Immune regulation nanobody, its nucleic acid coding sequence and application

Abstract

The present invention provides an immune regulatory nanobody that specifically binds to a programmed cell death ligand 1. The present invention also provides a nucleic acid coding sequence of the immune regulatory nanobody, the use of the immune regulatory nanobody for treating cancer and immune-related diseases, and a method for detecting the expression level of PD-L1.

Description

Technical Field

[0001] The present invention relates to the technical field of immune regulatory nanobodies, and particularly to an immune regulatory nanobody, its nucleic acid coding sequence, and its application. 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 malfunction of the mechanism that controls cell division and proliferation. The population suffering from cancer worldwide has a tendency to increase continuously. Cancer is one of the top ten causes of death among Chinese people and has ranked first among 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 eliminating or controlling tumors. However, the current tumor treatment methods still have problems of ineffective treatment and strong side effects, and even may give rise to other immune-related diseases.

[0004] Programmed cell death ligand 1 (PD-L1) has been found to be expressed on the cell surface of a variety of solid tumors. Therefore, some researchers have been committed to developing PD-L1 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 immune regulatory nanobody that specifically binds to a programmed cell death ligand 1 (PD-L1). The immune regulatory 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 an amino acid sequence of a heavy chain variable domain (VHH) of the immune regulatory nanobody.

[0008] In one embodiment of the present invention, the immune regulatory nanobody is conjugated to a fragment crystallizable region (Fc region).

[0009] In one embodiment of the present invention, the immune regulatory 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 immune regulatory nanobody blocks the interaction and / or binding of the PD-L1 to a receptor of the PD-L1.

[0011] In one embodiment of the present invention, the receptor is programmed cell death protein-1 (PD-1).

[0012] Another object of the present invention is to provide an isolated nucleic acid encoding an amino acid sequence of an immune regulatory 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.4, 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 immune regulatory nanobody as described above and a pharmaceutically acceptable carrier.

[0014] Another object of the present invention is to provide the use of an immune regulatory 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 PD-L1, which comprises administering an immune regulatory 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 immune regulatory nanobody of the present invention lies in: it is proved by surface plasmon resonance binding assay (SPR binding assay) that the anti-PD-L1 nanobody effectively binds to the PD-L1 protein with KD within 0.27 and 0.41 nM respectively; the PD-1 / PD-L1 axis blockade of the anti-PD-L1 nanobody is determined by the PD-1 / PD-L1 Blockade Bioassay kit, which proves that the anti-PD-L1 nanobody blocks the PD-L1 / PD-1 signal transduction in the PD-L1, APC / PD-1 effector co-culture system; the anti-PD-L1 nanobody enhances the cytotoxicity of γδT cells-induced on tumor cells (MDA-MB-231); it is proved by Western blotting that the anti-PD-L1 nanobody restores the proliferation of OKT3 (anti-CD3 monoclonal antibody)-induced T cells after PD-L1 conjugation, and it is proved by flow cytometry analysis and immunocytochemistry analysis that the anti-PDL-1 nanobody can be used to detect the expression of PD-L1 in cell samples, thereby achieving the utility of treating cancer and immune-related diseases. In particular, compared with the conventional antibody that has the disadvantages of low yield and ineffective results because the gene needs to be transfected into cells through a vector to express the antibody function, the immune regulatory 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 PD-L1.

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

[0019]

Brief Description of the Drawings

[0020] Figure 1A and Figure 1BSurface plasmon resonance binding analysis results of anti-PD-L1 nanobody are shown, where clone represents the clone, nb represents the nanobody, and RU represents the response unit, which is the CM5 chip coated with recombinant PD-L1 protein (Sino Biological, Cat: 10084-H05H).

[0021] Figure 2 Results of PD-1 / PD-L1 axis blockade of anti-PD-L1 nanobody measured by PD-1 / PD-L1 Blockade Bioassay kit are shown, where clone represents the clone, PD-L1nb represents the anti-PD-L1 nanobody, and Atezolizumab is an IgG1 isotype fully humanized anti-PD-L1 monoclonal antibody used for the treatment of urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer, small cell lung cancer, and hepatocellular carcinoma.

[0022] Figure 3 T cell proliferation analysis results of anti-PD-L1 nanobody are shown, where ** indicates p < 0.01; *** indicates p < 0.001.

[0023] Figure 4 Results showing that anti-PD-L1 nanobody enhances γδT (GDT)-induced cytotoxicity against human breast cancer cell line MDA-MB-231 are shown. The leftmost group represents the group treated with γ-δT cells alone without any antibody combination; * indicates p < 0.05; ** indicates p < 0.01.

[0024] Figure 5 Western blotting results of anti-PD-L1 nanobody are shown. The arrow indicates the position of the PD-L1 protein, which is the molecular weight position consistent with the commercial antibody. The cell line used is non-small cell lung cancer cell line H1975 (NCI-H1975[H-1975, H1975], ATCC), mAb represents monoclonal antibody, and Nb represents nanobody. The commercial antibody is a PD-L1 monoclonal antibody (66248-1-Ig, Proteintech). The primary antibody in the commercial antibody group is a PD-L1 monoclonal antibody (1:2500), and the secondary antibody is anti-rabbit-horseradish peroxidase (anti-Rab-HRP) (1:10000). The concentration of the anti-PD-L1 nanobody in the experimental group is 1 ng / ml, and the secondary antibody is anti-VHH-HRP (1:10000).

[0025] Figure 6A and Figure 6B showing the flow cytometry analysis results of anti-PD-L1 nanobody, wherein Figure 6A in (a), nb represents nanobody, Ab represents antibody, and FITC represents fluorescein isothiocyanate, Figure 6A in (b), mAb represents monoclonal antibody, H1975 is a non-small cell lung cancer cell line, and Alexa Fluor 488-H is a bright green fluorescent dye that is excited by a laser beam with a wavelength of 488 nm, Figure 6B in (a), A549 is a human non-small cell lung cancer cell line, Figure 6B in (b), MDA-MB-231 is a human breast cancer cell line.

[0026] Figure 7 showing the immunocytochemical analysis results of anti-PD-L1 nanobody, wherein H1975 is a non-small cell lung cancer cell line, clone represents a clone, the concentration of the anti-PDL-1 nanobody is 1 ng / ml, the secondary antibody is anti-VHH-fluorescein (FITC) (1:5000), pan-cadherin is a cell membrane marker, and the co-localization of pan-cadherin and PD-L1 can be used to explain that membrane-bound PD-L1 is detected by a specific nanobody. Embodiments

[0027] Definitions

[0028] The numerical values used herein are approximate values, and 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-programmed cell death ligand 1 (PD-L1) nanobody (NB)" and "immune regulatory nanobody" can be 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, human leukocyte antigen-G (HLA-G), 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 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 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 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 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" refer to 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" can be used interchangeably with "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide".

[0037] Example 1. Preparation of Anti-PD-L1 Nanobody

[0038] In this example, the preparation process of anti-programmed cell death ligand 1 (PD-L1) 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) to 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). Bind the supernatant 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 protocol based on 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 the plasmid of PD-L1 or PD-L1 multispecific nanobody was inoculated into 5 mL of medium containing 50 μg / mL ampicillin and cultured overnight at 37 °C. Then, 1 mL of this preculture 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 selected plant of the anti-PD-L1 nanobody are shown in Table 1. The amino acid sequence of the anti-PD-L1 nanobody selected plant #1 is SEQ ID NO.1; the amino acid sequence of the anti-PD-L1 nanobody selected plant #14 is SEQ ID NO.2; the amino acid sequence of the anti-PD-L1 nanobody selected plant #67 is SEQ ID NO.3; the nucleotide sequence encoding the amino acid sequence of the anti-PD-L1 nanobody selected plant #1 is SEQ ID NO.4; the nucleotide sequence encoding the amino acid sequence of the anti-PD-L1 nanobody selected plant #14 is SEQ ID NO.5; the nucleotide sequence encoding the amino acid sequence of the anti-PD-L1 nanobody selected plant #67 is SEQ ID NO.6.

[0041] Table 1

[0042]

[0043] Example 2. Results of the surface plasmon resonance binding assay (SPR binding assay) of the anti-PD-L1 nanobody

[0044] In this example, the operating procedure of the surface plasmon resonance binding assay (SPR binding assay) of the anti-PD-L1 nanobody is as follows. The CM5 or NTA chip is subjected to SPR analysis by BIAcore T200 (Biacore - GE Healthcare, Piscataway, NJ). Briefly, protein (PD-L1 recombinant protein) samples are diluted in a 10 mM buffer solution (pH 4.0, 5.5 or 6.0) at a concentration range of 20 μg / mL to obtain maximum surface retention for immobilization on the chip. Follow the surface preparation process and select ligands (PD-L1 or PD-L1 multispecific nanobody, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 nM) to coat on the chip. Then comes regeneration scouting and surface performance test. After regeneration scouting and surface performance test, then select a regeneration method to run the experiment. Then select BINDING ANALYSIS and DIRECT BINDING to study protein binding. Select KINETIC ANALYSIS and select MASS TRANSFER for kinetic analysis of the binding experiment. After that, perform data analysis and determination of kinetic constants.

[0045] The results of the surface plasmon resonance binding assay of the anti-PD-L1 nanobody are shown in Figure 1A and Figure 1B , where clone represents the clone, nb represents the nanobody, and RU represents the response unit. From Figure 1A and Figure 1B it can be seen that the anti-PD-L1 nanobody effectively binds the PD-L1 protein with KD within 0.27 and 0.41 nM respectively.

[0046] Example 3. Determination of the PD-1 / PD-L1 axis blockade of the anti-PD-L1 nanobody by a PD-1 / PD-L1 Blockade Bioassay kit

[0047] In this example, the operating procedure of the PD-1 / PD-L1 axis blockade of the anti-PD-L1 nanobody determined by a PD-1 / PD-L1 Blockade Bioassay kit is as follows. Dilute 1x104 The PD-L1 aAPC / CHO-K1 cells were seeded in 96-well plates overnight. The next day, 1x10 4 PD-1 effector cells were added to the wells containing PD-L1 aAPC / CHO-K1 cells. Subsequently, different concentrations of anti-PD-L1 nanobody (clone #1 or clone #67) or atezolizumab (a IgG1 isotype fully humanized anti-PD-L1 monoclonal antibody used for the treatment of urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer, small cell lung cancer and hepatocellular carcinoma) were added. After 6 hours, Bio-Glo TM reagent was added and luminescence was measured using the Discover system, and the data was fitted to a 4PL curve using Sigmaplot software.

[0048] The results of this example are shown in Figure 2 , where clone represents the clone, and atezolizumab is a IgG1 isotype fully humanized anti-PD-L1 monoclonal antibody used for the treatment of urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer, small cell lung cancer and hepatocellular carcinoma. As can be seen from Figure 2 , anti-PD-L1 nanobody clones #1 and #67 blocked PD-L1 / PD-1 signal transduction in the PD-L1, APC / PD-1 effector co-culture system.

[0049] Example 4. Results of T cell proliferation assay of anti-PD-L1 nanobody

[0050] In this example, the procedure for the T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation assay of anti-PD-L1 nanobody was as follows. 1x10 6 PBMC cells were seeded in 12-well plates, with or without recombinant human PD-L1 (10 μg / ml, SinoBiological, Cat. 10084-H05H). Then, 1 μg / ml of anti-PD-L1 nanobody (clone #1 or clone #67) was added. After 7 days, the total cell number was recorded, and then stained with FITC-conjugated anti-CD3 monoclonal antibody (Cat#11-0037-42), and then analyzed by flow cytometry. The number of CD3 positive cells was calculated as the percentage of CD3 cells (%) × total cell number, and the group with anti-CD3 monoclonal antibody alone was 100%.

[0051] The results of T cell proliferation analysis of anti-PD-L1 nanobody are shown in Figure 3 , where ** indicates p < 0.01; *** indicates p < 0.001. As shown by Figure 3 , the anti-PD-L1 nanobody clones #1 and #67 enhanced γδ T cell-induced cytotoxicity against tumor cells (MDA-MB-231).

[0052] Example 5. Evaluation of the utility of anti-PD-L1 nanobody in enhancing gamma delta T (GDT)-induced cytotoxicity against human breast cancer cell line MDA-MB-231

[0053] In this example, the experimental procedure for the anti-PD-L1 nanobody to enhance γδ T (GDT)-induced cytotoxicity against human breast cancer cell line MDA-MB-231 (purchased from American Type Culture Collection, ATCC) is as follows. Peripheral blood mononuclear cells (PBMCs), natural killer cells or γδ T cells are used as effector cells. The target cells (tumor cell line) and effector cells are co-cultured at 37 °C for 24 to 72 hours at a specified effector / target (E:T) ratio from 1:1 to 50:1. For live / dead cell viability analysis, all tumor cells are stained with green-fluorescent calcein-AM before co-culture and then stained with red-fluorescent ethidium homodimer-1 to label dead cells after co-culture. According to the manufacturer's instructions (ThermoFisher Scientific), dead tumor cells are identified as green-fluorescent + / red-fluorescent + cells. The cell killing rate is expressed as a percentage of the total cell population.

[0054] The procedure for primary γδ T cell expansion is as follows. Human PBMCs (1x10 7) Cultured for 2 weeks in X-VIVO15 medium (Lonza, Basel, Switzerland) containing zoledronic acid (5 μM) and 1000 IU / ml IL-2 and supplemented with 10% platelet-rich plasma (PRP). The cell number was recorded, and the purity and potency were determined by flow cytometry using fluorescently conjugated CD3, Vγ9, Vδ2, and NKG2D antibodies.

[0055] The procedure for primary natural killer cell expansion is as follows. Human natural killer cells were isolated from PBMCs by a negative selection kit according to the manufacturer's instructions (STEMCELL Technologies Vancouver Canada). The natural killer cells were cultured for 3 weeks in X-VIVO15 medium (Lonza, Basel, Switzerland) containing CD355 and CD2 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) and 500 IU / ml IL-2 (PeproTech, Rocky Hill, USA) and supplemented with 10% platelet-rich plasma (PRP). The cell number was recorded, and the purity was determined by flow cytometry using fluorescently conjugated CD56 and CD16 antibodies for natural killer cells.

[0056] 1x10 5 MDA-MB-231 cells were seeded in a 12-well plate overnight. The next day, 3x10 5 primary γδT cells were added to the wells containing MDA-MB-231 cells. Then, 1 μg / ml anti-PD-L1 nanobody (clone #1 or clone #67) or 10 μg / ml atezolizumab was added. After 48 hours, the specific lysis of primary γδT cells against MDA-MB-231 cells was determined by live / dead cell-mediated cytotoxicity assay using flow cytometry analysis.

[0057] The results of anti-PD-L1 nanobody enhancing γδT (GDT)-induced cytotoxicity against human breast cancer cell line MDA-MB-231 are shown in Figure 4 , where the leftmost group represents the treatment group of pure γ-δT cells without combination with any antibody; * indicates p < 0.05; ** indicates p < 0.01. The results of this example show that anti-PD-L1 nanobody can enhance γδT cell-induced cytotoxicity against tumor cells (MDA-MB-231).

[0058] Results of Western blotting of anti-PD-L1 nanobody

[0059] In this example, the procedure for Western blotting of anti-PD-L1 nanobody is as follows. Cells were obtained in PRO-PREP protein extraction solution (iNtRON, Taipei City, Taiwan Province, China) containing protease inhibitor mixture, and vigorously shaken at 4 °C for 15 minutes, then centrifuged. The supernatant was collected, and the protein concentration was measured using Bio-Rad BCA reagent (Bio-Rad Hercules, CA, USA). 30 μg of each sample lysate was electrophoresed on SDS-polyacrylamide gel and then electroblotted onto PVDF membrane. After adding 5% BSA in TBST blocking, the membrane was incubated with the primary antibody (dissolved in TBST) overnight at 4 °C. Then, it was 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 was incubated with SuperSignal West Pico ECL reagent (Pierce Biotechnology, Rockford, IL, USA) for 1 minute, and then chemiluminescence was detected by exposure to Kodak-X-Omat film.

[0060] The results of Western blotting of anti-PD-L1 nanobody are shown in Figure 5 , where the arrow indicates the position of PD-L1 protein, the molecular weight position consistent with the commercial antibody; the cell line used was non-small cell lung cancer cell line H1975 (NCI-H1975 [H-1975, H1975], ATCC), mAb represents monoclonal antibody, Nb represents nanobody; the commercial antibody was PD-L1 monoclonal antibody (66248-1-Ig, Proteintech), the primary antibody in the commercial antibody group was PD-L1 monoclonal antibody (1:2500), and the secondary antibody was anti-rabbit-horseradish peroxidase (anti-Rab-HRP) (1:10000); the concentration of the anti-PD-L1 nanobody in the experimental group was 1 ng / ml, and the secondary antibody was anti-VHH-HRP (1:10000). The results of this example showed that anti-PD-L1 nanobody clones #1 and #67 restored OKT3 (anti-CD3 monoclonal antibody)-induced T cell proliferation after PD-L1 binding.

[0061] Example 7. Results of flow cytometric analysis of anti-PD-L1 nanobody

[0062] In this example, the procedure for flow cytometric analysis of anti-PD-L1 nanobody is as follows. The anti-PD-L1 nanobody (1 ng / ml) was pre-stained with FITC fluorophore using the Fastlink fluorophore labeling kit (Abnova), and the procedure was carried out according to the instructions in the operation manual. MDA-MB-231, A549 or H1975 cells were stained with FITC-conjugated anti-PD-L1 monoclonal antibody (1:500, BD Pharmingen, clone MIH1, Cat.: 558065) or FITC-conjugated anti-PD-L1 nanobody (1 ng / ml) in PBS containing 1% BSA for 45 minutes. After washing with PBS, the cells were analyzed by flow cytometry using the FL1 channel.

[0063] The results of flow cytometric analysis of anti-PD-L1 nanobody are shown in Figure 6A and Figure 6B , where Figure 6A in (a) of Figure 6A nb represents nanobody, Ab represents antibody, FITC represents fluorescein isothiocyanate, Figure 6B in (b) of Figure 6B mAb represents monoclonal antibody, H1975 is a non-small cell lung cancer cell line, and Alexa Fluor 488-H is a bright green fluorescent dye that is excited by laser light at a wavelength of 488 nm.

[0064] Example 8. Results of immunocytochemistry analysis of anti-PD-L1 nanobody

[0065] In this example, the procedure for immunocytochemistry analysis of anti-PD-L1 nanobody is as follows. Tumor cells (1×10 5)Inoculate on the cover glass in a 6-well plate and culture overnight. After the designated treatment, fix the cells in 1% paraformaldehyde, wash with PBS, permeabilize with 0.1% Triton X-100 in PBS containing 0.5% BSA for 30 minutes, block with 2% BSA, and incubate with a specific antibody (diluted in 2% BSA / PBS (PBST) containing 0.05% Tween-20). After washing, incubate the cells with a fluorescein-conjugated secondary antibody, wash with PBST, and mount with an aqueous mounting medium containing an anti-fading agent and 4',6-diamidino-2-phenylindole (DAPI). Analyze the images under a Leica TCS SP8 X confocal microscope (Leica).

[0066] The results of immunocytochemical analysis of the anti-PD-L1 nanobody are shown in Figure 7 , where H1975 is a non-small cell lung cancer cell line, clone represents the selected clone, the concentration of the anti-PDL-1 nanobody is 1 ng / ml, the secondary antibody is anti-VHH-fluorescein isothiocyanate (FITC) (1:5000), pan-cadherin is a cell membrane marker, and the co-localization of pan-cadherin and PD-L1 can be used to explain that membrane-bound PD-L1 is detected by a specific nanobody. The results of this example show that the selected clone #1 of the anti-PDL-1 nanobody can be used to detect the expression of PD-L1 in cell samples.

[0067] In summary, the immune-regulatory nanobody of the present invention (i.e., anti-PD-L1 antibody) was proven by surface plasmon resonance binding assay (SPR binding assay) to effectively bind to the PD-L1 protein with KD within 0.27 and 0.41 nM respectively. The PD-1 / PD-L1 axis blockade of the anti-PD-L1 nanobody was measured by the Blockade Bioassay kit, which demonstrated that the anti-PD-L1 nanobody blocked PD-L1 / PD-1 signal transduction in the PD-L1, APC / PD-1 effector co-culture system. The anti-PD-L1 nanobody enhanced the cytotoxicity of γδ T cells-induced against tumor cells (MDA-MB-231). Western blotting was used to prove that the anti-PD-L1 nanobody restored OKT3 (anti-CD3 monoclonal antibody)-induced T cell proliferation after PD-L1 conjugation. Flow cytometry analysis and immunocytochemistry analysis were used to prove that the anti-PD-L1 nanobody could be used to detect the expression of PD-L1 in cell samples, thereby achieving the efficacy 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 immune-regulatory nanobody of the present invention can be directly administered to the required individuals for treatment after large-scale preparation in vitro. In addition, the present invention can also achieve the utility of detecting the expression level of PD-L1.

[0068] 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> Affiliated Hospital of China Medical University, Taiwan <120> Immune-Regulatory Nanobody and Its Nucleic Acid Coding Sequence and Application <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Anti-PD-L1 Nanobody Clone #1 <400> 1 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ser Asp Gly Ser Asn Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Ile Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ser Arg Cys Pro Asp Ile Tyr Cys Gly Gly Gln Tyr Thr Tyr Arg Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Anti-PD-L1 Nanobody Clone #14 <400> 2 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Asn Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Gln Thr Ser Gly Phe Gly Ser Asp Arg Tyr 20 25 30 Glu Ile Gly Trp Tyr Arg Gln Ile Pro Gly Trp Cys Glu Lys Val Ser 35 40 45 Thr Ile Ser Asp Thr Gly Thr Thr Phe Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Ala Ile Ser Gln Asp Thr Ala Lys Asn Leu Val Tyr Leu Gln 65 70 75 80 Met Asp Arg Leu Lys Pro Gln Asp Thr Ala Arg Tyr Tyr Cys Ala Ala 85 90 95 Ile Thr Thr Pro Ala Arg Asn Asn Gly Val Leu Asn Ala Leu Ser Arg 100 105 110 Leu Leu Lys Cys Leu Asn Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 115 120 125 Val Thr Val Ser Ser 130 <210> 3 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Anti-PD-L1 nanobody clone #67 <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Val Asn Ser Asp Gly Ser Asn Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Ile Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ser Arg Cys Pro Asp Ile Tyr Cys Gly Gly Gln Tyr Thr Tyr Arg Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Anti-PD-L1 nanobody cloning strain #1 <400> 4 catgtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagaatc 60 tcctgtgcag cctctggatt caccttcagt agccgtgcca tgagctgggt ccgccaggct 120 ccagggaagg gactcgagtg ggtctcaacc attaatagtg atggtagtaa cacatactat 180 tcagactccg tgaaggaccg attcaccatc tccagagaca acgccatcaa cacgctgtat 240 ctgcaattga acagcctgaa aactgaggac acggccatgt attactgttc ccgttgtccc 300 gatatttact gcggaggaca atatacgtat cggggccagg ggacccaggt cactgtctcc 360 tca 363 <210> 5 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Anti-PD-L1 Nanobody Cloning Strain #14 <400> 5 catgtgcagc tggtggagtc tgggggaggc tcggtgcaga atggagggtc tctgcgactc 60 tcctgccaaa cctctggatt tggttctgat cgttatgaaa tcggctggta tcgccaaatt 120 cccggctggt gcgagaaggt ttcaactatc agtgacaccg gcaccacatt ctatgcagac 180 tccgtgaagg gccgcttcgc catctcccaa gacaccgcca agaatctggt atatctgcaa 240 atggacaggt tgaaaccaca ggacacggcc cggtattatt gtgcggctat aaccacccct 300 gccaggaata atggcgtcct gaacgctctg agtcgattat tgaagtgctt aaatccatat 360 aactactggg gccaggggac ccaggtcacc gtctcctca 399 <210> 6 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Anti-PD-L1 nanobody cloning strain #67 <400> 6 caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagaatc 60 tcctgtgcag cctctggatt caccttcagt agccgtgcca tgagctgggt ccgccaggct 120 ccagggaagg gactcgagtg ggtctcaacc gttaatagtg atggtagtaa cacatactat 180 tcagactccg tgaaggaccg attcaccatc tccagagaca acgccatcaa cacgctgtat 240 ctgcaattga atagcctgaa aactgaggac acggccatgt attactgttc ccgttgtccc 300 gatatttact gcggaggaca atatacgtat cggggccagg ggacccaggt cactgtctcc 360 tca 363 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 of anti-PD-L1 nanobody cloning strain #1 <400> 7 Gly Phe Thr Phe Ser Ser Arg Ala 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 of anti-PD-L1 nanobody clone #1 <400> 8 Ile Asn Ser Asp Gly Ser Asn Thr 1 5 <210> 9 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CDR3 of anti-PD-L1 nanobody clone #1 <400> 9 Ser Arg Cys Pro Asp Ile Tyr Cys Gly Gly Gln Tyr Thr Tyr 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR1 of anti-PD-L1 nanobody clone #14 <400> 10 Thr Ser Gly Phe Gly Ser Asp Arg Tyr 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 of anti-PD-L1 nanobody clone #14 <400> 11 Thr Ile Ser Asp Thr Gly Thr Thr 1 5 <210> 12 <211> 28 <212> PRT <213> Artificial sequence <220> <223> CDR3 of anti-PD-L1 nanobody cloning strain #14 <400> 12 Ala Ala Ile Thr Thr Pro Ala Arg Asn Asn Gly Val Leu Asn Ala Leu 1 5 10 15 Ser Arg Leu Leu Lys Cys Leu Asn Pro Tyr Asn Tyr 20 25 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 of anti-PD-L1 nanobody cloning strain #67 <400> 13 Gly Phe Thr Phe Ser Ser Arg Ala 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 of anti-PD-L1 nanobody cloning strain #67 <400> 14 Val Asn Ser Asp Gly Ser Asn Thr 1 5 <210> 15 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CDR3 of anti-PD-L1 nanobody cloning strain #67 <400> 15 Ser Arg Cys Pro Asp Ile Tyr Cys Gly Gly Gln Tyr Thr Tyr 1 5 10

Claims

1. An immune-regulating nanobody, characterized in that, The immune regulatory nanobody specifically binds to programmed death ligand 1 (PD-L1), and the immune regulatory nanobody is composed of the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.3; wherein the immune regulatory nanobody is produced and purified from the E. coli strain HB2151.

2. An isolated nucleic acid, characterized in that, Encoding the immune regulatory nanobody as claimed in claim 1, the isolated nucleic acid is composed of the nucleotide sequence of SEQ ID NO.4 or SEQ ID NO.

6.

3. A pharmaceutical composition, characterized in that, Comprising the immune regulatory nanobody as claimed in claim 1 and a pharmaceutically acceptable carrier.

4. Use of the immune regulatory nanobody as claimed in claim 1 in the preparation of a reagent for detecting the expression level of PD-L1 in a test sample.

5. The use according to claim 4, characterized in that, Wherein the test sample is urine, sputum or body fluid.

6. The use according to claim 4, characterized in that, Wherein the test sample is blood or saliva.

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