Anti-T cell nanobody, its nucleic acid coding sequence and application

Abstract

The present invention provides an anti-T cell nanobody that specifically binds to CD3ε. The present invention also provides the nucleic acid coding sequence of the anti-T cell nanobody, the use of the anti-T cell nanobody for treating cancer, immunomodulation and activating immune cells, and a method for detecting the expression level of CD3ε.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-T cell nanobodies, and particularly to an anti-T cell nanobody, its nucleic acid coding sequence, and their 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 that controls cell division and proliferation. The number of people suffering from cancer worldwide has been on the rise. 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 eliminating or controlling tumors. However, the current tumor treatment methods still have problems such as ineffective treatment and strong side effects, and even may give rise to other immune-related diseases.

[0004] CD3ε (CD3 epsilon) is a transmembrane protein found on T cells and has been found to be associated with tumors and the regulation of immune functions. Therefore, some researchers have been committed to developing CD3ε as a target molecule for identifying tumors and regulating immune functions and finding out whether these target molecules have the potential to become anti-cancer drugs or immunomodulatory drugs.

[0005] To solve the above problems, those skilled in the art urgently need to develop novel and more effective pharmaceuticals for treating cancer, immunomodulation, and activating immune cells to benefit the large group of people in need. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an anti-T cell nanobody that specifically binds to a CD3ε (CD3 epsilon). The anti-T cell 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 an embodiment of the present invention, the amino acid sequence is an amino acid sequence of a heavy chain variable domain (VHH) of the anti-T cell nanobody.

[0008] In one embodiment of the present invention, the anti-T cell nanobody further comprises a fragment crystallizable region (Fc region).

[0009] In one embodiment of the present invention, the anti-T cell 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-T cell nanobody activates and / or aggregates CD3ε-positive cells.

[0011] Another object of the present invention is to provide an isolated nucleic acid encoding the amino acid sequence of the anti-T cell 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.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the anti-T cell nanobody as described above and a pharmaceutically acceptable carrier.

[0013] Another object of the present invention is to provide the use of the anti-T cell nanobody as described above for the preparation of a medicament for treating cancer, immunomodulation, and activating immune cells.

[0014] Another object of the present invention is to provide a method for detecting the expression level of CD3ε, comprising administering the anti-T cell nanobody as described above to a sample to be tested.

[0015] In one embodiment of the present invention, the sample to be tested is blood, urine, sputum, saliva, body fluid, tumor, organ, tissue, or cell.

[0016] In summary, the efficacy of the anti-T cell nanobody of the present invention lies in: it is demonstrated by T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation and activation assay that the anti-CD3ε nanobody can promote the aggregation, proliferation and activation of T cells, enhance the proliferation of CD3-positive T cells in PBMC, enhance the proliferation of CD3-positive T cells in γδT (GDT) cells; it is demonstrated by Western blotting that the anti-CD3ε nanobody can recognize the CD3ε protein in the cell lysate of human T cells; it is demonstrated by immunohistochemistry staining (IHC staining) and flow cytometric analysis that the anti-CD3ε nanobody can detect the expression of CD3ε in cell samples by flow cytometric analysis; it is demonstrated by surface plasmon resonance binding assay (SPR binding assay) that the anti-CD3ε nanobody effectively binds to the CD3ε / CD3δ heterodimer with a KD within 0.5056 nM, and it is demonstrated by immunocytochemical analysis that the anti-CD3ε nanobody can be used to detect the expression of CD3ε in cell samples, thereby achieving the effects of treating cancer, immunomodulation and activating immune cells. In particular, the anti-T cell nanobody of the present invention can play a role in regulating immune function and activating immune cells. 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-T cell 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 effect of detecting the expression level of CD3ε.

[0017] The following will further describe the embodiments of the present invention. The following listed examples 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 determined by the scope defined in the claims. Brief description of the drawings

[0018] Figure 1A and Figure 1BShow the results of the proliferation and activation assay of T cells (i.e., peripheral blood mononuclear cells, PBMCs) expressing anti-CD3ε nanobody.

[0019] Figures 2A to 2C Show the results of the evaluation of the efficacy of anti-CD3ε nanobody in enhancing the proliferation of CD3-positive T cells in PBMCs, where CD3e nb represents anti-CD3ε nanobody and Vehicle represents the carrier.

[0020] Figures 3A to 3D Show the results of the evaluation of the efficacy of anti-CD3ε nanobody in enhancing the proliferation of CD3-positive T cells in gamma delta T (GDT) cells, where CD3e nb represents anti-CD3ε nanobody, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0021] Figure 4 Show the results of Western blotting of anti-CD3ε nanobody, where the upper row numbers represent the amount (μg) of T cell protein lysate. (a) Using the traditional antibody #ab135372, which is an anti-CD3 antibody, the primary antibody concentration is 10 μg / ml (1:1000), and the secondary antibody is anti-rabbit-horseradish peroxidase (HRP) (1:1000); (b) Using anti-CD3ε nanobody (i.e., heavy chain variable domain (VHH) nanobody), the primary antibody concentration is 1 μg / ml (1:1000), and the secondary antibody is anti-VHH-HRP (1:1000).

[0022] Figure 5 Show the results of immunohistochemistry staining (IHC staining) of anti-CD3ε nanobody, where CD3e nb represents anti-CD3ε nanobody and SP7 is the traditional anti-CD3 antibody.

[0023] Figure 6Flow cytometry analysis results showing anti-CD3ε nanobody, where in (a), FSC-A represents forward scatter area, SSC-A represents side scatter area, and fcs represents flow cytometry standard; in (b), Alexa Fluor 488-A is a bright green fluorescent dye that is excited by a laser beam with a wavelength of 488 nm.

[0024] Figure 7 Results of surface plasmon resonance binding assay (SPR binding assay) of anti-CD3ε nanobody to CD3ε / CD3δ heterodimer, where the analysis concentrations are 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM, 1.953 nM, the association time is 120 seconds, the dissociation time is 600 seconds, and Kd: 5.056x10 -10 = 0.5056 nM, using an NTA chip coated with CD3ε / CD3δ heterodimer recombinant protein (ACROBiosystems, Cat: CDD-H52W1).

[0025] Figure 8 Results of immunocytochemistry analysis of anti-CD3ε nanobody, where the concentration of anti-CD3ε nanobody is 1 ng / ml, SP7 is a conventional anti-CD3 antibody (1:500, MA1-90582, Invitrogen), and the secondary antibody is anti-VHH-fluorescein (FITC) (1:5000). Embodiments

[0026] Definitions

[0027] The numerical values used herein are approximate values, and all experimental data are expressed within the range of ±20%, preferably within the range of ±10%, and most preferably within the range of ±5%.

[0028] As used herein, the term "second antibody" refers to 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 engager (BiKE), a triple specific killer cell engager (TriKE), or any bispecific antibody. Preferably, the second antibody may include, but is not limited to: antibodies against 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, human leukocyte antigen-G (HLA-G), epithelial cell adhesion molecule (EpCAM), mesothelin, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), glycoprotein 100 (gp100), and mucin 1 (Muc1).

[0029] As used herein, the terms "CD3e" and "CD3ε" are used interchangeably.

[0030] As used herein, the terms "CD3e nanobody", "CD3e nb", "CD3e Nb", "CD3e nanobody", "anti-CD3ε nanobody", and "anti-T cell nanobody" are used interchangeably.

[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, by techniques well known to those skilled in the art, into a dosage form suitable for parenteral administration, 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 contain a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may contain 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 contains 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 a deoxyribonucleotide sequence or ribonucleotide sequence in single-stranded or double-stranded form, and contain 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-CD3ε nanobody

[0038] In this embodiment, the preparation process of anti-CD3ε (CD3 epsilon) 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 (10 L - 20 L) containing the resistance at a ratio of 1:100 and culture at 37 °C and 220 r / min. When 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 binds 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 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] The nanobody was produced and purified from Escherichia coli (E. coli). To produce E. coli in the form of nanobody, refer to Microb Cell Fact. 2019 Mar 11;18(1):47. Briefly, the E. coli strain HB2151 was used. The plasmid pET (Creative Biolab) encoding ampicillin resistance was used for cytoplasmic protein production. The E. coli HB2151 newly transformed with the CD3ε or CD3ε 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 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, the 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 a 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 with a molecular weight cut-off of 3.5 - 5 kDa ( Laboratories) was used to remove imidazole and change the buffer by dialysis against PBS.

[0040] The CD3ε VHH was A human single-domain antibody library (Creative Biolabs) was generated. Briefly, after four rounds of panning with CD3ε antigen binding / washing / elution, approximately 100 colonies were picked, and then positive single phages were selected by phage enzyme-linked immunosorbent assay (ELISA). The positive colonies were sequenced by the Sanger method to obtain the nanobody sequences.

[0041] The colony projects for which DNA sequencing was performed are shown in Table 1 below.

[0042] Table 1

[0043]

[0044] The phagemid of colony #2 was sequenced by the Sanger method, and then all DNA sequences were translated into the corresponding encoded amino acids by computer simulation. Subsequently, colony #2 was selected because the DNA of colony #2 and the corresponding CDR amino acids were identical to the amino acid sequences of the nanobody phagemid colonies. The amino acid sequences of the anti-CD3ε nanobodies are SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the nucleotide sequences are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. Next, a competitive enzyme-linked immunosorbent assay (competitive ELISA) was performed on colony #2 as follows. The phagemid of colony #2 was amplified in Escherichia coli, and then the supernatant was collected. After coating and washing the CD3ε recombinant protein (0.2 μg), 250 μl of TB medium containing the phagemid of colony #2, mock TB medium, and PBS control were added to each well. The next day, the supernatant was removed, washed with PBST, and then reacted with an anti-M13 phage horseradish peroxidase (HRP)-conjugated secondary antibody for 2 hours. After washing, TMB substrate (for detecting HRP activity) (50 μl) was added, and the signal was detected using a 450 nm channel by an ELISA reader. The results are shown in Table 2 below.

[0045] Table 2

[0046]

[0047] Example 2. Analysis Results of T Cell Proliferation and Activation of Anti-CD3ε Nanobody

[0048] In this example, the operation process of the analysis of T cell (i.e., peripheral blood mononuclear cell, PBMC) proliferation and activation of anti-CD3ε nanobody is as follows. 1×10 6 PBMC cells were seeded in 12-well plates in the presence or absence of anti-CD3ε nanobody (1 μg / ml) or the clinical CD3ε monoclonal antibody OKT3 (10 mg / ml, Invitrogen, Cat: MA1-10175). Then, 50 IU / ml of IL-2 and 2 μg / ml of IL-15 (Sino Biological, Cat No: 10360-H07E) were added. After 5 or 7 days, the total cell number was recorded, and then stained with FITC-conjugated CD3 monoclonal antibody (OKT3, 11-0037-42, eBioscience), and then analyzed by flow cytometry. The photos were taken with a microscope at 40x. The number of CD3-positive cells was calculated as the percentage of CD3 cells (%) × total cell number.

[0049] The analysis results of T cell (i.e., peripheral blood mononuclear cell) proliferation and activation of anti-CD3ε nanobody are shown in Figure 1A and Figure 1B . It can be seen from Figure 1A and Figure 1B that the anti-CD3ε nanobody of the present invention can promote T cell aggregation, proliferation and activation.

[0050] Example 3. Evaluation of the Efficacy of Anti-CD3ε Nanobody in Enhancing the Proliferation of CD3-Positive T Cells in PBMC

[0051] In this example, the operation process of evaluating the efficacy of anti-CD3ε nanobody in enhancing the proliferation of CD3-positive T cells in PBMC is as follows. 1×10 6PBMC cells were seeded in 12-well plates, in the presence or absence of anti-CD3ε nanobody (10, 100, 1000, 5000 ng / ml). Then, 50 IU / ml of IL-2 (Gibco, PHC0021) and 2 μg / ml of IL-15 (Sino Biological, Cat No: 10360-H07E) were added. After 3 or 7 days, the total cell number was recorded, and then stained with FITC-conjugated anti-CD3 monoclonal antibody (OKT3, 11-0037-42, eBioscience), and then analyzed by flow cytometry. Photos were taken with a microscope at 40x. The number of CD3-positive cells was calculated as the percentage of CD3 cells (%) × total cell number.

[0052] Results of the evaluation of the effect of anti-CD3ε nanobody on enhancing the proliferation of CD3-positive T cells in PBMCs are shown in Figures 2A to 2C , where CD3e nb represents anti-CD3ε nanobody and Vehicle represents the carrier. As can be seen from Figures 2A to 2C , the anti-CD3ε nanobody significantly stimulated the proliferation of CD3-positive T cells in PBMCs in a cluster formation manner.

[0053] Example 4. Evaluation of the effect of anti-CD3ε nanobody on enhancing the proliferation of CD3-positive T cells in gamma delta T (GDT) cells

[0054] In this example, the procedure for evaluating the effect of anti-CD3ε nanobody on enhancing the proliferation of CD3-positive T cells in gamma delta T (GDT) cells is as follows. 1×10 6 primary gamma delta T (GDT) cells were seeded in 12-well plates, in the presence or absence of anti-CD3ε nanobody (10, 100, 1000, 5000 ng / ml). Then, 50 IU / ml of IL-2 (Gibco, PHC0021) and 2 μg / ml of IL-15 (Sino Biological, Cat No: 10360-H07E) were added. After 3 or 7 days, the total cell number was recorded, and then stained with FITC-conjugated anti-CD3 monoclonal antibody (OKT3, 11-0037-42, eBioscience), and then analyzed by flow cytometry. Photos were taken with a microscope at 40x. The number of CD3-positive GDT cells was calculated as the percentage of CD3 GDT cells (%) × total cell number.

[0055] Results of the evaluation of the effect of anti-CD3ε nanobody on enhancing the proliferation of CD3-positive T cells in gamma delta T (GDT) cells are shown in Figures 3A to 3D, where CD3e nb represents the anti-CD3ε nanobody. The results of this example show that the anti-CD3ε nanobody can effectively enhance γδT cell proliferation in a dose-dependent manner.

[0056] Example 5. Results of Western blotting analysis of anti-CD3ε nanobody

[0057] In this example, the procedure for Western blotting analysis of the anti-CD3ε nanobody is as follows. Cells were obtained in PRO-PREP protein extraction solution (iNtRON, Taipei City, Taiwan Province, China) containing a protease inhibitor mixture and shaken vigorously at 4 °C for 15 minutes, then centrifuged. The supernatant was collected, and the protein concentration was then measured using Bio-Rad BCA reagent (Bio-Rad Hercules, CA, USA). 30 μg of each sample lysate was electrophoresed on an SDS-polyacrylamide gel and then electroblotted onto a 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.

[0058] The results of Western blotting analysis of the anti-CD3ε nanobody are shown in Figure 4 , where the upper row numbers represent the amount (μg) of T cell protein lysate. (a) Using the conventional antibody #ab135372, which is an anti-CD3 antibody, the primary antibody concentration was 10 μg / ml (1:1000), and the secondary antibody was anti-rabbit-HRP (1:1000); (b) Using the anti-CD3ε nanobody (i.e., the heavy chain variable domain (VHH) nanobody), the primary antibody concentration was 1 μg / ml (1:1000), and the secondary antibody was anti-VHH-HRP (1:1000). The results of this example show that by Western blotting analysis, the anti-CD3ε nanobody can recognize the CD3ε protein in the cell lysate of human T cells.

[0059] Results of immunohistochemistry staining (IHC staining) of anti-CD3ε nanobody

[0060] In this example, the operation process of immunohistochemistry staining (IHC staining) of anti-CD3ε nanobody is as follows. Human PBMC 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 performed by microwave at 99 °C. The sections were washed with H2O2 and treated for 15 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. Next, 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. The quantification of staining was performed independently by optical microscopy (Nikon, Japan) at magnifications of 40× and 400×.

[0061] The results of immunohistochemistry staining of anti-CD3ε nanobody are shown in Figure 5 , where CD3e nb represents anti-CD3ε nanobody and SP7 is a conventional anti-CD3 antibody. The results of this example show that anti-CD3ε nanobody can be used to detect the expression of CD3ε by immunohistochemistry staining.

[0062] Example 7. Results of flow cytometric analysis of anti-CD3ε nanobody

[0063] In this example, the operation process of flow cytometric analysis of anti-CD3ε nanobody is as follows. Human PBMCs were stained with FITC-conjugated CD3ε nanobody (1 μg / ml) and OKT3 antibody (anti-CD3 monoclonal antibody) (10 μg / ml) on ice for 45 minutes. After washing, the cells were analyzed by flow cytometry using the FL1 channel.

[0064] The results of flow cytometric analysis of anti-CD3ε nanobody are shown in Figure 6, where in (a), FSC-A represents the forward scatter area, SSC-A represents the side scatter area, and fcs represents the flow cytometry standard. In (b), Alexa Fluor488-A is a bright green fluorescent dye that is excited by a laser beam with a wavelength of 488 nm. From Figure 6 It can be seen that the anti-CD3ε nanobody can detect the expression of CD3ε in cell samples by flow cytometry analysis.

[0065] Example 8. Results of the surface plasmon resonance binding assay (SPR binding assay) of the anti-CD3ε nanobody to the CD3ε / CD3δ heterodimer

[0066] In this example, the operating procedure for the surface plasmon resonance binding assay (SPR binding assay) of the anti-CD3ε nanobody to the CD3ε / CD3δ heterodimer is as follows. For CM5 or NTA chips, research grade SPR analysis was performed using a Biacore T200 (Biacore - GE Healthcare, Piscataway, NJ). Briefly, protein (CD3ε / CD3δ recombinant protein) samples were diluted in a 10 mM buffer solution (pH 4.0, 5.5, or 6.0) in a concentration range of 20 μg / mL to obtain maximum surface retention for immobilization on the chip. Following the surface preparation process and ligand selection (CD3ε or CD3ε multispecific nanobody, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 nM) were coated on the chip. Then came regeneration scouting and surface performance testing. After regeneration scouting and surface performance testing, a regeneration method was then selected to run the experiment. Then BINDING ANALYSIS and DIRECT BINDING were selected to study protein binding. KINETIC ANALYSIS was selected and MASS TRANSFER was chosen for kinetic analysis of the binding experiment. After that, data analysis and determination of kinetic constants were performed.

[0067] The results of surface plasmon resonance binding analysis (SPR binding assay) of the anti-CD3ε nanobody to the CD3ε / CD3δ heterodimer are shown in Figure 7 , where the analysis concentrations were 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, 3.90625 nM, 1.953 nM), the association time was 120 seconds, the dissociation time was 600 seconds, Kd: 5.056×10 -10 = 0.5056 nM, using the coated CD3ε / CD3δ heterodimer recombinant protein (ACROBiosystems, Cat: CDD-H52W1), NTA chip. As can be seen from Figure 7 , the anti-CD3ε nanobody effectively binds to the CD3ε / CD3δ heterodimer with a KD within 0.5056 nM.

[0068] Example 9. Results of immunocytochemistry analysis of the anti-CD3ε nanobody

[0069] In this example, the procedure for immunocytochemistry analysis of the anti-CD3ε nanobody is as follows. 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 (prepared in 2% BSA / PBS (PBST) containing 0.05% Tween-20). After washing, the cells were incubated with the fluorescein-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). Images were analyzed under a Leica TCS SP8 X confocal microscope (Leica).

[0070] The results of immunocytochemistry analysis of the anti-CD3ε nanobody are shown in Figure 8, wherein the concentration of the anti-CD3ε nanobody is 1 ng / ml, SP7 is a conventional anti-CD3 antibody (1:500, MA1-90582, Invitrogen), and the secondary antibody is anti-VHH-fluorescein (FITC) (1:5000). The results of this example show that, through immunocytochemical analysis, the anti-CD3ε nanobody can be used to detect the expression of CD3ε in cell samples.

[0071] In summary, the anti-T cell nanobody (i.e., anti-CD3ε antibody) of the present invention proves that the anti-CD3ε nanobody can promote the aggregation, proliferation, and activation of T cells, enhance the proliferation of CD3-positive T cells in PBMC, enhance the proliferation of CD3-positive T cells in γδT (GDT) cells, prove that the anti-CD3ε nanobody can recognize the CD3ε protein in the cell lysate of human T cells through Western blotting, prove that the anti-CD3ε nanobody can detect the expression of CD3ε in cell samples through flow cytometric analysis by immunohistochemistry staining (IHC staining) and flow cytometric analysis, prove that the anti-CD3ε nanobody effectively binds to the CD3ε / CD3δ heterodimer with a KD within 0.5056 nM through surface plasmon resonance binding assay (SPR binding assay), and prove that the anti-CD3ε nanobody can be used to detect the expression of CD3ε in cell samples through immunocytochemical analysis, thereby achieving the effects of treating cancer, immunomodulation, and activating immune cells. In particular, the anti-T cell nanobody of the present invention can play a role in regulating immune function and activating immune cells. Compared with the conventional antibody, which has the disadvantages of low yield and ineffective results due to the need to transfect genes into cells through a vector to express antibody functions, the anti-T cell nanobody of the present invention can be directly administered to individuals in need after large-scale preparation in vitro for treatment. In addition, the present invention can also achieve the effect of detecting the expression level of CD3ε.

[0072] The above is only illustrative and not restrictive. Any equivalent modification or change 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> Anti-T cell nanobody, its nucleic acid coding sequence and application <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 1 Gly Val Ile Phe Lys Asn Glu Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 2 Ala Ser Pro Gly Gly Thr Ile Thr 1 5 <210> 3 <211> 23 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 3 Ala Leu Asp Pro Ser Thr Thr Ser Trp Ser Ile Ile Arg His Gly Pro 1 5 10 15 Ser Leu Trp Arg Tyr Ser Gly 20 <210> 4 <211> 24 <212> DNA <213> Artificial sequence <220> <223> CDR1 <400> 4 ggagtcatct ttaagaacga gtac 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CDR2 <400> 5 gcttcgcctg gtggaacgat taca 24 <210> 6 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> CDR3 <400> 6 gcgttggatc cctcgactac gtcatggtct atcatccgcc acggtccatc gctttggcgt 60 tatagcggc 69

Claims

1. An anti-T cell nanobody, characterized in that, The anti-T cell nanobody specifically binds to CD3ε, and the anti-T cell nanobody comprises a CDR1 with the amino acid sequence of SEQ ID NO.1, a CDR2 with the amino acid sequence of SEQ ID NO.2, and a CDR3 with the amino acid sequence of SEQ ID NO.

3.

2. An isolated nucleic acid, characterized in that, Encoding the anti-T cell nanobody according to claim 1, the isolated nucleic acid comprises SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, wherein SEQ ID NO.4 encodes the amino acid sequence of SEQ ID NO.1, SEQ ID NO.5 encodes the amino acid sequence of SEQ ID NO.2, and SEQ ID NO.6 encodes the amino acid sequence of SEQ ID NO.

3.

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

4. Use of the anti-T cell nanobody according to claim 1 in the preparation of a reagent for detecting the expression level of CD3ε in a sample to be tested.

5. The use according to claim 4, characterized in that, The sample to be tested is body fluid, tumor, organ, tissue or cell.

6. The use according to claim 4, characterized in that, The sample to be tested is blood, urine, sputum or saliva.

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