Anti-NKG2D nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-NKG2D nano antibody as well as a preparation method and application thereof. The method comprises the following steps: constructing and expressing NKG2D protein, immunizing alpaca to obtain immune alpaca PBMC (peripheral blood mononuclear cells), extracting RNA (ribonucleic acid) of the PBMC, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), obtaining an antibody gene segment through PCR (polymerase chain reaction), and constructing the antibody gene segment on a phage expression vector; the method comprises the following steps: carrying out electrotransformation on a vector with an antibody gene segment to an SS320 electrotransformation competent state to obtain an NKG2D immune antibody library, carrying out two rounds of NKG2D protein panning and Clone ELISA detection to obtain an antibody combined with NKG2D protein, carrying out sequencing analysis, selecting a unique sequence, and carrying out high-throughput expression through a mammalian cell high-throughput expression system to obtain an antibody protein. And carrying out FACS detection by using an NKG2D high-expression cell line to finally obtain the antibody combined with the NKG2D. The method disclosed by the invention is simple and quick to operate; a mammalian cell expression system induces high-efficiency expression of the antibody, processing modification can be performed after translation, and the activity of the antibody is closer to that of a natural antibody.

Description

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

[0002] The present invention belongs to the technical field of antibody preparation, and specifically relates to an anti-NKG2D nano antibody and a preparation method and application thereof. Background Art

[0003] In the past few years, camelid single-domain antibodies (nanobodies) have been shown to be the basis for the generation of innovative therapeutic approaches for various human diseases. Nanobodies can bind to epitopes that are inaccessible to conventional antibodies, and they are very suitable for the generation of blocking or agonist molecules. In addition, because they are relatively small proteins (15KD), they allow the generation of multivalent, multispecific or multi-heterotopic structures, which can show obvious advantages over traditional monoclonal antibodies. (2-4) .

[0004] At present, the research and use of immune checkpoint inhibitors have been quite extensive, among which PD-1 and its ligand PDL-1 (1) Immune checkpoint inhibitors represented by NKG2D have shown good tumor immunotherapy effects, but there are also problems with low patient response rates and drug resistance. In addition, new treatments for activating the immune system should be sought to improve the clinical benefits of patients with advanced tumors. NKG2D is a C-type lectin-like receptor molecule composed of homodimers. (6) In humans, NKG2D is usually 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 of NK cells, and 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. The activation of NK cells is regulated by the balance between the activating receptors and inhibitory receptors on their surface. NKG2D can activate NK cells by binding to NKG2D ligands on the tumor surface, thereby playing a role in monitoring and clearing tumor immunity. (5) , This makes it an important target in tumor immunotherapy. Tumor immunotherapy is a biological treatment method that aims to overcome the tumor 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] Targeting NKG2D therapy can also effectively reduce various inflammatory injuries, and NKG2D blocking antibodies can delay NK cell-mediated airway allergic reactions (12) , colitis, etc. Blocking antibodies against NKG2D have been shown to significantly increase clinical remission in patients with Crohn's disease. Targeting and blocking NKG2D will be another new mechanism of action for the treatment of patients with moderate to severe Crohn's disease. (11) NKG2D is also involved in the pathogenesis of some autoimmune or inflammatory diseases, such as diabetes (13) , Celiac disease, Multiple sclerosis (14) , rheumatoid arthritis (15) As well as the occurrence of acute hepatitis, the blocking and inhibition of NKG2D can effectively protect the body from the occurrence of disease or reduce inflammatory damage.

[0006] WingKeung Chan et al. (16) The study showed that CS1-NKG2D bispecific antibody promoted CS1 + Multiple myeloma cells and NKG2D + Enhancement of the immune synapse between cytolytic innate and antigen-specific effector cells also in turn activates these immune cells to improve clearance of multiple myeloma. Sebastian Lutz et al. (17) They also studied the use of novel NKG2D-directed bispecific antibodies to enhance antibody-mediated killing of malignant B cells by NK cells and T cells, and obtained CD20-NKG2D bispecific antibodies that recruited cytotoxic lymphocytes into lymphoma cells and triggered NK cell-mediated lymphoma cell lysis. They also proposed some combination approaches that may be applicable to existing immunotherapies.

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

[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 the present invention is to overcome the deficiencies of the prior art and provide an anti-NKG2D nanobody and a preparation method and application thereof.

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

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

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

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

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

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

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

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

[0035]

[0036] Further, the anti-NKG2D nanobody has an amino acid sequence selected from any one 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 the second aspect of the present invention, a nucleic acid encoding the anti-NKG2D nanobody as described in the first aspect is provided.

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

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

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

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

[0042] In the present invention, the alpaca immunization, the primers used in the step of amplifying the antibody gene fragment, the panning method, and the construction of the NKG2D high-expression cell line are obtained after optimization in conventional operations.

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

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

[0045] In the eighth aspect of the present invention, there is provided the use of the anti-NKG2D nanobody as described in the first aspect in the preparation of a product for detecting NKG2D.

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

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

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

[0049] 2) The antibody preparation method of the present invention is simple and rapid to operate; and the mammalian cell expression system induces efficient expression of antibodies, which can be processed and modified after translation, and their activity is closer to that of natural antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the titer of NKG2D protein immune serum.

[0051] Figure 2 RNA agarose gel electrophoresis.

[0052] Figure 3 This is the electrophoresis diagram of the first round of PCR products.

[0053] Figure 4 The electrophoresis diagram of the second-round PCR products. 1-13 are the bands amplified by 13 pairs of second-round PCR primers.

[0054] Figure 5 Cell binding experiment results. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0056] The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0057] Example 1

[0058] In this embodiment, the method for preparing NKG2D nanobody comprises the following steps:

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

[0060] 2) Immunize the alpaca three times with a mixture of the modified antigen obtained in step 1 and Freund's adjuvant to obtain alpaca PBMC cells: use an emulsified mixture of human NKG2D / His protein (i.e., the modified amino acids obtained in step 1) and Freund's complete adjuvant to prime the alpaca, and boost immunize twice with human NKG2D / His protein and Freund's incomplete adjuvant on days 21 and 42. One week after each immunization, collect blood to detect the Anti-NKG2D / His serum titer; one week after the third immunization, collect 50 mL of blood for screening and library construction.

[0061] 3) Anti-NKG2D / His serum titer was detected by ELISA. The detection process was as follows: 2 μg / mL NKG2D / His protein was coated on the ELISA plate overnight at 4°C, 3% BSA blocking solution was added, blocking for 1 hour, washing 3 times, 100 μL of serum obtained after three immunizations (the control was pre-immunization alpaca serum) was added to each well with a 2-fold gradient dilution (i.e., each well was diluted to 2 times of the previous well), incubated at 37°C for 1.5 hours, washed twice, and 1:10000 horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody was added to each well, incubated at 37°C for 1 hour, washed 5 times, TMB substrate was added, incubated at 37°C for 5-10 minutes, 0.1M H2SO4 was added to stop the reaction, and OD450nm was measured. When the OD450 value of the sample to be tested is more than 3 times that of the negative control, the antiserum titer is judged to be positive. The results are as follows: Figure 1 As shown, Figure 1 The titer of the antiserum after 3 immunizations is shown to be 102400. This shows that the antigen can induce alpacas to produce high-titer antiserum specific for NKG2D protein, and the antibody library can be constructed using 3 immunization PBMCs.

[0062] 4) The collected blood was diluted twice with PBS, PBMC cells were separated with lymphocyte separation solution (Ficoll reagent), and Trizol was added to dissolve the cells to obtain a cell concentration of 10 7 / ml PBMC cells, stored at -80 degrees.

[0063] 5) Extract RNA from PBMCs using Trizol method, determine RNA concentration and perform agarose gel analysis, such as Figure 2, 28S, 18S and 5S small RNA bands can be seen, indicating that the RNA integrity is good; the ratio of 28S and 18S RNA is 2:1, indicating that the RNA is not degraded. Use according to the amount required for reverse transcription, and store the remaining at -80℃.

[0064] 6) RNA was reverse transcribed into cDNA using oligo(dT) (the reverse transcription kit was TaKaRa-SMARTcribeReverse Transcript), and the first and second rounds of PCR were performed using nested PCR to obtain the target gene fragment. Figure 3 This is the result of the first round of PCR. The target fragment is about 750 bp. Figure 4 The second round of PCR results showed that the target band was about 400 bp. The target gene fragment was cloned into a phage expression vector to obtain a phage vector with an antibody fragment, which was electrotransformed into an SS320 electrotransformation competent medium, and the bacteria were collected to obtain an NKG2D immune phage antibody library with a library capacity of 1.1E+09 cfu.

[0065] 7) The NKG2D phage antibody library was panned, and the obtained library bacteria were packaged into phages and the titer was measured, which was 1E+13 cfu, which was 1 Input phage. Coat human NKG2D / His protein in an immunotube overnight at 4°C, and coat the negative screening tube with 3% BSA at the same time. On the second day, block the positive and negative screening tubes and the antibody library phage to be put in with 3% BSA at the same time, block for 1 hour, remove the blocking solution of the negative screening tube, and then transfer the blocked phage to the negative screening tube and incubate at room temperature for 1 hour. Remove the blocking solution of the positive screening tube, transfer the phage in the negative screening tube to the positive screening tube, incubate at room temperature for 1 hour, wash the immunotube 8 times with 0.1% PBST, add 1ml of trypsin for 20 minutes to elute, this is 1Output phage, use 0.5ml of 1Output phage to infect 5ml SS320 with an OD600 of 0.4-0.6, after infection at 37°C for 30 minutes, retain 300ul of supernatant after centrifugation, resuspend, and then apply to the plate (with ampicillin and tetracycline), and culture at 37°C overnight to obtain 1Output library bacterial plate; at the same time, use 1Output Phage was diluted 10 times to infect SS320, and the plate was spotted. The titer of 1Outputphage was measured, which was 2E+07cfu. The 1Outputphage library bacteria were packaged as phage, and the obtained phage was 2Input phage. At the same time, its titer was measured, which was 1E+13cfu, and the next round of screening was carried out. In the second round, the amount of human NKG2D / His protein coating and the input amount of 2Input phage were reduced. The selection process was the same as the first round, and the concentration of 2Output phage obtained was 1E+09cfu. According to the results, phage was enriched in the second round of selection, so the phage obtained in the second round was selected. After infecting SS320, the plate was coated, and the monoclonal culture was selected for clone ELISA.

[0066] 8) Clone ELISA: Coat human at 4℃ overnight NKG2D / His protein was added to a 96-well ELISA plate. The next day, 1% BSA was added to block the plate at room temperature for 1 hour, and the plate was washed three times with 0.05% PBST. The cultured monoclonal bacterial solution was centrifuged and the supernatant was added. The last column was a negative control. The plate was incubated at room temperature for 1 hour, washed three times with 0.05% PBST, and the 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, TMB colorimetric solution was added, and the color was developed for 5-10 minutes. The stop solution was added to stop the reaction, and the OD450 value was detected by an ELISA instrument. The non-positive clones with a value greater than 3 times that of the negative control were as shown in Tables 1-1 to 1-4. The shaded part is the one with an OD450 value greater than three times that of the negative control, which can be used as a positive clone. A total of 115 monoclonal clones that combined with NKG2D protein were selected and sequenced. The results showed that there were 53 uinque sequences, which were replaced on the fusion human FC eukaryotic expression vector for purification and expression. Finally, 18 sequences were successfully expressed.

[0067] The amino acid sequences of the 18 anti-NKG2D nanoantibodies are: 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 as 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, SEQID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48 and SEQ ID NO.49 respectively.

[0068] Table 1-1 Monoclonal ELISA results after panning

[0069]

[0070]

[0071] Table 1-2 Monoclonal ELISA results after selection

[0072]

[0073] Table 1-3 Monoclonal ELISA results after selection

[0074]

[0075]

[0076] Table 1-4 Monoclonal ELISA results after selection

[0077]

[0078] 9) Cell function detection: cell binding (FACS). The cultured NKG2D overexpressing cells were prepared with MACS buffer to a cell number of 2E+06 cells / ml, and 50ul / well cell suspension was added to a 96-well plate. The prepared antibody was diluted 4 times with MACS buffer, and 50ul antibody dilution was added to the designated wells of the 96-well plate. Mix well, add positive control and negative control at the same time, incubate at 4°C in the dark for 60min, wash 3 times with MACS buffer, add 100ul fluorescent secondary antibody (1:1000) to resuspend the cells, incubate at 4°C in the dark for 30min, wash 3 times with MACS buffer, add 200ul MACS buffer to resuspend the cells, measure on a flow cytometer, and perform data analysis. Figure 5 As a result, the results showed that all 18 antibodies bound to cells overexpressing NKG2D.

[0079] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-NKG2D nanobody, characterized in that: The anti-NKG2D nanobody includes a framework region and a complementary determining region; the complementary determining region includes CDR1, CDR2, and CDR3, the complementary determining region CDR1 sequence is shown in SEQ ID NO.11, the complementary determining region CDR2 sequence is shown in SEQ ID NO.12, and the complementary determining region CDR3 sequence is shown in SEQ ID NO.

13.

2. The anti-NKG2D nanobody according to claim 1, characterized in that The anti-NKG2D nanobody has an amino acid sequence as shown in SEQ ID NO.

17.

3. A nucleic acid encoding the anti-NKG2D nanobody as claimed in claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid sequence of the anti-NKG2D nanobody is shown as SEQ ID NO.

35.

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

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

7. A kit comprising a Nanobody as claimed in claim 1 or 2 or a biomaterial as claimed in any one of claims 4 to 6.

8. Use of the anti-NKG2D nanobody as claimed in claim 1 or 2 in the preparation of a product for detecting NKG2D.

9. Use of the anti-NKG2D nanobody according to claim 1 or 2 in the preparation of a product binding to NKG2D.

Images