An antibody capable of specifically binding to nkp30 and uses thereof

Abstract

The present application relates to an antibody capable of specifically binding to NKp30 and application thereof. The antibody or antigen-binding fragment thereof of the present application comprises a VHH and a constant region, the VHH comprises HCDR1, HCDR2 and HCDR3, and the VHH is connected to the N terminal or C terminal of the constant region through a linker L1. Experimental results show that the antibody of the present application has good binding activity with NKp30.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antibody or its antigen-binding fragment that can specifically bind to NKp30 and its applications. Background Technology

[0002] The human NKp30 (Natural cytotoxicity triggering receptor 3) protein is encoded by the NCR3 gene and belongs to the family of natural cytotoxicity triggering receptors (NCRs). It is an activating receptor on the surface of NK cells.

[0003] NK cells are the body's main "warriors" responsible for killing aging, virus-infected, and tumor cells. NKp30 is expressed in all resting and activated NK cells, various effector NKT cells, γδT- cells, and MAIT cells, and can activate tumor-killing cells such as NK cells and γδT- cells, thereby killing tumors.

[0004] NKp30 is a key trigger receptor for killing certain tumors, such as melanoma MEL15. NKp30 can also synergize with other activating receptors to enhance NK cell activity. Therefore, developing specific antibodies targeting tumor-associated antigens and NKp30 to bridge tumor cells and NK cells, and activating only NK cells in the tumor microenvironment, may help reduce side effects and avoid decreased sensitivity to abnormal cells that have lost MHC molecules due to systemic hyperactivation of NK cells. Summary of the Invention

[0005] The present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to NKp30, the antibody or antigen-binding fragment thereof comprising a VHH and a constant region, the VHH comprising HCDR1, HCDR2 and HCDR3, and the VHH being connected to the N-terminus or C-terminus of the constant region via a linker L1.

[0006] In some embodiments, the antibody or its antigen-binding fragment contains two or more VHHs.

[0007] In some embodiments, the antibody or its antigen-binding fragment comprises four VHHs.

[0008] In some implementations, two of the four VHHs are connected to the N-terminus of the CH2 portion of the constant region, and the other two are connected to the C-terminus of the CH3 portion of the constant region.

[0009] In some implementations, the four VHHs are combined into two groups by connecting them in series, and these two groups are respectively connected to the N end of the CH2 portion in the constant region.

[0010] In some implementations, the VHHs are connected by connector L2.

[0011] In some implementations, connector L1 and connector L2 may be the same or different.

[0012] In some embodiments, the linker L1 and / or the linker L2 independently have an amino acid sequence as shown in (GyS)x, where x and y are independently integers selected from 1 to 6; preferably, the linker L1 and / or the linker L2 are independently (G4S)2, (G4S)3, (G4S)4, (G3S)3, (G2S)3 or (GS)3.

[0013] In some embodiments, the constant region of the antibody or its antigen-binding fragment includes an Fc region. In some embodiments, the constant region of the antibody or its antigen-binding fragment does not include CH1.

[0014] In some implementations, the Fc region is selected from IgG, IgA, IgD, IgE, IgM and their variants.

[0015] In some implementations, the Fc region is selected from IgG1, IgG2, IgG3, IgG4 and their variants.

[0016] In some implementations, the Fc region contains one or more amino acid mutations, and the mutations are amino acid substitutions, insertions, or deletions.

[0017] In some implementations, the VHH is a camel antibody, a shark antibody, or a humanized single-domain antibody.

[0018] In some embodiments, the antibody or its antigen-binding fragment HCDR1 is as shown in SEQ ID NO: 1, or is a sequence having at least 80% identity with SEQ ID NO: 1; HCDR2 is as shown in SEQ ID NO: 2, or is a sequence having at least 80% identity with SEQ ID NO: 2; HCDR3 is as shown in SEQ ID NO: 3, or is a sequence having at least 80% identity with SEQ ID NO: 3.

[0019] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or an antigen-binding fragment thereof.

[0020] The present invention also provides a multifunctional fusion protein comprising the antibody or its antigen-binding fragment.

[0021] In some embodiments, the multifunctional fusion protein further comprises one or more second antibodies or antigen-binding fragments thereof that specifically bind to other antigens.

[0022] In some implementations, the other antigens are selected from tumor-associated antigens (TAAs) or immune checkpoints.

[0023] In some embodiments, the other antigens are selected from GPC3, CD19, CD20 (MS4A1), CD22, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, and caspase. -8 (caspase-8), CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM , EphA2, Fra-1, FOLR1, GAGE ​​protein, GD2, GD3, GloboH, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, IL13Rα2 , LMP2, κ-Light, LeY, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MART-1, mesothelin, ML-IAP, MOv-γ, Muc1, Muc2, Muc3, M uc4, Muc5, Muc16, MUM1, Ras, RGS5, Rho, ROR1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Tonkin's antigen, TRP-1, TRP-2, tyrosinase and urolysin-3, 5T4, PD-L1, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, ILT4, TREM2, LAG3, CD27, CD24, B7H3 or B7H4.

[0024] In some implementations, cytokines are also included.

[0025] In some embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-2 Rα, IL-2 Rβ, IL-3, IL-3Rα, IL-4, IL-4 Rα, IL-5, IL-5 Rα, IL-6, IL-6 Rα, IL-7, IL-7 Rα, IL-8, IL-9, IL-9 Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11 Rα, IL-12, IL-12 Rα, IL-12 Rβ2, IL-12 Rβ1, IL-13, IL-13 Rα, IL-13 Rα2, IL-14, IL-15, IL-15Rα sushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21 Rα, IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFNγ, IFNα, or GM-CSF.

[0026] The present invention also provides the use of the antibody or its antigen-binding fragment or the multifunctional fusion protein in the preparation of a medicament for treating cancer.

[0027] In some implementations, the cancer is selected from human astroblastoma, human pharyngeal carcinoma, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, Ewing tumor, extraosseous myxoid chondrosarcoma, fibrous dysplasia of bone, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, etc. Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid adenoma, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, or uterine cancer.

[0028] The present invention also provides the use of the antibody or its antigen-binding fragment or the multifunctional fusion protein in the preparation of a medicament for treating autoimmune diseases.

[0029] In some implementations, the autoimmune disease is selected from graft-versus-host disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, colitis, psoriasis, autoimmune uveitis, pemphigus, epidermolysis bullosa, or type I diabetes.

[0030] In some implementations, the use is achieved through one or more of tumor immunotherapy, cell therapy, or gene therapy.

[0031] The present invention also provides the use of the antibody or its antigen-binding fragment or the multifunctional fusion protein in the preparation of a medicament for inducing the proliferation or activation of NK cells, effector NKT cells, γδT- cells or MAIT cells.

[0032] The present invention also provides a method for inducing the proliferation or activation of NK cells, effector NKT cells, γδT- cells or MAIT cells, comprising administering the antibody or its antigen-binding fragment or the multifunctional fusion protein to a subject.

[0033] The present invention also provides a method for in vitro expansion or activation of NK cells, effector NKT cells, γδT- cells or MAIT cells, comprising using the antibody or its antigen-binding fragment or the multifunctional fusion protein.

[0034] The present invention also provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0035] The present invention also provides a pharmaceutical composition comprising the multifunctional fusion protein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0036] The present invention also provides an antibody-drug conjugate comprising the antibody or its antigen-binding fragment and the conjugated drug.

[0037] In some embodiments, the conjugated drug is selected from cytotoxins, small molecule chemical drugs, or immunotoxins.

[0038] Abbreviations and Terminology Definitions

[0039] The following abbreviations are used in this article. VHH: Antibody heavy chain variable region; CDR: Complementarity-determining region in the immunoglobulin variable region; IgG: Immunoglobulin G.

[0040] The term "antibody" refers to a naturally occurring immunoglobulin or an immunoglobulin prepared through partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma or serum where the antibody is naturally present, or from the culture supernatant of antibody-producing hybridoma cells, animal immune serum, or through phage library screening. Alternatively, they can be partially or completely synthesized using techniques such as gene recombination. Preferred antibodies include, for example, antibodies to isotypes of immunoglobulins or subtypes of these isotypes. Human immunoglobulins are known to include nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgG1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention may include IgG1, IgG2, IgG3, and / or IgG4.

[0041] The term "antigen-binding fragment" refers to a polypeptide fragment of an antibody, such as a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical fragmentation of the intact antibody.

[0042] The term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antigen-binding fragment) linked to one or more conjugated drugs (which may optionally be therapeutic agents or cytotoxic agents). Its structure typically consists of three parts: an antibody or antibody-like ligand, a drug moiety, and a linker that conjugates the antibody or antibody-like ligand and the drug. ADCs typically have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs conjugated to the antibody.

[0043] The term "peptide" refers to an amino acid chain of any length, regardless of modifications such as phosphorylation or glycosylation. The term peptide includes proteins and fragments thereof. Peptides can be "exogenous," meaning they are "heterogeneous," i.e., derived from a host cell, such as human peptides produced by bacterial cells. Peptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right, from the amino terminus to the carboxyl terminus. Following standard nomenclature, amino acid residue sequences are named with three-letter or single-letter codes.

[0044] The term "VHH" refers to a single antigen-binding polypeptide containing only a heavy chain variable region (VHH), derived from the variable domain of a naturally occurring heavy chain molecule lacking a light chain, to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camel species such as camels, llamas, vicuñas, dromedaries, alpacas, and guanacos. Other species outside the camel family, such as sharks, can produce naturally occurring heavy chain molecules lacking a light chain, and such VHHs are within the scope of this application.

[0045] The term "Fc region" generally refers to the domain that contains the constant CH2 and CH3 regions of immunoglobulins (e.g., antibodies).

[0046] The term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell to which they are introduced. Some vectors can direct the expression of nucleic acids to which they are operatively linked.

[0047] The term "pharmaceutically acceptable carrier" includes any standard drug carrier, such as phosphate-buffered saline solutions, water and emulsions, as well as various types of wetting agents.

[0048] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a control polypeptide sequence after sequence alignment and, where necessary, nicking to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, like BLAST software or the FASTA package.

[0049] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical to those in the control polypeptide sequence is more than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0050] Various methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the location of structural cyclic regions; the IMGT system defines CDRs based on sequence variability and location within variable domain structures; AbM is defined using Oxford Molecular's AbM antibody modeling software, representing a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in several ways. Unless otherwise specified, the CDRs in this patent are defined by Kabat.

[0051] The term "specific" means that one of the molecules involved in specific binding does not exhibit significant binding to any molecules other than one or more of its binding partner molecules. Furthermore, the term is also used when a domain containing an antibody-variable region is specific to a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody-variable region is contained in several different antigens, an antigen-binding molecule containing the domain containing the antibody-variable region can bind to various antigens having said epitope.

[0052] The term "epitope" refers to an antigenic determinant within an antigen, and specifically to the antigenic site to which a domain of an antigen-binding molecule containing an antibody variable region, as disclosed in this specification, binds. Therefore, an epitope can be defined based on its structure. Alternatively, an epitope can be defined based on the antigen-binding activity of the antigen-binding molecule that recognizes it. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that form it; when the epitope is a glycan, it can be determined by its specific glycan structure.

[0053] The term "positive control" refers to a natural or engineered cell or antibody that can bind to or express a target protein. In this article, the positive control refers to a single-target positive control.

[0054] The term "negative control" refers to the use of immunoglobulins of the same species, subtype, dose, and subtype as the experimental sample, along with the same labeling, in the same experiment. This is used to eliminate the background influence of non-specifically bound samples on experimental values, serving as a more illustrative control of the experimental results.

[0055] The term "linker" is defined and described as follows: when forming the antibodies or antigen-binding fragments or fusion proteins of the present invention, a linker may be used, but is not mandatory. When a linker is used, its chemical structure may not be critical, as it primarily functions as a spacer. A linker may consist of amino acids linked together by peptide bonds. The present invention contemplates linkers of any length or composition. The linkers described herein are exemplary, and the present invention also contemplates linkers that are much longer and include additional residues. The terms "linker" and "linker" are used interchangeably. Attached Figure Description

[0056] Figure 1 An exemplary antibody with a symmetrical structure as described in this invention comprises four VHHs capable of specifically recognizing NKp30, wherein two VHHs are linked by a linker and attached to the N-terminus of Fc.

[0057] Figure 2 An exemplary antibody with a symmetrical structure as described in this invention comprises four VHHs capable of specifically recognizing NKp30, wherein the VHHs are respectively linked to the N-terminus and C-terminus of Fc via linkers.

[0058] Figure 3 The binding activity of antibodies A and B of the present invention with NKp30 is shown.

[0059] Figure 4 The binding activity of antibodies B1 and B2 of the present invention with NKp30 is shown. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations and not for limiting the scope of protection of the present invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0061] Example 1: Construction of NKp30 antibodies with different structures

[0062] The NKp30 antibodies A and B of the present invention are respectively formulated according to... Figure 1 and Figure 2 The structural constructions shown are numbered as shown in Table 1. The full-length sequences of NKp30 antibodies A and B of this invention are SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0063] Table 1. NKp30 antibody sequence listing

[0064] Antibody A Antibody B Heavy chain variable region VHH SEQ ID NO: 4 SEQ ID NO: 4 HCDR1 SEQ ID NO: 1 SEQ ID NO: 1 HCDR2 SEQ ID NO: 2 SEQ ID NO: 2 HCDR3 SEQ ID NO: 3 SEQ ID NO: 3 L1 SEQ ID NO: 5 SEQ ID NO: 5 and 6 L2 SEQ ID NO: 6 - full length SEQ ID NO: 7 SEQ ID NO: 8

[0065] Example 2: Transient expression and antibody purification

[0066] The pcDNA3.1 vector was used as the dedicated vector for expressing the antibody heavy chain. The nucleotide sequences of the antibody A and antibody B expression heavy chains were obtained by codon optimization and gene synthesis by General Biotechnology (Anhui) Co., Ltd., and inserted into the HindIII-XhoI restriction site of the pcDNA3.1 vector. The plasmids were then extracted to obtain pcDNA3.1-antibody A and pcDNA3.1-antibody B expression plasmids.

[0067] ExpiCHO cells were cultured at 37℃, 8% CO2, and 100 rpm until the cell density reached 6 × 10⁶ cells / year. 6Cells / mL. The constructed vectors pcDNA3.1-antibody A and pcDNA3.1-antibody B were transfected into the above cells using liposomes at a plasmid concentration of 1 μg / mL. The liposome concentration was determined according to the ExpiCHO™ Expression System kit. Cells were cultured at 32℃, 5% CO2, and 100 rpm for 7-10 days. Feeding was performed 18-22 h after transfection and again between 5-8 days. The culture products were centrifuged at 4000 rpm, filtered through a 0.22 μm filter, and the supernatant was collected. Antibody A and Antibody B proteins were purified using a Protein A affinity column. The protein expression levels in the culture supernatant and the purity of the purified proteins are shown in Table 2. It can be seen that antibody A and antibody B have high expression levels and good production potential, and the purity after purification is good, especially that of antibody B, which shows a significant advantage in purity.

[0068] Table 2

[0069] Instantaneous 7-day expression level One-step affinity SEC-HPLC purity Antibody A 0.546g / L 80.78% Antibody B 0.682g / L 90.54%

[0070] Example 3: Experiment on the binding activity of the antibody of the present invention with NKp30

[0071] The solutions used in the examples are prepared as follows.

[0072] Coating solution (1×PBS, pH 7.4): Take one packet of phosphate buffer (1×PBS, pH 7.4), add 1000mL of water to dissolve, mix well, and set aside.

[0073] Washing solution (1×PBST, containing 0.05% Tween 20): Dissolve 500mL of Tween 20 in 1000mL of 1×PBS and mix well.

[0074] Blocking solution and sample diluent (3% skim milk powder): Weigh 3g of skim milk powder and dissolve it in 100mL of 1×PBST. Mix well and set aside.

[0075] Termination solution (1 mol / L HCl): Measure 83 mL of concentrated hydrochloric acid, dilute with water to 1000 mL, mix well, and set aside.

[0076] Human NKp30 Protein, His tag (manufacturer: KACTUS, catalog number: NKP-HM430) was diluted to 0.2 μg / mL with coating buffer (1×PBS, pH 7.4), and 100 μL / well was used to coat the microplate. The plate was incubated overnight at 4°C. The liquid in the wells was discarded, and the plate was washed three times with washing buffer (1×PBST, containing 0.05% Tween 20) at 300 μL / well. 3% skim milk powder was added to the microplate at 300 μL / well, and the plate was incubated at 37°C for 1 h. The blocking buffer in the wells was discarded, and the plate was washed three times with washing buffer at 300 μL / well. The test samples (antibodies A and B of this invention) were pre-diluted with 3% skim milk powder to 40 nM at the NKp30 target concentration. This initial concentration was then used for 3-fold dilution, resulting in a total of 9 dilutions. Add 100 μL / well of each concentration gradient of the test sample to the microplate. Include one blank well with only diluent. Make two replicates for each sample and blank well. Incubate at 37°C for 1 hour. Discard the liquid in the wells, wash three times with a plate washer, and blot dry on a plate. Dilute the detection antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific) (manufacturer: Jackson Immuno, catalog number: 109-035-098) 1:10000 with sample diluent. Add 100 μL / well to the microplate and incubate at 37°C for 45 minutes. Wash six times with a plate washer and blot dry on a plate. Add 100 μL / well of TMB single-component chromogenic solution to the microplate and incubate at 37°C in the dark for 3-4 minutes. Stop the reaction by adding 100 μL / well of stop solution (1 mol / L HCl). The absorbance (OD) was measured at 450 nm using an ELISA reader. 450 The result is as follows: Figure 3 As shown, both antibody A and antibody B of the present invention have good binding activity with NKp30.

[0077] Example 4: Experiment on the binding activity of the antibody of the present invention with NKp30

[0078] Human NKp30 Protein, His tag (manufacturer: KACTUS, catalog number: NKP-HM430) was diluted to 0.2 μg / mL with coating buffer (1×PBS, pH 7.4), and 100 μL / well was used to coat the microplate. The plate was incubated overnight at 4°C. The liquid in the wells was discarded, and the plate was washed three times with washing buffer (1×PBST, containing 0.05% Tween 20) at 300 μL / well. 3% skim milk powder was added to the microplate at 300 μL / well, and the plate was incubated at 37°C for 1 hour. The blocking buffer in the wells was discarded, and the plate was washed three times with washing buffer at 300 μL / well. Two different batches of antibody B of this invention, namely antibody B1 and antibody B2, were pre-diluted with 3% skim milk powder to a NKp30 target concentration of 40 nM. These were then used as initial concentrations for 3-fold dilution, resulting in a total of 11 dilutions. Add 100 μL of each concentration gradient of NKp30 protein to each well of the microplate. Include one blank well with only diluent. Make two replicates for both the test sample and the blank. Incubate at 37°C for 1 hour. Discard the liquid from the wells, wash the plate three times, and pat dry on a microplate paper. Dilute the detection antibody (Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (manufacturer: Jackson Immuno, catalog number: 109-036-098) 1:10000 with diluent, add 100 μL / well to the microplate, and incubate at 37°C for 45 min. Wash 6 times with a plate washer and blot dry on a plate paper. Add 100 μL / well of TMB single-component chromogenic solution to the microplate and incubate at 37°C in the dark for 3-4 min. Stop the reaction by adding 100 μL / well of stop solution (1 mol / L HCl). Measure the absorbance (OD) at 450 nm using a microplate reader. 450 The result is as follows: Figure 4 As shown, both antibody B1 and antibody B2 of the present invention have good binding activity with NKp30.

[0079] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. An antibody or its antigen-binding fragment capable of specifically binding to NKp30, characterized in that, The antibody or its antigen-binding fragment comprises a VHH and a constant region, wherein the VHH comprises HCDR1, HCDR2 and HCDR3, and the VHH is connected to the N-terminus or C-terminus of the constant region via a linker L1.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment contains two or more VHHs.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment contains four VHHs.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, Two of the four VHHs are connected to the N-terminus of the CH2 portion in the constant region, and the other two are connected to the C-terminus of the CH3 portion in the constant region.

5. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The four VHHs are combined into two groups by connecting them in series, and these two groups are respectively connected to the N end of the CH2 part in the constant region.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The VHHs are connected by connector L2.

7. The antibody or its antigen-binding fragment according to claim 1 or 6, characterized in that, The connector L1 and the connector L2 may be the same or different.

8. The antibody or its antigen-binding fragment according to claim 7, characterized in that, The linker L1 and / or the linker L2 independently have an amino acid sequence as shown in (GyS)x, wherein x and y are independently integers selected from 1 to 6; preferably, the linker L1 and / or the linker L2 are independently (G4S)2, (G4S)3, (G4S)4, (G3S)3, (G2S)3 or (GS)3.

9. The antibody or antigen-binding fragment thereof according to claims 1-8, characterized in that, The constant region of the antibody or its antigen-binding fragment includes the Fc region.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The Fc region is selected from IgG, IgA, IgD, IgE, IgM and their variants.

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