 nanobody verified by SDS-PAGE in Example 2 of the present invention;

[0114] Figure 5 This is a graph showing the EC50 test results of CD27-VHH95 binding to CD27 in Example 3 of the present invention;

[0115] Figure 6 This is a result diagram of the ELISA test for the specificity of CD27-VHH95 antibody in Example 3 of the present invention;

[0116] Figure 7 This is a diagram showing the binding activity detection results of CD27-VHH95-mFc in Example 3 of the present invention;

[0117] Figure 8 This is a graph showing the affinity test results of CD27-VHH95-mFc in Example 3 of the present invention;

[0118] Fig. 9 This is a graph showing the test results of verifying the cell binding activity of CD27-VHH95-mFc on Raji cells in Example 4 of the present invention;

[0119] Fig.10 This is a graph showing the results of the determination of the CD27 antibody in Example 4 of the present invention specifically recognizing the CD27+ cell population in PBMC;

[0120] Fig.11 This is a graph showing the results of Western Blot testing of the specificity of the CD27-VHH95-mFc antibody in Example 4 of the present invention, wherein M is maker, purchased from thermo with a catalog number of 26616, 1 is human CXCR5 protein, 2 is human CD27 protein, 3 is BSA, the predicted size of human CD27 protein is 43.2KD, the predicted size of human CXCR5 protein is about 60KD, and PC is a commercially available CD27 monoclonal antibody (Abcam, Ab256583). DETAILED DESCRIPTION

[0121] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples are reagents and materials obtained from commercial sources unless otherwise specified.

[0122] Example 1 Construction of phage display library after immunization

[0123] 1. Animal immunization

[0124] The CD27 antigen (Sino-Bio, 10039-H08H) was fully mixed with an equal volume of Freund's adjuvant (Sigma) and injected subcutaneously at two points on the neck of the alpaca for immunization. The immunization dose was 200 μg each time, and the interval between each immunization was two weeks. About 5 mL of venous peripheral blood was collected before immunization as a plasma titer control before immunization. In order to obtain a better immunization effect, a total of seven immunizations were performed, and the immunization effect was detected in real time.

[0125] The titer was tested by ELISA, and the results were as follows Figure 1 As shown in the figure, it can be seen that after 7 immunizations, the titer of antigen-specific serum in alpacas increased significantly, reaching 1:10 5 dilution, indicating that abundant CD27-specific antibodies were formed in the immunized animals.

[0126] 2. Obtaining total RNA and cDNA from alpaca peripheral blood mononuclear cells (PBMC)

[0127] Collect 30mL of whole blood sample from immunized alpacas, separate and purify lymphocytes using lymphocyte separation solution (GE, 17-1440-02) and density gradient centrifugation, wash the cells with PBS two to three times, add 1mL TRIzol (INVITROGEN, 15596-018) and mix well to fully lyse the cells, let stand at room temperature for 10 minutes, add 0.2 times the volume of chloroform, mix vigorously for 15 seconds, and centrifuge at 12000rpm and 4℃ for 10 minutes. Pipette the upper aqueous phase into a new 1.5mL centrifuge tube, add an equal volume of isopropanol, and place in a -20℃ refrigerator for sedimentation for 30 minutes. Centrifuge at 12000rpm and 4℃ for 10 minutes, discard the supernatant, add 75% ethanol, and centrifuge at 12000rpm and 4℃ for 10 minutes. Discard the supernatant and turn it upside down on paper to dry the inner wall liquid of the tube. Add 50 μL of DEPC water to dissolve the RNA precipitate and obtain the total RNA of alpaca peripheral blood mononuclear cells (PBMC). The total RNA concentration was determined by Nanodrop 2000, and the purity of the total RNA was identified by agarose gel electrophoresis.

[0128] cDNA reverse transcription: The total RNA of alpaca peripheral blood mononuclear cells (PBMC) obtained above was used to synthesize cDNA of alpaca peripheral blood mononuclear cells according to the operating instructions of the SuperScript™III First-Strand Synthesis Super Mix (Invitrogen) kit.

[0129] 3. Phage library construction

[0130] (1) Obtain the target fragment by performing two-step PCR amplification.

[0131] 1) First round of PCR

[0132] The first round of PCR was performed using Primer 1 and Primer 2 as primers (sequences are shown in Table 1), and the alpaca peripheral blood mononuclear cell cDNA obtained above was used as a template to enrich the alpaca heavy chain antibody V region (VHH) by Nest-PCR. The reaction system is shown in Table 2, and the reaction conditions are shown in Table 3.

[0133] Table 1

[0134]

[0135] Table 2

[0136]

[0137] Table 3

[0138]

[0139] 2) Second round of PCR

[0140] The second round of PCR was performed using Primer 3 and Primer 4 as primers (sequences are shown in Table 1), the template DNA was the amplification product obtained in the first round of PCR, the amplification system was shown in Table 4, and the amplification program was shown in Table 5.

[0141] Table 4

[0142]

[0143] Table 5

[0144]

[0145] After the PCR reaction was completed, the target fragment was recovered using QIAgen gel purification kit (Qiagen) according to the operating procedures. The recovered product was measured for concentration and purity using Nanodrop 2000 and stored at -20°C.

[0146] (2) Enzyme digestion, ligation and electroporation of the target fragment and vector to obtain a phage library

[0147] The target fragment obtained in step (1) and the phagemid vector pMECS were double-digested with NotI and PstI (purchased from NEB) restriction enzymes, respectively. The digestion system is shown in Table 6. The digestion was carried out at 37°C for 1 hour. The digestion products were then subjected to agarose gel electrophoresis, the gel was cut, and the digested VHH fragment and phagemid vector were respectively recovered using an agarose gel recovery kit (QIAGEN, 20051). The digested VHH fragment and phagemid vector were added with T4 DNA ligase (NEB) and ligated overnight at 22°C. The ligation system is shown in Table 7.

[0148] Table 6

[0149]

[0150] Table 7

[0151]

[0152] The ligation product was purified by PCR Purification Kit (QIAGEN), and 5 μL of the purified product was electroporated into 30 μL TG competent cells. The number of clones in different bacterial solution dilution gradients was counted the next day. It was evaluated that the constructed phage library reached 10 7CFU / mL or more, randomly select 24 clones for bacterial liquid PCR, bacterial liquid PCR primers MP57 (sequence: TTATGCTTCCGGCTCGTATG (SEQ ID NO: 17)) and GIII (sequence: CCACAGACAGCCCTCATAG (SEQ ID NO: 18)), run program: 95℃, 5min; enter the cycle stage: (94℃, 30S; 58℃, 45S; 72℃, 45S) amplification 22 cycles; 72℃, 5min. The amplified product was identified by 1% agarose gel electrophoresis and the insertion rate was 100%. In addition, 20 clones were randomly selected for sequencing, with a positive rate of 100%, which can be used for nano-antibody development.

[0153] Example 2 Development of CD27 Nanobodies

[0154] (1) Nanoantibody phage library screening

[0155] After the phage library was rescued from the bacterial solution obtained in Example 1 using helper phage M13K07 (NEB), its titer was measured to reach 5×10 14 pfu / mL. Take out the ELISA plate coated with CD27 recombinant protein (Sino-Qiao, 10039-H08H) overnight, add the phage library, and perform enrichment and panning. Add 100μL 1.5% triethylamine to the phage enriched with CD27 recombinant protein, incubate for 30min at room temperature, and add 100μL pH7.4 Tris HCl (Sangong) to neutralize; the collected phage is the CD27 nanoantibody phage library obtained by affinity panning; take 10μL phage to infect logarithmic phase TG1 cells and spread on the plate, count the number of clones after panning the next day, and continue to amplify the remaining phage.

[0156] Phage ELISA test results Figure 2 As shown in the figure, it can be seen that after three rounds of phage panning, CD27-specific phage was enriched 1000 times.

[0157] (2) Identification of target antigen-specific clones by ELISA

[0158] In order to obtain CD27 nanoantibody monoclonal strains, 96 monoclones were randomly selected from the phage clone plate after the third round of enrichment and inoculated into 1 mL of culture medium. They were cultured at 37°C until the logarithmic phase and induced overnight with 1 mM IPTG. Meanwhile, 100 ng CD27 protein (Sino-Qiao, 10039-H08H) was coated on the ELISA plate and incubated overnight at 4°C. The next day, the bacterial pellet was collected by centrifugation and broken. After centrifugation at 3,700 g for 15 min, the supernatant was collected. Meanwhile, the ELISA plate was taken and blocked with 2% BSA at room temperature for 1 h. The supernatant of monoclonal broken was added to each well of the experimental group, and the supernatant of blank TG1 bacterial solution was added to each well of the control group. The plates were incubated at room temperature for 1 h. The plates were washed with PBST for 5 times, and the antibody of Anti-HA (HRP) (abcam) diluted 1:2500 was added at room temperature for 1 h. The plates were washed with PBST for 5 times. The substrate was added and the absorbance was read on the microplate reader. When the ratio of the absorbance to the control well was greater than 2 (Base line), it was determined to be a positive clone.

[0159] The results are as follows Figure 3 As shown, from Figure 3 As can be seen from Figures A, B, C and D, 86 positive clones were obtained with a positive rate of 89.58%. The positive clones were subjected to Sanger sequencing to obtain 4 candidate nanoantibody sequences, and the CD27 nanoantibody CD27-VHH95 with the best effect was obtained through screening.

[0160] The amino acid sequence of CD27-VHH95 is composed of the framework region (FR1, FR2, FR3 and FR4) and the complementarity determining region (CDR1, CDR2 and CDR3). The nucleotide sequence of the screened CD27 nanoantibody was compared with the IGH reference gene of Alpaca in the International Immunogenetics Database (IMGT), and the obtained amino acid comparison sequence is shown in Table 8.

[0161] Table 8

[0162]

[0163] Nucleic acid molecules encoding the amino acid sequences of CD27-VHH95 complementarity determining regions (CDR1, CDR2 and CDR3):

[0164] CDR1: GGAAGCTCTGGACAATCAATGCC (SEQ ID NO: 8);

[0165] CDR2: ATGACTAATGGTGGTAGTACA (SEQ ID NO: 9);

[0166] CDR3: AATGCAGATCTGCGGCCCCATAGCGACCCTAGATATGAGTATGACTAC (SEQ ID NO: 10).

[0167] Full-length CD27-VHH95 nanobody heavy chain variable region amino acid sequence:

[0168] QVQLQESGGGLVQPGGSLRLACAASGSLWTINAMGWYRQAPGKQRELVATMTNGGSTNYADSVKGRFTISRDYAKNTVYLQMNSLKPEDTAVYYCNADLRPHSDPRYEYDYWGQGTQVTVSS (SEQ ID NO: 11).

[0169] Nucleotide sequence encoding the full-length CD27-VHH95 nanobody:

[0170] caggtgcagctgcaggagtctgggggaggcttggtgcagcctgggggatctctgagactcgcctgtgcagcctctggaagcctctggacaatcaatgccatgggctggtaccgccaggctccagggaagcagcgcgagttggtcgcaactatgactaatggtggtagtacaaactatgcagactcc gtgaagggccgattcaccatctccagagactacgccaagaacaccgtgtatctgcaaatgaacagcctgaaacctgaagacacggccgtctattactgtaatgcagatctgcggccccatagcgaccctagatatgagtatgactactggggccaggggacccaggtcaccgtctcctcag (SEQID NO: 12).

[0171] (3) Inducible expression and purification of CD27-VHH95 nanobody

[0172] The positive clones containing the CD27-VHH95 nanobody sequence obtained in step (2) above were streaked onto LB (containing ampicillin) solid plates and grown overnight at 37°C to obtain single clones. The next day, positive clones were picked and cultured overnight at 37°C and 220 rpm. On the third day, 1 mL of overnight culture solution and 400 μL of 1 M MgCl 2Solution, 1mL 20% glucose solution, 200μL ampicillin (100mg / mL) were added to 200mL TB medium, and amplified at 37℃, 220rpm for 4h, and then 1mM IPTG was added to induce expression and cultured overnight. On the fourth day, the bacterial precipitate was collected by centrifugation at 6000g for 15min, and 6mL TES (containing Tris-HCl pH 8.0 with a final concentration of 0.2M, EDTA with a final concentration of 0.5mM, and sucrose with a concentration of 0.5M) was added, and lysed at 300rpm, 4℃ for 1h, and 12mL 1 / 4 TES was added to continue lysis. After 3~4h, centrifugation was performed at 8000g for 10min to collect the supernatant of the broken bacterial cells.

[0173] Nanoantibodies are obtained by affinity purification of bacterial supernatants through Ni columns. The steps are as follows: the gravity column is first washed twice with ethanol at a mass concentration of 20%, and then washed twice with ultrapure water; the Ni filler is first washed twice with ultrapure water, and then washed twice with PBS, and the washed 1mL Ni filler is incubated with the crushed supernatant of the above CD27 nanoantibody expressing bacteria at 4°C, 300rpm overnight. The next day, the incubated mixture is added to the Ni column at a flow rate of 1mL / min; 5 times the column volume of affinity solution A (PBS) is used to wash away impurities, and then the target protein is eluted with an equal volume of affinity solution B (150mM or 250mM imidazole) in turn, and the eluate is collected; finally, the expression and purification of the CD27 nanoantibody is detected by SDS-PAGE. Figure 4 As shown, there is a target band only at the 15KD band, the purity of the nanobody reaches more than 90%, the yield is as high as 10 mg / mL, and the nanobody is successfully and correctly expressed.

[0174] (4) Affinity activity detection of CD27 nanoantibodies

[0175] In order to evaluate the binding activity of the nanobody to the human CD27 protein, the CD27-VHH95 nanobody purified in the above (3) induced expression and purification of CD27-VHH95 nanobody was used for verification by ELISA and SPR, respectively.

[0176] 1) The ELISA validation process is as follows: 100 ng CD27 protein (Sino-Bio, 10039-H08H) was used to coat the ELISA plate, and the plate was incubated overnight at 4°C; the next day, 2% BSA was added to block the plate at room temperature for 2 h; the purified CD27 nano antibody was diluted 3 times, with the highest concentration of 200 nM, and added to the control group and experimental group, respectively, at room temperature for 1 h; washed 10 times with PBST, and Anti-HA (HRP) antibody was added at room temperature for 1 h; washed 5 times with PBST, and the substrate was added. After reacting for 10 min, the stop solution was added. The absorbance value was read on an ELISA reader.

[0177] The results are as follows Figure 5 As shown in the figure, it can be seen that the CD27-VHH95 nanobody has a good binding ability to the CD27 protein, and the EC50 calculated by the fitting curve reaches 17.9nM.

[0178] 2) The SPR validation process is as follows: Place the CD27-coupled chip in the Biacore T200 instrument, perform a 2-fold gradient dilution of CD27-VHH95 as the analyte, flow rate is 30µL / min, binding 120s, dissociation 240s. The parameters of the interaction between CD27-VHH95 and the antigen CD27 are: Ka (1 / Ms), Kd (1 / s), KD (M), Rmax (RΜ). The results showed that the affinity of the nanobody to the CD27 protein reached 6.52×10 -8 M, indicating that the candidate nanoantibody has good CD27 protein binding activity.

[0179] (4) CD27 nanoantibody specific detection

[0180] In order to verify the specificity of CD27-VHH95 nanoantibody, 100 ng of CD27 protein (Sino Biological, 10039-H08H) was used as the experimental group and 100 ng of other irrelevant proteins (including human CXCR5 protein (Sino Biological, 18301-H82H), human CD40 protein (Sino Biological, 10774-H08H), human CD117 protein (ACRO, CD7-H52H4), human CD3 protein (Sino Biological, 10977-H08S), human CD20 protein (11007-H07H2)) was used as the control group. The ELISA plate was coated at 4°C overnight; 2% BSA was added at room temperature for 2 hours the next day to block; 1 μg / mL CD27 nanoantibody was added to the control group and experimental group, respectively, and incubated at room temperature for 1 hour; Wash 5 times with PBST, add Anti-HA (HRP) antibody, incubate at room temperature for 1 hour; wash 5 times with PBST, add substrate, react for 10 minutes, add stop solution, and read the OD450nm absorbance on a microplate reader. Figure 6 As shown in the figure, it can be seen that the nano antibody CD27-VHH95 specifically recognizes the CD27 protein and does not bind to other proteins. The results show that the CD27 nano antibody obtained by the present invention has good specificity for the human CD27 protein.

[0181] Example 3 Monovalent Nanobody Bivalent Transformation, Eukaryotic Expression and Purification

[0182] The nanobody CD27-VHH95 in Example 2 was modified, and the gene sequence encoding the nanobody CD27-VHH95 was seamlessly cloned into the pcDNA3.1 vector containing the Fc of mouse IgG1 (the amino acid sequence of the IgG Fc fragment sequence is shown in SEQ ID NO: 15, and the nucleotide sequence encoding the amino acid sequence of mouse IgG Fc is shown in SEQ ID NO: 16), and transfected into HEK 293T cells cultured in advance to the logarithmic phase for antibody expression. The culture conditions were: 37°C, 8% CO 2 , relative humidity ≥ 80%, 150 rpm shaking culture; after 5-6 days, the culture supernatant was collected and purified by protein A affinity chromatography to obtain the CD27 bivalent antibody CD27-VHH95-mFc (the amino acid sequence of the CD27-VHH95-mFc antibody is shown in SEQ ID NO: 13, and the nucleotide sequence encoding the CD27-VHH95-mFc antibody is shown in SEQ ID NO: 14) protein; the antibody band size was 80 KDa under SDS-PAGE non-reducing conditions, and the band size was about 40 KDa under reducing conditions, which was in line with expectations.

[0183] Full-length heavy chain antibody (variable region + IgG Fc framework region) amino acid sequence:

[0184] QVQLQESGGGLVQPGGSLRLACAASGSLWTINAMGWYRQAPGKQRELVATMTNGGSTNYADSVKGRFTISRDYAKNTVYLQMNSLKPEDTAVYYCNADLRPHSDPRYEYDYW GQGTQVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 13).

[0185] Nucleic acid molecule encoding the amino acid sequence of a full-length heavy chain antibody (variable region + IgG Fc framework region):

[0186]

[0187] The amino acid sequence of murine IgG Fc is as follows:

[0188] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:15).

[0189] The nucleotide sequence of murine IgG Fc:

[0190] gccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaataa(SEQID NO: 16)。

[0191] CD27-VHH95-mFc antibody activity evaluation:

[0192] (1) EC50 assay

[0193] The detection method is as follows: the experimental group was coated with 100 ng of CD27 protein (Sino-Bio, 10039-H08H), and the control group was coated with coating solution, and incubated at 4°C overnight; the next day, 2% BSA was added to block at room temperature for 2 hours; CD27-VHH95-mFc was diluted 5 times, with the highest concentration of 18nM, and added to the control group and the experimental group respectively, at room temperature, and incubated for 1 hour; washed 10 times with PBST, and Anti-mouse Fc (HRP) (Invitrogen, 31437) antibody was added, at room temperature for 1 hour; washed 5 times with PBST, substrate was added, reacted for 5 minutes, and then stop solution was added. The absorbance value was read on an ELISA reader.

[0194] The results are as follows Figure 7 As shown in the figure, it can be seen that the affinity of CD27-VHH95-mFc is 0.018nM, and the binding activity is increased by more than 100 times compared with the nanobody.

[0195] (2) SPR detection

[0196] The detection method is as follows: Place the CD27-coupled chip in the Biacore T200 instrument, perform a 2-fold gradient dilution of CD27-VHH95-mFc as the analyte, flow rate is 30 µL / min, binding is 120s, and dissociation is 240s. The parameters of the interaction between the bivalent antibody and the antigen CD27 are: Ka (1 / Ms), Kd (1 / s), KD (M), Rmax (RΜ).

[0197] The results are as follows Figure 8 As shown in the figure, the affinity of CD27-VHH95-mFc is 5.54×10 -9 M level, the affinity is increased by more than 10 times compared with the nano antibody, and the dissociation constant is increased by 100 times, indicating that the binding ability of CD27-VHH95-mFc antibody to antigen is further improved. In summary, the results show that the CD27 antibody has good development potential and application value.

[0198] Example 4 Application of CD27-VHH95-mFc Antibody in Flow Cytometry and Western Blot Detection

[0199] 1. Application of the CD27-VHH95-mFc antibody prepared in Example 3 in flow cytometry

[0200] (1) CD27-VHH95-mFc binding activity test on Raji cells

[0201] Take 10mL of 1640 (10% FBS) cultured Raji cells with a cell viability of 99%, centrifuge at 400g for 5min. Collect the cells, resuspend the cells in working buffer (PBS + 4% FBS), and wash the cells at 400g for 5min. Take 2.5×10 5 cells, add 33nM CD27-VHH95-mFc, final volume 100μL, 4℃, bind for 1h. 400×g, 5min, add working buffer to wash twice / 1mL. Add 200μL 2% BSA (diluted with PBS), block for 1h at 4℃. 400×g, 5min, wash twice with working buffer / 1mL. Add 1:200 diluted Anti-mouse Fc-FITC (Invitrogen, A21202), bind at 4℃ in the dark for 1h. 400×g, 5min, wash twice with working buffer / 1mL. Resuspend in 200μL working buffer and detect on flow cytometer. Take an equimolar amount of commercial CD27 flow cytometry antibody (Abcam) as a positive control, and the negative control is Raji cells without CD27-VHH95-mFc antibody treatment. The test results are as follows Fig. 9 As shown in the figure, it can be seen that the cell binding activity of the negative control is 0.44%, and the cell binding activity of CD27-VHH95-mFc is 98.63%, which is comparable to the cell binding activity of the positive control antibody (99.68%).

[0202] (2) CD27-VHH95-mFc for lymphocyte clustering

[0203] Take PBMC cells (Raiden), centrifuge at 400g for 5 minutes, collect the cells, resuspend the cells in working buffer (PBS + 4% FBS), wash the cells, and centrifuge at 400g for 5 minutes. The detection method is the same as (1) CD27-VHH95-mFc binding activity test with Raji cells, and the positive control is a commercialized CD27 flow cytometry antibody (Abcam).

[0204] Test results such as Fig.10 As shown in the figure, it can be seen that the negative control cell binding activity is 2.68%, and the CD27-VHH95-mFc cell binding activity is 49.37%, which is better than the cell binding activity of the positive control antibody. It has good application value and provides available candidate antibody molecules for the development of CD27 flow cytometry detection antibodies.

[0205] 2. CD27-VHH95-mFc for Western Blot

[0206] Take 50ng CD27 protein (Sino-Bio, 10039-H08H) and irrelevant control proteins, such as CXCR5 protein (Sino-Bio, 18301-H82H) and BSA (VETEC, V900933-100G), and perform SDS-PAGE. Cut the nitrocellulose membrane of appropriate size, activate it with methanol for 5s, wash it with PBST three times, cover the separation gel on the nitrocellulose membrane, transfer it with GenScript instrument, and place the nitrocellulose membrane after transfer in 2% BSA (PBST dilution) solution at room temperature for 2h at 60rpm. Wash it with PBST five times, add 3μg / mL CD27-VHH95-mFc antibody and positive antibody (commercially available rabbit CD27 antibody, Abcam, Ab256583), incubate it at room temperature for 1h, and wash it with PBST five times. Anti-mouse IgG (HRP) (SAB, L3032) or Anti-rabbit IgG (HRP) (genscript, A011856-200) were added respectively, incubated at room temperature and 60 rpm for 1 hour, washed 5 times with PBST, and developed.

[0207] Test results such as Fig.11 As shown in the figure, it can be seen that the CD27-VHH95-mFc antibody of the present invention can specifically detect human CD27 and does not bind to unrelated proteins; PC (commercially available rabbit CD27 antibody) detection can detect binding to human CD27, but there is also binding to unrelated protein human CXCR5. The above results show that the antibody-specific CD27-VHH95-mFc of the present invention is better than the commercially available positive antibody, and can be applied to both flow detection and Western Blot detection. The application value of this antibody is better than the commercially available positive antibody.

[0208] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-CD27 nanobody or an antigen-binding fragment thereof, characterized in that: The Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3; The amino acid sequence of the CDR1 is GSLWTINA; The amino acid sequence of the CDR2 is MTNGGST; The amino acid sequence of the CDR3 is NADLRPHSDPRYEYDY.

2. The anti-CD27 nanobody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region comprises: a1) SEQ ID NO: 11; or a2) an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identical to SEQ ID NO: 11 and has the same function as the protein shown in SEQ ID NO:

11.

3. A bivalent anti-CD27 nanobody, characterized in that: The bivalent Nanobody comprises the Nanobody as claimed in claim 1 or 2; and Heavy chain constant region; the heavy chain constant region is mouse IgG Fc.

4. A recombinant protein, characterized in that It consists of the anti-CD27 nanobody or its antigen-binding fragment according to any one of claims 1-2 or the anti-CD27 bivalent nanobody according to claim 3, and a tag sequence.

5. A biomaterial associated with the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the anti-CD27 bivalent nanobody according to claim 3, or the recombinant protein according to claim 4, wherein the biomaterial comprises at least one of b1) to b8): b1) a nucleic acid molecule encoding the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the anti-CD27 bivalent nanobody according to claim 3, or the recombinant protein according to claim 4; b2) an expression cassette comprising the nucleic acid molecule described in b1); b3) a vector comprising the nucleic acid molecule described in b1); b4) a vector comprising the expression cassette described in b2); b5) a transgenic cell line comprising the nucleic acid molecule described in b1); b6) a transgenic cell line comprising the expression cassette described in b2); b7) a transgenic cell line comprising the vector described in b3); b8) A transgenic cell line comprising the vector described in b4).

6. A conjugate, characterized in that: Comprising: at least one of the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the anti-CD27 bivalent nanobody according to claim 3, and the recombinant protein according to claim 4; and a coupling portion, wherein the coupling portion comprises at least one of a detectable label, a gold nanoparticle, and a nanomagnetic particle.

7. A solid phase carrier, characterized in that It comprises: at least one of the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the anti-CD27 bivalent nanobody according to claim 3, and / or the recombinant protein according to claim 4.

8. Use of at least one of (1) to (6) in the preparation of a product; (1) The anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2; (2) The anti-CD27 bivalent nanobody according to claim 3; (3) The recombinant protein according to claim 4; (4) The biomaterial according to claim 5; (5) The conjugate according to claim 6; (6) The solid phase carrier according to claim 7; The product comprises at least one of a drug, a detection reagent, a detection plate, a test kit, and a detection chip; The drug has at least one of the functions d1)-d2): d1) Treatment of tumors expressing CD27; d2) Treatment and / or prevention of autoimmune diseases; The tumors include solid tumors and non-solid tumors; the solid tumors are: cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, lung cancer, fibrosarcoma, skin cancer, kidney cancer, testicular cancer, penile cancer, multiple myeloma and / or malignant melanoma; the non-solid tumors are: leukemia and / or malignant lymphoma; The autoimmune disease is at least one of multiple sclerosis, rheumatoid arthritis, ulcerative colitis, systemic lupus erythematosus, autoimmune encephalomyelitis, Sjögren's syndrome and psoriatic arthritis; The detection reagent, detection plate, detection chip or kit has at least one of the functions e1)-e2): e1) Detection of CD27; e2) Lymphocyte clustering.

9. A product, characterized in that The product comprises at least one of f1) to f5): f1) the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2; f2) The anti-CD27 bivalent Nanobody according to claim 3; f3) The recombinant protein according to claim 4; f4) the conjugate according to claim 6; f5) The solid phase carrier according to claim 7; The product comprises at least one of a drug, a detection reagent, a detection plate, a test kit, and a detection chip.

10. A method for preparing the anti-CD27 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the anti-CD27 bivalent nanobody according to claim 3, and / or the recombinant protein according to claim 4, characterized in that: The preparation method comprises the following steps: preparing by culturing the transgenic cell line described in claim 5.

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