An anti-NKG2D nanobody, its preparation method and application

Abstract

This invention belongs to the field of antibody preparation technology, specifically relating to an anti-NKG2D nanobody, its preparation method, and its application. The method is as follows: NKG2D protein expression is constructed, and alpacas are immunized to obtain immunized alpaca PBMC cells. RNA is extracted from the PBMC cells, reverse transcribed into cDNA, and antibody gene fragments are obtained by PCR. These fragments are then constructed into a phage expression vector. The obtained vector containing the antibody gene fragment is electroporated into SS320 electrocompetent cells to obtain an NKG2D immune antibody library. After two rounds of NKG2D protein panning and Clone ELISA detection, antibodies binding to NKG2D protein are obtained. After sequencing analysis, unique sequences are selected and expressed in a mammalian cell high-throughput expression system to obtain antibody proteins. The antibodies are then detected using an NKG2D high-expression cell line via FACS, ultimately yielding antibodies binding to NKG2D. The method of this invention is simple and rapid; the mammalian cell expression system induces highly efficient antibody expression, and the translated antibodies can be further modified, resulting in activity closer to that of natural antibodies.

Description

[0001] This application is a divisional application of application number "202410520392.8" filed on April 28, 2024, entitled "An anti-NKG2D nanobody and its preparation method and application". Technical Field

[0002] This invention belongs to the field of antibody preparation technology, specifically relating to an anti-NKG2D nanobody, its preparation method, and its application. Background Technology

[0003] Over the past few years, camel single-domain antibodies (nanobodies) have proven to be fundamental to the development of innovative treatments for a variety of human diseases. Nanobodies can bind to epitopes that are inaccessible to conventional antibodies, making them ideal for generating blocking or agonist molecules. Furthermore, their small protein size (15KD) allows for the creation of multivalent, multispecific, or multi-heterotopic structures, demonstrating significant advantages over traditional monoclonal antibodies. (2-4) .

[0004] Currently, the research and use of immune checkpoint inhibitors are quite extensive, among which PD-1 and its ligand PDL-1 are the most prominent. (1) Immune checkpoint inhibitors, such as [specific examples of inhibitors], have shown promising efficacy in tumor immunotherapy, but they also suffer from low patient response rates and drug resistance. Therefore, novel treatments that activate the immune system should be sought to improve clinical benefits for patients with advanced cancer. NKG2D is a C-type lectin-like receptor molecule composed of homodimers. (6) In the human body, NKG2D is typically expressed on NK cells, CD8 T cells, γδ T cells, and some CD4 T cells, but not in normal tissues. (7-8) NKG2D is the main activating receptor for NK cells. Its ligands are major histocompatibility complex class I-associated proteins A and B (MICA / B) and REAT1 family UL-16 binding proteins (ULBP1-6). (9-10) NK cells are the first line of defense in anti-tumor immunity. NK cell activation is regulated by the balance between activating and inhibitory receptors on their surface. NKG2D can activate NK cells by binding to NKG2D ligands on the tumor surface, thereby exerting a surveillance and clearance function in the tumor immune system. (5) , This makes it an important target in tumor immunotherapy. Tumor immunotherapy is a biological treatment method that aims to overcome the tumor's immune escape mechanism and reactivate immune cells to kill and eliminate tumor cells. Compared with traditional radiotherapy and chemotherapy, tumor immunotherapy has fewer toxic side effects and more lasting efficacy.

[0005] Targeted NKG2D therapy can also effectively reduce various inflammatory damages, and NKG2D blocking antibodies can delay NK cell-mediated airway allergic reactions. (12) Colitis, etc. Antibodies targeting NKG2D have been shown to significantly increase clinical remission in patients with Crohn's disease. Targeting and blocking NKG2D will be another novel mechanism of action for treating moderate to severe Crohn's disease. (11) NKG2D is also involved in the pathogenesis of some autoimmune diseases or inflammations, such as diabetes. (13) Abdominal diseases, multiple sclerosis (14) Rheumatoid arthritis (15) In addition to preventing the occurrence of acute hepatitis, blocking and inhibiting NKG2D can effectively protect the body from disease or reduce inflammatory damage.

[0006] WingKeung Chan et al. (16) Studies have shown that the CS1-NKG2D bispecific antibody promotes CS1 + Multiple myeloma cells and NKG2D + The enhanced immune synapses between innate and antigen-specific effector cells, which in turn activate these immune cells to improve the clearance of multiple myeloma. (Sebastian Lutz et al.) (17) They also investigated a novel NKG2D-directed bispecific antibody that enhances antibody-mediated killing of malignant B cells by NK and T cells. The resulting CD20-NKG2D bispecific antibody recruited cytotoxic lymphocytes to lymphoma cells, triggering NK cell-mediated lymphoma cell lysis. They also proposed several combination approaches that may be applicable to existing immunotherapies.

[0007] Nevertheless, for example in tumor immunotherapy, there is still a need for antibodies that target NKG2D, and to optimize and improve the efficacy and safety of these antibodies.

[0008] 1. Dong Y, Sun Q, Zhang

[0009] 2..Arbabi-Ghahroudi M.Camelid single-domain antibodies:historicalperspective and future outlook.Front Immunol.2017;8:1589

[0010] 3.Del Bano J,Chames P,Baty D,Kerfelec B.Taking up cancerimmunotherapy challenges:bispecific antibodies,the path forward?Antibodies.2016;5(1):1.doi:10.3390 / antib5010001.

[0011] 4.Steeland S,Vandenbroucke RE,Libert C.Nanobodies as therapeutics:bigopportunities for small antibodies.Drug Discov Today.2016;21:1076–1113.

[0012] 5.FanJ,ShiJ,ZhangY,etal.NKG2D discriminates diverse ligands throughselectively mechano-regulated ligand conformational changes[J / OL].EMBOJ 2022,41(2):e107739[2023-08-28].

[0013] 6.Vivier E,Tomasello E,Paul P.Lymphocyte activation via NKG2D:towardsa new paradigm in immune recognition?Curr Opin Immunol,2002;14(3):306-311.

[0014] 7.Raulet DH:Roles of the NKG2D immunoreceptor and its ligands.NatRevImmunol 2003,3:781–790.[PubMed:14523385]

[0015] 8.Zhang J,Basher F,Wu JD:NKG2D ligands in tumor immunity:two sidesofa coin.Front Immunol 2015,6:p97.

[0016] 9.Eagle RA,Trowsdale J:Promiscuity and the single receptor:NKG2D.NatRev Immunol 2007,7:737–744.[PubMed:17673918]

[0017] 10.El-Gazzar A,Groh V,Spies T:Immunobiology and conflicting rolesofthe human NKG2D lymphocyte receptor and its ligands in cancer.J Immunol2013,191:1509–1515.[PubMed:23913973]

[0018] 11.Allez,M.;Petryka,R.;Skolnick,B.E.;Wisniewska-Jarosinska,M.A.Mo1213efficacy and safety of NNC0142-0002,a novel human monoclonalantibodytargeting NKG2D:A randomized,double-blind,single-dose phase2 trial inpatients with crohn’s disease.Gastroenterology 2014,146,S-587.[CrossRef]

[0019] 12.Haworth,O,Cernadas,M.,and Levy,B.D.(2011).NK cells are effectorsforresolvin E1 in the timely resolution of allerglc airwayinflammation.JImmunol 186,6129-6135.

[0020] 13.Van Belle,T.L.,Ling,E.,Haase,C.,Bresson,D.,Urso,B.,and vonHerrath,M.G.(2012).NKG2D blockade faciltates diabetes prevention byantigen-specificTregs in a virus-induced model of diabetes.Journal ofautoimmunity.

[0021] 14.Saikali,P.,Antel,J.P.,Newcombe,J.,Chen,Z.,Freedman,M.,Blain,M.,Cayrol,R.,Prat,A.,Hall,J.A.,and Arbour,N(2007).NKG2D-mediatedcytotoxicitytoword oligodendrocytes suggests a mechanism for tissueinjury in multiplesclerosis.The Journal of neuroscience:the officialjournal of the society forNeuroscience 27,1220-1228.

[0022] 15.Andersson,A.K.,Sumariwalla,P.E.,McCann,F.E.,Amjadi,P.,Chang,

[0023] C.,McNamee,K.,Tornehave,D.,Haase,C.,Agerso,H.Stennicke,V.W.,etal.(2011b).Blockade of NKG2D ameliorates disease in mice with collagen-inducedarthritis:a potential pathogenic role in chronic inflammatoryarthritis.Arthritis and rheumatism 63,2617-2629.

[0024] 16.Wing,KC,Siwen,K,Youssef,Y.,Erin NG,etal.A CS1-NKG2D bispecificantibody collectively activates cytolytic immune cells against multiplemyeloma.Cancer Immunol Res.2018July;6(7):776–787.doi:10.1158 / 2326-6066.CIR-17-0649.

[0025] 17.Sebastian L.,Katja.K.,Anca-Maria A.,Lea E.etal.,Novel NKG2D-directed bispecific antibodies enhance antibody-mediated killing of malignan(2023),doi:10.3389 / fimmu.2023.1227572. Summary of the Invention

[0026] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-NKG2D nanobody, its preparation method, and its application.

[0027] In a first aspect of the present invention, an anti-NKG2D nanobody is provided, the anti-NKG2D nanobody comprising a framework region and a complementarity-determining region; the complementarity-determining region comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 are selected from any one of the following groups:

[0028] 1) The complementary decision region CDR1 sequence is shown in SEQ ID NO.1, the complementary decision region CDR2 sequence is shown in SEQ ID NO.2, and the complementary decision region CDR3 sequence is shown in SEQ ID NO.3;

[0029] 2) The complementary decision region CDR1 sequence is shown in SEQ ID NO.1, the complementary decision region CDR2 sequence is shown in SEQ ID NO.4, and the complementary decision region CDR3 sequence is shown in SEQ ID NO.3;

[0030] 3) The complementary decision region CDR1 sequence is shown in SEQ ID NO.5, the complementary decision region CDR2 sequence is shown in SEQ ID NO.6, and the complementary decision region CDR3 sequence is shown in SEQ ID NO.7;

[0031] 4) The complementary decision region CDR1 sequence is shown in SEQ ID NO.8, the complementary decision region CDR2 sequence is shown in SEQ ID NO.9, and the complementary decision region CDR3 sequence is shown in SEQ ID NO.10;

[0032] 5) The complementary decision region CDR1 sequence is shown in SEQ ID NO.11, the complementary decision region CDR2 sequence is shown in SEQ ID NO.12, and the complementary decision region CDR3 sequence is shown in SEQ ID NO.13.

[0033] 6) The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 are at least 90% identical to the amino acid sequences of any one set of complementarity-determining regions CDR1, CDR2 and CDR3 in 1)-5).

[0034] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 in groups 1-5 are as follows:

[0035]

[0036] Furthermore, the anti-NKG2D nanobody has an amino acid sequence selected from any of the following: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31.

[0037] In a second aspect of the invention, a nucleic acid encoding an anti-NKG2D nanobody as described in the first aspect is provided.

[0038] Furthermore, the nucleic acid sequence of the anti-NKG2D nanobody is shown in SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48 or SEQ ID NO.49.

[0039] In a third aspect of the invention, a vector containing the nucleic acid as described in the second aspect is provided. Further, the vector is -pComb3XSS.

[0040] In a fourth aspect of the invention, a host cell containing the vector as described in the third aspect is provided. Further, the host cell is TG1 or SS320; preferably, SS320 host cell is used.

[0041] In a fifth aspect of the present invention, a method for preparing anti-NKG2D nanobodies as described in the first aspect is provided, comprising the following steps: constructing an expression of NKG2D protein, immunizing alpacas to obtain immunized alpaca PBMC cells, extracting RNA from PBMC cells, reverse transcribing it into cDNA, obtaining antibody gene fragments by PCR, and constructing them into a phage expression vector; electroporating the obtained vector containing the antibody gene fragments into SS320 electrocompetent cells to obtain an NKG2D immune antibody library, obtaining antibodies binding to NKG2D protein through two rounds of NKG2D protein panning, detecting them by Clone ELISA, selecting unique sequences after sequencing analysis, performing high-throughput expression using a mammalian cell high-throughput expression system to obtain antibody proteins, performing FACS detection using an NKG2D high-expression cell line, and finally obtaining antibodies binding to NKG2D.

[0042] In this invention, the primers used for antibody gene fragment amplification, the panning method, and the construction of the NKG2D high-expression cell line are all obtained through optimization of routine operations.

[0043] In a sixth aspect of the invention, a product or kit is provided containing nanobodies as described in the first aspect or biomaterials as described in the second, third or fourth aspects.

[0044] In a seventh aspect of the invention, the use of the anti-NKG2D nanobody as described in the first aspect is provided in the preparation of a medicament for treating various inflammatory injuries, autoimmune diseases, and tumor immunotherapy.

[0045] In an eighth aspect of the invention, the application of the anti-NKG2D nanobody as described in the first aspect in the preparation of products for detecting NKG2D is provided.

[0046] In a ninth aspect of the invention, the use of the anti-NKG2D nanobody as described in the first aspect is provided in the preparation of a product bound to NKG2D.

[0047] The present invention has the following technical effects:

[0048] 1) The antibody of the present invention has a small molecular weight, high specificity, simple structure, is easy to engineer, and has the characteristic of binding to NKG2D.

[0049] 2) The antibody preparation method of the present invention is simple and fast; and the mammalian cell expression system induces efficient antibody expression, which can be processed and modified after translation, and its activity is closer to that of natural antibodies. Attached Figure Description

[0050] Figure 1 The serum titer for NKG2D protein immunity.

[0051] Figure 2 This is an image of RNA agarose gel electrophoresis.

[0052] Figure 3 This is an electrophoresis image of one round of PCR products.

[0053] Figure 4 This is an electrophoresis image of the products from the second round of PCR. 1-13 show the bands amplified by the 13 pairs of primers used in the second round of PCR.

[0054] Figure 5 The results are from the Cell binding experiment. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0056] The reagents or instruments used, whose manufacturers are not specified, are all conventional products that can be purchased through legitimate channels.

[0057] Example 1

[0058] In this embodiment, the preparation method of NKG2D nanobody includes the following steps:

[0059] 1) Based on the gene and protein sequence information of NKG2D (NCBI Reference Sequence: NP_031386.2), the immunogen NKG2D was expressed, and a His-tag was linked to its C-terminus to obtain modified amino acids for subsequent purification and detection;

[0060] 2) Alpaca were immunized three times with the modified antigen and Freund's adjuvant mixture obtained in step 1 to obtain alpaca PBMC cells: Alpaca were primarily immunized with an emulsion mixture of human NKG2D / His protein (i.e., the modified amino acids obtained in step 1) and Freund's complete adjuvant. On days 21 and 42, alpaca were boosted with human NKG2D / His protein and Freund's incomplete adjuvant. One week after each immunization, blood was collected to detect the serum titer of Anti-NKG2D / His. One week after the third immunization, 50 mL of blood was collected for screening and library construction.

[0061] 3) The anti-NKG2D / His serum titer was detected by ELISA. The detection procedure was as follows: NKG2D / His protein at a concentration of 2 μg / mL was coated onto an ELISA plate overnight at 4°C. 3% BSA blocking buffer was added, and the plate was blocked for 1 hour. After washing three times, 100 μL of serum obtained after three immunizations (control: pre-immunization alpaca serum) was added to each well in a 2-fold serial dilution (i.e., each well was diluted twice the concentration of the previous well). The plate was incubated at 37°C for 1.5 hours, washed twice, and horseradish peroxidase-labeled Goatanti-Alpaca IgG (H+L) secondary antibody diluted 1:10000 was added to each well. The plate was incubated at 37°C for 1 hour, washed five times, and then TMB substrate was added. The plate was incubated at 37°C for 5-10 minutes, and the reaction was stopped by adding 0.1 M H2SO4. The OD450 value was measured. When the OD450 value of the test sample was more than three times that of the negative control, the antiserum titer was considered positive. The results are as follows: Figure 1 As shown, Figure 1 The antiserum titer after three immunizations is shown to be 102400. This demonstrates that the antigen can induce alpacas to produce high-titer antiserum specifically targeting the NKG2D protein, and antibody libraries can be constructed using 3-immunized PBMCs.

[0062] 4) Dilute the collected blood twice with PBS, separate PBMCs using lymphocyte separation medium (Ficoll reagent), and then dissolve them with Trizol to obtain a cell concentration of 10. 7 / ml of PBMC cells, stored at -80 degrees Celsius.

[0063] 5) RNA was extracted from PBMCs using the Trizol method, and the RNA concentration was determined and identified by agarose gel electrophoresis. Figure 2If 28S, 18S, and 5S small RNA bands are visible, it indicates good RNA integrity; a 28S to 18S RNA ratio of 2:1 indicates no RNA degradation. Use the amount required for reverse transcription, and store any remaining amount at -80℃.

[0064] 6) RNA was reverse transcribed into cDNA using oligo(dT) (using the TaKaRa-SMARTcribe Reverse Transcript kit). Nested PCR was then performed in the first and second rounds to obtain the target gene fragment. Figure 3 These are the results of the first round of PCR; the target fragment is around 750bp. Figure 4 The results of the second round of PCR show that the target band is approximately 400 bp. The obtained target gene fragment was cloned into a phage expression vector to obtain a phage vector containing the antibody fragment. This vector was then electroporated into SS320 electroporation competent cells, and the cells were collected to obtain an NKG2D immune phage antibody library with a capacity of 1.1E+09 cfu.

[0065] 7) Patch the NKG2D phage antibody library, package the obtained library bacteria into phages, and determine its titer, which is 1E+13cfu, which is 1Input phage. Human NKG2D / His protein was coated overnight at 4°C in immunotubes. Simultaneously, negative sieve tubes were coated with 3% BSA. The next day, positive and negative sieve tubes, along with the antibody library phage to be added, were blocked with 3% BSA for 1 hour. The blocking solution in the negative sieve tubes was removed, and the blocked phage was transferred to the negative sieve tubes and incubated at room temperature for 1 hour. The blocking solution in the positive sieve tubes was removed, and the phage in the negative sieve tubes was transferred to the positive sieve tubes and incubated at room temperature for 1 hour. The immunotubes were washed 8 times with 0.1% PBST, and eluted with 1 ml of trypsin for 20 minutes, resulting in a 1-output phage. 0.5 ml of the 1-output phage was used to infect 5 ml of SS320 (OD600 0.4-0.6) at 37°C for 30 minutes. After centrifugation, 300 μl of supernatant was collected for resuspending, and then plated (with ampicillin and tetracycline added). The plates were incubated overnight at 37°C to obtain 1-output library plates. Simultaneously, 1-output phage was used... Phage was serially diluted 10-fold to infect SS320 cells, spotted onto plates, and the titer of 1 Output phage was measured to be 2E+0.7 CFU. Phage was then packaged into 2 Input phages, and their titer was measured to be 1E+1.3 CFU, which were then used for the next round of screening. In the second round, the amount of human NKG2D / His protein coating and the amount of 2 Input phages were reduced. The panning process was the same as the first round, and the concentration of the obtained 2 Output phages was 1E+0.9 CFU. Based on the results, phage enrichment was observed in the second round of panning, so the phages obtained in the second round were selected. After infecting SS320 cells, the phages were plated, single clones were selected and cultured, and clone ELISA was performed.

[0066] 8) Clone ELISA: Overnight human body at 4°C NKG2D / His protein was added to a 96-well microplate. The next day, 1% BSA was added for blocking at room temperature for 1 hour. After washing three times with 0.05% PBST, the cultured single-clone culture was centrifuged, and the supernatant was added. The last column was the negative control. The plate was incubated at room temperature for 1 hour, washed three times with 0.05% PBST, and diluted secondary antibody anti-M13 (1:10000) was added. The plate was incubated at room temperature for 40 minutes, washed six times with 0.05% PBST, and TMB chromogenic solution was added. The plate was incubated for 5-10 minutes, and the stop solution was added to stop the reaction. The OD450 value was measured using a microplate reader. Clones with an OD450 value greater than three times that of the negative control were not positive. The results are shown in Tables 1-1 to 1-4. The shaded areas are those with an OD450 value greater than three times that of the negative control, which can be considered as positive clones. A total of 115 single clones that bound to NKG2D protein were selected. Sequencing analysis showed that there were 53 uinque sequences. These sequences were replaced with a human FC eukaryotic expression vector for purification and expression. Ultimately, 18 sequences were successfully expressed.

[0067] The amino acid sequences of the 18 anti-NKG2D nanobody are as follows: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. The nucleotide sequences are shown in SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48 and SEQ ID NO.49.

[0068] Table 1-1 Results of Monoclonal ELISA after selection

[0069]

[0070]

[0071] Table 1-2 Results of Monoclonal ELISA After Selection

[0072]

[0073] Table 1-3 Results of Monoclonal ELISA after selection

[0074]

[0075]

[0076] Table 1-4 Results of Monoclonal ELISA After Selection

[0077]

[0078] 9) Cell function assay: Cell binding (FACS). Cultured NKG2D overexpressing cells were prepared to a cell count of 2E+06 cells / ml using MACS buffer. 50 μL of cell suspension was added to each well of a 96-well plate. The prepared antibody was serially diluted 4-fold with MACS buffer, and 50 μL of antibody dilution was added to each well of the plate. Positive and negative controls were added. The plates were incubated at 4°C in the dark for 60 min. After washing three times with MACS buffer, the cells were resuspended in 100 μL of fluorescent secondary antibody (1:1000). The plates were incubated at 4°C in the dark for 30 min. After washing three times with MACS buffer, the cells were resuspended in 200 μL of MACS buffer. The cells were then analyzed using flow cytometry. Figure 5 The results showed that all 18 antibodies bound to cells overexpressing NKG2D.

[0079] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. An anti-NKG2D Nanobody, characterized in that, The anti-NKG2D nanobody comprises a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2, CDR3, the complementarity determining region CDR1 sequence is shown as SEQ ID NO. 8, the complementarity determining region CDR2 sequence is shown as SEQ ID NO. 9, and the complementarity determining region CDR3 sequence is shown as SEQ ID NO.

10.

2. The anti-NKG2D Nanobody according to claim 1, characterized in that, The amino acid sequence of the anti-NKG2D nanobody is shown as SEQ ID NO.

31.

3. A nucleic acid encoding the anti-NKG2D nanobody according to claim 1 or 2.

4. A vector containing the nucleic acid according to claim 3.

5. A host cell containing the vector according to claim 4.

6. A kit containing the nanobody according to claim 1 or 2.

7. Use of the anti-NKG2D nanobody according to claim 1 or 2 in the preparation of a product for detecting NKG2D.

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