Anti-tumor antigen nanobody, nucleic acid coding sequence thereof and application

Abstract

The present invention provides an anti-tumor antigen nanobody that specifically binds to a human leukocyte antigen-G. The present invention also provides a nucleic acid coding sequence of the anti-tumor antigen nanobody, the use of the anti-tumor antigen nanobody for treating cancer and immune-related diseases, and a method for detecting the expression level of HLA-G.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor antigen nanobodies, and particularly to an anti-tumor antigen nanobody, its nucleic acid coding sequence, and its applications. Background Art

[0002] Cancer, also known as malignant tumor, is the abnormal proliferation of cells, and these proliferating cells may invade other parts of the body. It is a disease caused by the disorder of the mechanism controlling cell division and proliferation. The population suffering from cancer worldwide shows an increasing trend. Cancer is one of the top ten causes of death among Chinese people and has ranked first in the top ten causes of death for many consecutive years.

[0003] Conventional tumor treatment methods include surgical treatment, radiotherapy, chemotherapy, and targeted therapy, etc. Tumor immunotherapy is another method for treating tumors other than the above-mentioned treatment methods. It activates the patient's own immune system, uses tumor cells or tumor antigen substances to induce specific cellular and humoral immune responses in the body, enhances the body's anti-cancer ability, and prevents the growth, spread, and recurrence of tumors, so as to achieve the purpose of removing or controlling tumors. However, the current tumor treatment methods still have problems such as ineffective treatment and strong side effects, and may even give rise to other immune-related diseases.

[0004] Human leukocyte antigen-G (HLA-G) has been found to be highly expressed in a variety of solid tumors and has the property of inhibiting immune cells. Therefore, some researchers have been committed to developing HLA-G as a target molecule for identifying tumors and finding out whether these target molecules have the potential to become anti-cancer drugs.

[0005] To solve the above problems, those skilled in the art urgently need to develop novel and more effective pharmaceuticals for treating cancer and immune-related diseases to benefit the vast population in need. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide an anti-tumor antigen nanobody that specifically binds to a human leukocyte antigen-G (HLA-G). The anti-tumor antigen nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and any combination of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0007] In one embodiment of the present invention, the amino acid sequence is the amino acid sequence of a heavy chain variable domain (VHH) of the anti-tumor antigen nanobody.

[0008] In one embodiment of the present invention, the anti-tumor antigen nanobody is conjugated to a fragment crystallizable region (Fc region).

[0009] In one embodiment of the present invention, the anti-tumor antigen nanobody is conjugated to a second antibody to form a bispecific T-cell engager (BiTE), a trispecific T-cell engager (TriTE), a bispecific killer cell engager (BiKE), a trispecific killer cell engager (TriKE), or any bispecific antibody.

[0010] In one embodiment of the present invention, the anti-tumor antigen nanobody blocks the interaction and / or binding of HLA-G with a receptor of HLA-G.

[0011] In one embodiment of the present invention, the receptor is killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) or leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1).

[0012] Another object of the present invention is to provide an isolated nucleic acid encoding the amino acid sequence of an anti-tumor antigen nanobody as described above, the isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and any combination of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising an anti-tumor antigen nanobody as described above and a pharmaceutically acceptable carrier.

[0014] Another object of the present invention is to provide the use of an anti-tumor antigen nanobody as described above for the preparation of a medicament for treating cancer and immune-related diseases.

[0015] Another object of the present invention is to provide a method for detecting the expression level of HLA-G, comprising administering an anti-tumor antigen nanobody as described above to a sample to be tested.

[0016] In an embodiment of the present invention, the sample to be tested is blood, urine, sputum, saliva or body fluid.

[0017] In summary, the efficacy of the anti-tumor antigen nanobody of the present invention lies in: demonstrating by competitive enzyme linked immunosorbent assay (competitive ELISA) that the anti-HLA-G nanobody blocks the interaction between HLA-G and its receptors, killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) and leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) within 50% of the IC50 blocking activity, enhancing the cytolysis and cytotoxicity of natural killer cells (NK cells) against the human breast cancer cell line MDA-MB-231, demonstrating by Western blotting that the anti-HLA-G nanobody can recognize the HLA-G protein in the cytolysis products of the human cancer cell lines MDA-MB-231 and A549 cells, flow cytometric analysis, demonstrating by immunocytochemical analysis that the anti-HLA-G nanobody can recognize the HLA-G protein on the cell membrane, the expression of HLA-G co-localizes with the plasma membrane marker Pan-Cadherin on MDA-MB-231 and A549 cells, and demonstrating by immunohistochemistry staining (IHC staining) that the anti-HLA-G antibody can be used to detect the expression of HLA-G, thereby achieving the utility of treating cancer and immune-related diseases. In particular, compared with conventional antibodies that have the disadvantages of low yield and ineffective results due to the need to transfect genes into cells through carriers to express antibody functions, the anti-tumor antigen nanobody of the present invention can be directly administered to individuals in need after large-scale preparation in vitro. In addition, the present invention can also achieve the utility of detecting the expression level of HLA-G.

[0018] The following will further illustrate the embodiments of the present invention. The following listed examples are used to clarify the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to that defined by the claims.

[0019]

Brief Description of Drawings

[0020] Figures 1A to 1H Shows the blockade of the HLA-G / killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) or leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) axis by anti-HLA-G nanobody determined by competitive enzyme linked immunosorbent assay (competitive ELISA), where LILRB1 represents leukocyte immunoglobulin-like receptor subfamily B member 1; KIR2DL4 represents killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4; nb represents nanobody; 87G represents a commercial anti-HLA-G monoclonal antibody.

[0021] Figure 2 Is a data graph showing that anti-HLA-G nanobody enhances the cytolysis of human breast cancer cell line MDA-MB-231 by natural killer cells (NK cells), where HLA-GmAB (87G) represents a commercial anti-HLA-G monoclonal antibody (87G).

[0022] Figure 3A Shows the Western blot analysis results of anti-HLA-G nanobody, using the human breast cancer cell line MDA-MB-231 as the cell line and HLA-G (E8N9C) as the commercial antibody Rabbit mAb#79769. The numbers in the upper row represent the amount (μg) of cell lysate of MDA-MB-231 cell line. The concentration of the primary antibody is 1 ng / ml. The secondary antibody of the commercial antibody group is anti-rabbit-horseradish peroxidase (anti-Rab-HRP) (1:1000). The anti-HLA-G nanobody is a heavy chain variable domain (VHH) nanobody (1 ng / ml). The secondary antibodies of the experimental groups (#9, #20) are anti-VHH-HRP (1:1000).

[0023] Figure 3B Showing the Western blot analysis results of anti-HLA-G nanobody. The cell line used is human non-small cell lung cancer cell line A549. The commercial antibody is HLA-G (E8N9C). Rabbit mAb#79769. The numbers in the upper row represent the amount (μg) of cell lysate of A549 cell line. The concentration of the primary antibody is 1 ng / ml. The secondary antibody of the commercial antibody group is anti-rabbit-horseradish peroxidase (1:1000). The anti-HLA-G nanobody is a heavy chain variable domain (VHH) nanobody (1 ng / ml). The secondary antibodies of the experimental groups (#9, #20) are anti-VHH-HRP (1:1000).

[0024] Figure 4 Showing the flow cytometry analysis results of anti-HLA-G nanobody. The amount of human breast cancer cell line MDA-MB-231 and human non-small cell lung cancer cell line A549 is 1×1 0 6. The commercial antibody (commercial Ab) is PE (#12-9957-42) (it is an anti-HLA-G monoclonal antibody, 0.25 μg in 100 μl PBS solution) and 87G. The anti-HLA-G nanobody is a heavy chain variable domain (VHH) nanobody (0.25 μg in 100 μl PBS solution). The secondary antibody is rabbit anti-camelid VHH, iFluor555 (0.5 μg in 100 μl PBS solution). MFI represents mean fluorescence intensity. Unstained represents not stained. Clone represents clone.

[0025] Figure 5A and 5BImmunocytochemical analysis showing anti-HLA-G nanobody, wherein Figure 5A The cell line used was the human breast cancer cell line MDA-MB-231, Figure 5B The cell line used was the human non-small cell lung cancer cell line A549. The commercial antibody 4H84 is a monoclonal anti-HLA-G antibody. The concentration of the anti-HLA-G nanobody was 1 ng / ml, and the secondary antibody was anti-VHH-fluorescein (FITC) (1:5000).

[0026] Figure 6 Immunohistochemical staining results showing anti-HLA-G nanobody, wherein the sample used was human placenta. The commercial antibody was 4H84 (which is a monoclonal anti-HLA-G antibody) (#sc-21799), with a concentration of 200 μg / ml and a working concentration of 4 μg / ml. The secondary antibody for the commercial antibody group was goat anti-rabbit HRP. DAB represents diaminobenzidine (the most sensitive and commonly used chromogenic reactant for horseradish peroxidase). The anti-HLA-G nanobody was a heavy chain variable domain (VHH) nanobody (working concentration 4 μg / ml). The antibodies in the experimental group (#9) included rabbit anti-camelid VHH antibody, biotin (0.5 μg in 100 μl PBS solution), and goat anti-rabbit HRP. Embodiments

[0027] Definitions

[0028] The numerical values used herein are approximate values. All experimental data are expressed within a range of ±20%, preferably within a range of ±10%, and most preferably within a range of ±5%.

[0029] As used herein, the terms "anti-human leukocyte antigen-G (HLA-G) nanobody (NB)" and "anti-tumor antigen nanobody" are used interchangeably.

[0030] As used herein, the term "second antibody" means an antibody that can be conjugated to a nanobody to form a bispecific T-cell engager (BiTE), a triple specific T-cell engager (TriTE), a bispecific killer cell enager (BiKE), a triple specific killer cell engager (TriKE), or any bispecific antibody. Preferably, the second antibody may include, but is not limited to: anti-CD3ε, CD3, programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), T-cell immunoglobulin domain and mucin domain 3 (Tim3), epidermal growth factor receptor (EGFR), EGFRvIII, human epidermal growth factor receptor 2 (Her2), B-cell maturation antigen (BCMA), CD19, CD20, CD34, CD16, Fc, epithelial cell adhesion molecule (EpCAM), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glycoprotein 100 (gp100), and mucin 1 (Muc1) antibodies.

[0031] As used herein, "treating" or "treatment" means alleviating, reducing, ameliorating, relieving or controlling one or more clinical signs of a disease or disorder, and lowering, stopping or reversing the progression of the severity of a condition or symptom being treated.

[0032] The pharmaceutical compositions according to the present invention can be manufactured into a dosage form suitable for parenteral administration by techniques well known to those skilled in the art, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0033] The pharmaceutical compositions according to the present invention can be administered by parenteral routes selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0034] The pharmaceutical product according to the present invention may include a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the professional competence and routine techniques of those skilled in the art.

[0035] The pharmaceutically acceptable carrier according to the present invention includes a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), sugar-containing solution, aqueous solution containing alcohol, and combinations thereof.

[0036] As used herein, terms such as "nucleic acid", "nucleic acid sequence", or "nucleic acid fragment" mean deoxyribonucleotide sequences or ribonucleotide sequences in single-stranded or double-stranded form, and include known naturally occurring nucleotides or artificial chemical mimics. As used herein, the term "nucleic acid" may be used interchangeably with "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide".

[0037] Example 1. Preparation of anti-HLA-G nanobody

[0038] In this embodiment, the preparation process of anti-human leukocyte antigen-G (HLA-G) nanobody (NB) is as follows. The production process of the heavy chain variable domain (VHH) is as follows. The VHH gene is constructed in the expression vector pET22b (Amp resistance) or pSB-init (CmR resistance); the plasmid is identified by restriction endonuclease digestion and sequencing verification. Add 1 μL of the identified plasmid (about 50 ng) into BL21(DE3), and incubate overnight at 37 °C. Inoculate a single colony into LB medium containing the resistance, and incubate the culture overnight at 37 °C and 220 r / min. Inoculate the overnight culture into fresh LB medium containing the resistance (10 L - 20 L) at a ratio of 1:100, and culture at 37 °C and 220 r / min. When the OD 600 reaches 0.8, cool to room temperature. Add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, and induce overnight at 20 °C and 220 r / min. Obtain the cells and supernatant after cell disruption by centrifugation (20 mM Tris pH8.0, 150 mM NaCl). The supernatant is bound to Ni-NTA beads (1 mL) by flow-through. Wash and elute the Ni-NTA beads with buffers containing appropriate gradients of imidazole (10 mM, 20 mM, 50 mM, 100 mM, 250 mM, and 500 mM). Analyze the eluted fraction by SDS-PAGE, and determine the subsequent purification scheme according to the protein purity and yield (ion exchange chromatography or gel filtration chromatography). The qualified protein is separated and purified by gel filtration chromatography, and the buffer is changed to PBS buffer. Analyze the protein components by SDS-PAGE, combine and concentrate the qualified components, filter through a 0.22 μm filter membrane, and aliquot. Then, store the protein at -20 °C or lower temperature.

[0039] Nanobodies were produced and purified from Escherichia coli (E. coli). To produce E. coli in the form of nanobodies, the reference Microb Cell Fact. 2019 Mar 11;18(1):47 was followed. Briefly, the E. coli strain HB2151 was used. The plasmid pET (Creative Biolab) encoding ampicillin resistance was used for cytoplasmic protein production. E. coli HB2151 newly transformed with pET-HLA-G or HLA-G multispecific nanobody plasmid was inoculated into 5 mL of medium containing 50 μg / mL ampicillin and cultured overnight at 37 °C. Then, 1 mL of this pre-culture was inoculated into 100 mL of medium and grown at 37 °C. After overnight culture, two EnPresso Boost tablets and an additional dose of glucose-releasing enzyme (0.6 U / L) were added to each 100 mL of culture. At the same time, recombinant nanobody protein expression was induced by adding 1 mM IPTG for 24 hours. Then the culture was collected and cooled on ice for 5 minutes, and then centrifuged at 6,000×g and 4 °C for 15 minutes. After removing the supernatant, the cell pellet was purified by immobilized metal affinity chromatography (IMAC) using high-capacity Myc-tag binding resin. Gravity-flow-based chromatography was performed under native conditions according to the manufacturer's protocol (Clontech Laboratories). Effective cell lysis was achieved by adding 1 mL of xTractor cell lysis buffer (Clontech Laboratories) to each 200 mg of bacterial cell pellet, supplemented with a protease inhibitor mixture without EDTA (Roche Diagnostics) and 25 U of endonuclease (Thermo Scientific Pierce). After acting on ice for 15 minutes and centrifuging at 10,000×g and 4 °C for 20 minutes to remove cell debris, the clarified supernatant was added to a gravity-flow column containing 1 mL of pre-packed resin and allowed to act at room temperature for 30 minutes. The column was washed twice with 20 and 40 mM imidazole concentrations before eluting the nanobody with an elution buffer containing 300 mM imidazole. The cellulose ester membrane ( Laboratories) was used to remove imidazole and change the buffer by dialysis against PBS.

[0040] The alignment and amino acid sequences of the complementarity determining regions (CDRs) of each clone of the anti-HLA-G nanobody are shown in Table 1. The amino acid sequence of the anti-HLA-G nanobody clone #9 is SEQ ID NO.1; the amino acid sequence of the anti-HLA-G nanobody clone #20 is SEQ ID NO.2; the amino acid sequence of the anti-HLA-G nanobody clone #33 is SEQ ID NO.3; the nucleotide sequence encoding the amino acid sequence of the anti-HLA-G nanobody clone #9 is SEQ ID NO.4; the nucleotide sequence encoding the amino acid sequence of the anti-HLA-G nanobody clone #20 is SEQ ID NO.5; the nucleotide sequence encoding the amino acid sequence of the anti-HLA-G nanobody clone #33 is SEQ ID NO.6.

[0041] Table 1

[0042] Clone CDR1 CDR2 CDR3 #9 GRTYSSNC IYTGGDGI AADPNRRRMGVGGSC #20 GFTVDDSD ITSGGGK VAPAWTGYGCT #33 AYTFSASG AATYTRSAKT AVARCAGRPDRSTLTSFAW

[0043] Example 2. Determination of the HLA-G / killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) or leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) axis blockade of the anti-HLA-G nanobody by competitive enzyme linked immunosorbent assay (competitive ELISA)

[0044] In this example, the operation process for measuring the blockade of the HLA-G / killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) axis by anti-HLA-G nanobody using competitive enzyme linked immunosorbent assay (competitive ELISA) is as follows. First, HLA-G recombinant protein (CAT#: TP305216, Origene) (0.2 μg / ml, 100 μl per well) was coated on a 96-well plate at 4 °C overnight. The next day, the coating buffer was removed, and blocked with 3% skim milk at room temperature for 2 hours. Then, it was washed 5 times with PBST (0.05% Tween dissolved in PBS). Different concentrations of anti-HLA-G nanobody (clone #9, #20 or #33) or commercial anti-HLA-G monoclonal antibody (87G) were added and incubated overnight at 4 °C. After washing 5 times, biotinylated KIR2DL4 (Sino Biological, Cat: 13052-H02S) (0.2 mg / ml, 100 μl per well) was added at room temperature for 2 hours. After washing 9 times, each well was incubated with 100 μl of PBST containing streptavidin-HRP conjugates (ThermoFisher, CatNo: N100, dilution titer: 5000:1) at room temperature for 2 hours. After washing 9 times with PBST, 50 μl of TMB substrate (for detecting HRP activity) (ThermoFisher, Cat No: N301) was added. Then, 50 μl of stop solution (ThermoFisher, Cat No: N600) was added to terminate the reaction, and then measured using an ELISA reader with a wavelength of 450 nm. The highest concentration of the commercial anti-HLA-G monoclonal antibody (87G) was set as 100% blocking of the KIR2DL4 / HLA-G interaction to calculate other reactions.

[0045] In addition, the operation process for measuring the blocking of the HLA-G / leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) axis by anti-HLA-G nanobody using competitive enzyme-linked immunosorbent assay (competitive ELISA) is generally the same as above, with the differences being that biotinylated KIR2DL4 is replaced by biotinylated LILRB1 (Sino Biological, Cat: 16014-H08H), and the highest concentration of the commercial anti-HLA-G monoclonal antibody (87G) is set to 100% to block the LILRB1 / HLA-G interaction for calculating other reactions.

[0046] The results of this example are shown in Figures 1A to 1H, where LILRB1 represents leukocyte immunoglobulin-like receptor subfamily B member 1; KIR2DL4 represents killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4; nb represents nanobody; 87G represents a commercial anti-HLA-G monoclonal antibody. The results of this example confirmed that the commercial anti-HLA-G monoclonal antibody (87G) blocked the interaction between HLA-G and one of its receptors, KIR2DL4, within 50% blocking activity at 41.4 ng / ml (IC50). Anti-HLA-G nanobody clone #9 blocked the interaction between HLA-G and one of its receptors, KIR2DL4, within 50% blocking activity at 6.14 ng / ml (IC50), and the blocking activity was normalized to 87G. Anti-HLA-G nanobody clone #20 blocked the interaction between HLA-G and one of its receptors, KIR2DL4, within 50% blocking activity at 814 ng / ml (IC50), and the blocking activity was normalized to 87G. Anti-HLA-G nanobody clone #33 blocked the interaction between HLA-G and one of its receptors, KIR2DL4, within 50% blocking activity at 53.3 ng / ml (IC50), and the blocking activity was normalized to 87G. The commercial anti-HLA-G monoclonal antibody (87G) blocked the interaction between HLA-G and one of its other receptors, LILRB1, within 50% blocking activity at 100.9 ng / ml (IC50). Anti-HLA-G nanobody clone #9 blocked the interaction between HLA-G and one of its other receptors, LILRB1, within 50% blocking activity at 0.825 ng / ml (IC50), and the blocking activity was normalized to 87G. Anti-HLA-G nanobody clone #20 blocked the interaction between HLA-G and one of its other receptors, LILRB1, within 50% blocking activity at 0.174 ng / ml (IC50), and the blocking activity was normalized to 87G. Anti-HLA-G nanobody clone #33 blocked the interaction between HLA-G and one of its other receptors, LILRB1, within 50% blocking activity at 0.074 ng / ml (IC50), and the blocking activity was normalized to 87G.

[0047] Example 3. Evaluation of the utility of anti-HLA-G nanobody in enhancing the cytolysis of human breast cancer cell line MDA-MB-231 by natural killer cells (NK cells)

[0048] In this example, the experimental procedure for evaluating the utility of anti-HLA-G nanobody in enhancing the cytolysis of human breast cancer cell line MDA-MB-231 (purchased from American Type Culture Collection (ATCC)) by natural killer cells (NK cells) is as follows. 1×10 5 MDA-MB-231 cells were seeded into 12-well plates and incubated overnight. The next day, 5×10 5 primary NK cells were added to the wells containing MDA-MB-231 cells. Then, 1 μg / ml anti-HLA-G nanobody (clone #9, #20 or #33) or 10 μg / ml commercial anti-HLA-G monoclonal antibody (87G) (87G, Thermo Fisher, Cat No: 14-9957-82) was added. After 48 hours, the specific lysis of primary NK cells against MDA-MB-231 cells was determined by live-dead cell-mediated cytotoxicity assay using flow cytometry analysis.

[0049] The results of the anti-HLA-G nanobody enhancing the cytolysis of human breast cancer cell line MDA-MB-231 by NK cells are shown in Figure 2 . The results of this example confirmed that anti-HLA-G nanobody clones #9, #20 and #33 enhanced the cytotoxicity of NK cells-induced against tumor cells (MDA-MB-231).

[0050] Example 4. Results of Western blotting of anti-HLA-G nanobody

[0051] In this example, the operation process of Western blotting of anti-HLA-G nanobody is as follows. Cells are obtained in PRO-PREP protein extraction solution (iNtRON, Taipei City, Taiwan Province, China) containing protease inhibitor mixture, and shaken vigorously at 4 °C for 15 minutes, and then centrifuged. The supernatant is collected, and then the protein concentration is measured using Bio-Rad BCA reagent (Bio-Rad Hercules, CA, USA). 30 μg of each sample lysate is electrophoresed on an SDS-polyacrylamide gel and then electroblotted onto a PVDF membrane. After adding 5% BSA in TBST blocking, the membrane is incubated with the primary antibody (dissolved in TBST) overnight at 4 °C. Then, it is washed 4 times and incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse or rabbit IgG (Upstate, Billerica, MA, USA) for two hours. After washing 4 times with TBST, the blot is incubated with SuperSignal West Pico ECL reagent (Pierce Biotechnology, Rockford, IL, USA) for 1 minute, and then chemiluminescence is detected by exposure to Kodak-X-Omat film.

[0052] The results of Western blotting of anti-HLA-G nanobody are shown in Figure 3A and Figure 3B , where Figure 3A the cell line used is the human breast cancer cell line MDA-MB-231, and the commercial antibody is HLA-G (E8N9C) Rabbit mAb#79769. The numbers in the upper row represent the amount (μg) of cell lysate of MDA-MB-231 cell line. The concentration of the primary antibody is 1 ng / ml. The secondary antibody for the commercial antibody group is anti-rabbit-horseradish peroxidase (anti-Rab-HRP) (1:1000). The anti-HLA-G nanobody is a heavy chain variable domain (VHH) nanobody (1 ng / ml). The secondary antibody for the experimental groups (#9, #20) is anti-VHH-HRP (1:1000).

[0053] Figure 3BThe cell line used was the human non-small cell lung cancer cell line A549 (purchased from the American Type Culture Collection (ATCC)), and the commercial antibody was HLA-G (E8N9C). Rabbit mAb #79769. The upper row numbers represent the amount (μg) of cell lysates of the A549 cell line. The concentration of the primary antibody was 1 ng / ml. The secondary antibody for the commercial antibody group was anti-rabbit-horseradish peroxidase (1:1000), and the anti-HLA-G nanobody was a heavy chain variable domain (VHH) nanobody (1 ng / ml). The secondary antibody for the experimental groups (#9, #20) was anti-VHH-HRP (1:1000). The results of this example showed that by Western blot analysis, anti-HLA-G nanobody clones #9 and #20 could recognize the HLA-G protein in the cell lysates of human cancer cell lines MDA-MB-231 and A549 cells.

[0054] Example 5. Results of flow cytometric analysis of anti-HLA-G nanobody

[0055] In this example, the operation process of flow cytometric analysis of anti-HLA-G nanobody is as follows. First, HLA-G recombinant protein (CAT#: TP305216, Origene) (0.2 μg / ml, 100 μl per well) was coated overnight at 4 °C on a 96-well plate. The next day, the coating buffer was removed and blocked with 3% skim milk for 2 hours at room temperature. Then, it was washed 5 times with PBST (0.05% Tween dissolved in PBS). Different concentrations of anti-HLA-G nanobodies (clone #9, #20 or #33) or commercial anti-HLA-G monoclonal antibody (87G) were added and incubated overnight at 4 °C. After washing 5 times, biotinylated KIR2DL4 (Sino Biological, Cat: 13052-H02S) (0.2 mg / ml, 100 μl per well) was added for 2 hours at room temperature. After washing 9 times, each well was incubated with 100 μl of PBST containing streptavidin-HRP conjugates (ThermoFisher, CatNo: N100, dilution titer: 5000:1) for 2 hours at room temperature. After washing 9 times with PBST, 50 μl of TMB substrate (for detecting HRP activity) (ThermoFisher, Cat No: N301) was added. Then, 50 μl of stop solution (ThermoFisher, Cat No: N600) was added to terminate the reaction, and the measurement was then performed using an ELISA reader at a wavelength of 450 nm. The highest concentration of the commercial anti-HLA-G monoclonal antibody (87G) was set as 100% blocking of the KIR2DL4 / HLA-G interaction to calculate other reactions.

[0056] The results of flow cytometric analysis of anti-HLA-G nanobody are shown in Figure 4 , where the amounts of human breast cancer cell line MDA-MB-231 and human non-small cell lung cancer cell line A549 are 1×10 6The commercial antibodies (commercial Ab) were PE (#12-9957-42) (which is a monoclonal antibody against HLA-G, 0.25 μg in 100 μl of PBS solution) and the commercial monoclonal antibody 87G. The anti-HLA-G nanobody was a heavy chain variable domain (VHH) nanobody (0.25 μg in 100 μl of PBS solution). The secondary antibody was rabbit anti-camelid VHH, iFluor555 (0.5 μg in 100 μl of PBS solution). MFI represents mean fluorescence intensity, unstained represents not stained, and clone represents clone.

[0057] Example 6. Results of immunocytochemistry analysis of anti-HLA-G nanobody

[0058] In this example, the operation procedure for the immunocytochemistry analysis of the anti-HLA-G nanobody is as follows. Tumor cells (1×10 5 ) were seeded on the coverslips of a 6-well plate and incubated overnight. After the designated treatment, the cells were fixed in 1% paraformaldehyde, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS containing 0.5% BSA for 30 minutes, blocked with 2% BSA, and incubated with the specific antibody (in 2% BSA / PBS (PBST) containing 0.05% Tween-20). After washing, the cells were incubated with the fluorophore-conjugated secondary antibody, washed with PBST, and mounted with an aqueous mounting medium containing an anti-fading agent and 4',6-diamidino-2-phenylindole (DAPI). The images were analyzed under a Leica TCS SP8 X confocal microscope (Leica).

[0059] The results of the immunocytochemistry analysis of the anti-HLA-G nanobody are shown in Figure 5A and 5B where Figure 5A the cell line used was the human breast cancer cell line MDA-MB-231, Figure 5BThe cell line used was the human non-small cell lung cancer cell line A549. The commercial antibody 4H84 is an anti-HLA-G monoclonal antibody. The concentration of the anti-HLA-G nanobody was 1 ng / ml, and the secondary antibody was anti-VHH-fluorescein (FITC) (1:5000). From Figure 5A and 5B It can be seen that by immunohistochemical analysis, the anti-HLA-G nanobody clones #9 and #20 can recognize the HLA-G protein on the cell membrane. Using the anti-HLA-G nanobody clones #9 and #20, the commercial antibody 4H84, and the commercial Pan-Cadherin antibody,

[0060] the expression of HLA-G was co-localized with the plasma membrane marker Pan-Cadherin on MDA-MB-231 and A549 cells.

[0061] Example 7. Results of immunohistochemical staining of anti-HLA-G nanobody

[0062] In this example, the procedure for immunohistochemical staining of the anti-HLA-G nanobody was as follows. Human placenta samples were fixed in 10% formaldehyde and embedded in paraffin. Sections (thickness = 3 μm) were stained with hematoxylin and eosin. For immunohistochemistry, antigen retrieval was carried out by microwave at 99°C. The sections were washed with H2O2 and incubated for 20 minutes to block endogenous peroxidase, and then soaked in 5% BSA for 30 minutes. Then the sections were incubated with the primary antibody overnight at 4°C. After washing, the sections were incubated with the diluted biotin-conjugated secondary antibody for 2 hours at room temperature or overnight at 4°C. Finally, the sections were incubated with the polymer for 10 minutes at room temperature and then stained with diaminobenzidine (DAB) (the most sensitive and commonly used chromogenic reactant for horseradish peroxidase), stained with hematoxylin and eosin, and fixed with neutral rubber. Quantification of the staining was performed independently by optical microscopy (Nikon, Japan) at magnifications of 40x and 400x.

[0063] The results of immunohistochemical staining of the anti-HLA-G nanobody are shown in Figure 6, where the sample used was human placenta, the commercial antibody was 4H84 (which is a monoclonal anti-HLA-G antibody) (#sc-21799), with a concentration of 200 μg / ml and a working concentration of 4 μg / ml. The secondary antibody for the commercial antibody group was goat anti-rabbit HRP. DAB represents diaminobenzidine (which is the most sensitive and commonly used chromogenic reactant for horseradish peroxidase). The anti-HLA-G nanobody was a heavy chain variable domain (VHH) nanobody (with a working concentration of 4 μg / ml). The antibodies in the experimental group (#9) included rabbit anti-camelid VHH antibody, biotin (0.5 μg dissolved in 100 μl of PBS solution), and goat anti-rabbit HRP. The results of this example confirmed that the anti-tumor antigen nanobody (i.e., anti-HLA-G antibody) can be used to detect the expression of HLA-G by immunohistochemical staining.

[0064] In summary, the anti-tumor antigen nanobody of the present invention (i.e., anti-HLA-G antibody) proves that the anti-HLA-G nanobody blocks the interaction between HLA-G and its receptors: killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 4 (KIR2DL4) and leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) within 50% of the IC50 of the competitive enzyme linked immunosorbent assay (competitive ELISA), enhances the cytolysis and cytotoxicity of natural killer cells (NK cells) against the human breast cancer cell line MDA-MB-231, proves that the anti-HLA-G nanobody can recognize the HLA-G protein in the cytolysis products of the human cancer cell lines MDA-MB-231 and A549 cells by Western blotting, flow cytometric analysis, proves that the anti-HLA-G nanobody can recognize the HLA-G protein on the cell membrane by immunocytochemical analysis, the expression of HLA-G co-localizes with the plasma membrane marker Pan-Cadherin on MDA-MB-231 and A549 cells, and proves that the anti-HLA-G antibody can be used to detect the expression of HLA-G by immunohistochemistry staining (IHC staining), thereby achieving the utility of treating cancer and immune-related diseases. In particular, compared with the conventional antibodies that have the disadvantages of low yield and ineffective results due to the need to transfect genes into cells through vectors to express antibody functions, the anti-tumor antigen nanobody of the present invention can be directly administered to the individuals in need after large-scale preparation in vitro for treatment. In addition, the present invention can also achieve the utility of detecting the expression level of HLA-G.

[0065] The above description is only illustrative and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the claims.  <110> China Medical University Hospital, Taiwan <120> Anti-tumor antigen nanobody, its nucleic acid coding sequence and application <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Artificial sequence <220> <223> Anti-HLA-G nanobody clone #9 <400> 1 Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Arg Thr Tyr Ser 20 25 30 Ser Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Asn Val Ala Ala Ile Tyr Thr Gly Gly Asp Gly Ile Thr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys Leu Lys Asn Met 65 70 75 80 Leu Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Asp Pro Asn Arg Arg Arg Met Gly Val Gly Gly Ser 100 105 110 Cys Leu Arg Ala Asn Phe Gly Pro Gly Gly Gln Gly Thr Gln Val Thr 115 120 125 Val Ser Ser Leu Glu 130 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Anti-HLA-G Nanobody Clone #20 <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Arg Pro Gly Glu 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Val Asp Asp Ser 20 25 30 Asp Met Ser Trp Tyr Arg Gln Ala Pro Gly Asp Glu Cys Glu Leu Val 35 40 45 Ser Ser Ile Thr Ser Gly Gly Gly Lys Tyr Tyr Ser His Pro Val Lys 50 55 60 Gly Arg Phe Thr Ile Phe Arg Asp Lys Gly Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Val Pro Glu Asp Thr Gly Val Tyr Tyr Cys Val 85 90 95 Ala Pro Ala Trp Thr Gly Tyr Gly Cys Thr Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Anti-HLA-G Nanobody Clone #33 <400> 3 Met Gly His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala 1 5 10 15 Gly Gly Ser Leu Lys Leu Ser Cys Val Thr Ser Ala Tyr Thr Phe Ser 20 25 30 Ala Ser Gly Asn Cys Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly 35 40 45 Arg Glu Gly Ile Ala Ala Thr Tyr Thr Arg Ser Ala Lys Thr Tyr Tyr 50 55 60 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys 65 70 75 80 Asn Thr Val Tyr Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala 85 90 95 Thr Tyr Tyr Cys Ala Val Ala Arg Cys Ala Gly Arg Pro Asp Arg Ser 100 105 110 Thr Leu Thr Ser Phe Ala Trp Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125 Ser Ser Leu Glu 130 <210> 4 <211> 399 <212> DNA <213> Artificial sequence <220> <223> Anti-HLA-G nanobody clone #9 <400> 4 atgggccagg tgcagctggt ggaaagcggc ggcggtagtg ttcaggcagg tggcagcctg 60 cgcctgagtt gtgaagcaag cggccgcacc tatagcagca attgcatggg ttggtttcgc 120 caggcaccgg gcaaagaacg cgaaaatgtg gcagcaatct ataccggcgg tgacggtatt 180 acctatgccg atagtgtgaa aggtcgtttt accattagtc aggataaact gaaaaacatg 240 ctgtatctgc agatggatag cctgaaaccg gaagataccg ccatgtatta ttgcgcagca 300 gatccgaatc gccgccgtat gggcgttggt ggcagctgcc tgcgcgccaa ttttggcccg 360 ggcggccagg gtacccaggt gaccgttagc agcctcgag 399 <210> 5 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Anti-HLA-G nanobody clone #20 <400> 5 gaagtgcagc tggttgaaag tggtggtggc agcgttcgcc cgggcgaaac cttacgtctg 60 agctgcaccg caagtggctt caccgtggat gatagcgata tgagctggta tcgccaggcc 120 ccgggtgatg aatgtgaact ggttagtagc attaccagtg gtggcggcaa atattatagt 180 catccggtga aaggccgctt caccatcttc cgtgataaag gtaaaaatac catgtatctg 240 cagatgaata gcctggttcc ggaagatacc ggcgtgtatt attgtgtggc cccggcctgg 300 accggctatg gttgcacctg gggccagggt acccaggtga ccgttagcag t 351 <210> 6 <211> 396 <212> DNA <213> Artificial sequence <220> <223> Anti-HLA-G nanobody clone #33 <400> 6 atgggccatg ttcagctggt tgaaagtggc ggtggcagtg tgcaggccgg tggtagtctg 60 aaactgagct gcgttaccag cgcatatacc ttcagcgcaa gcggtaattg tatgggctgg 120 ctgcgtcagg caccgggtaa aggccgcgaa ggtattgcag ccacctatac ccgtagtgcc 180 aaaacctatt atgcagatag tgttaaaggt cgcttcacca ttagtcagga taatgcaaaa 240 aataccgtgt atctgcagat gaatggtctg aaaccggaag ataccgccac ctattattgt 300 gcagtggcac gctgtgccgg tcgtccggat cgtagtaccc tgaccagctt cgcctggtgg 360 ggtcagggca cccaggtgac cgtgagcagc ctcgag 396

Claims

1. An anti-tumor antigen nanobody, characterized in that, The anti-tumor antigen nanobody specifically binds to human leukocyte antigen-G (HLA-G), and the amino acid sequence of the anti-tumor antigen nanobody is shown as SEQ ID NO.

1.

2. An isolated nucleic acid, characterized in that, Encoding the anti-tumor antigen nanobody according to claim 1, the nucleotide sequence of the isolated nucleic acid is SEQ ID NO.

4.

3. A pharmaceutical composition, characterized in that, Comprising the anti-tumor antigen nanobody according to claim 1 and a pharmaceutically acceptable carrier.

4. Use of the anti-tumor antigen nanobody according to claim 1 for the preparation of a medicament for treating cancer, characterized in that, The cancer is breast cancer or non-small cell lung cancer.

5. Use of the anti-tumor antigen nanobody according to claim 1 in the preparation of a kit for detecting the expression level of HLA-G in a sample to be tested.

6. The use according to claim 5, characterized in that, Wherein the sample to be tested is a body fluid.

7. The use according to claim 5, wherein The sample to be tested is blood, urine, sputum or saliva.

Images