Nano antibody with high affinity with human DR3 extracellular domain

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a nano antibody with high affinity with a human DR3 extracellular domain. The method comprises the following steps: immunizing alpaca by using a DR3 extracellular domain of an insect expression system, separating peripheral blood lymphocytes, performing total RNA extraction, performing reverse transcription, amplifying a nano-antibody sequence, and finally separating to obtain a nano-antibody named as H10. The SPR (Surface Plasmon Resonance) result shows that the nano antibody can be combined with the extracellular domain of human DR3 with high affinity, and the nano antibody provided by the invention is expected to provide an experimental factual basis for the treatment thought of DR3-related diseases.

Description

[0001] This application is a divisional application of the application with the application number 202310649138.3, the application date of June 2, 2023, and the invention name "A Nanobody Targeting the Extracellular Domain of DR3 and Its Application". Technical Field

[0002] The present invention relates to the field of biopharmaceutical technology, and in particular to a nanobody with high affinity for the extracellular domain of human DR3. Technical Background

[0003] The tumor necrosis factor receptor superfamily (TNFRSF) is one of the most important families in the immune system, widely regulating various immune responses such as the development of immune organs, co-stimulating lymphocytes, and determining the fate of lymphocytes. Among the TNFRSF family, death receptor 3 (DR3) has the highest amino acid sequence homology with TNFR1. DR3 is highly expressed in lymphocytes in the thymus and spleen and peripheral blood lymphocytes, and plays a certain pro-apoptotic role in the development of thymocytes, while in lymphocytes, it mainly plays a co-stimulatory role with its only ligand TL1A (TNF-like ligand 1A, TNFSF15). When the ligand TL1A activates the signaling pathway, the intracellular DR3 can recruit its downstream adaptor molecule TRADD (TNFR-associated death domain), and ultimately activate the NF-κB signal. The most important chronic autoimmune diseases related to TL1A-DR3 are inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). Studies have shown that the DR3-TL1A signaling pathway can play a pathogenic role in inflammatory bowel disease by promoting the immune responses of Th1 and Th17. For example, TL1A / DR3 can play a pro-inflammatory role in the intestine by activating ILC3 (Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation).

[0004] Given that the TL1A-DR3 signaling pathway plays a crucial role in autoimmune and inflammatory diseases, studies have shown that neutralizing TL1A with soluble DR3 protein can effectively alleviate colitis induced by α-CD40 and DSS, indicating that inhibiting the TL1A-DR3 interaction may be an effective therapeutic strategy for improving autoimmune diseases and local inflammation in target organs. In recent years, the incidence and prevalence of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease have been on the rise. However, current traditional drug treatments such as 5-aminosalicylic acid esters have uncertain efficacy and may also have a high recurrence rate, and are ineffective in patients with moderate to severe IBD. Ligand and receptor drugs targeting related signaling pathways have become the most promising targets for anti-IBD drug development. Developing an antibody that targets the extracellular domain of DR3 and blocks its activation of the signaling pathway by ligand TL1A will have great clinical significance. Summary of the Invention

[0005] To solve the problems in the prior art, one of the objectives of the present invention is to provide a nanobody targeting the extracellular domain of DR3, and the nanobody is any one of A2, A6, and H10. The A2, A6, and H10 all contain three antigen-binding complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0006] The amino acid sequences of the three antigen-binding complementarity-determining regions of A2 are respectively amino acid sequences with a homology of greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3;

[0007] The amino acid sequences of the three antigen-binding complementarity-determining regions of A6 are respectively amino acid sequences with a homology of greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6;

[0008] The amino acid sequences of the three antigen-binding complementarity-determining regions of H10 are respectively amino acid sequences with a homology of greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0009] The above homologous sequences also include amino acid sequences having one or more (preferably 1, 2, or 3) conservative amino acid mutations (preferably substitution, insertion, or deletion) compared with the sequences shown in the sequence listing.

[0010] Preferably, the amino acid sequences of A2, A6 and H10 are as follows:

[0011] Amino acid sequence of A2:

[0012] QVQLVESGGGLVQAGGSLRLSCAAS GDTICISAM GWYRQAPGKERE LVAGITSS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADLGSCPGVYE YEYWGQGTQVTVSS(SEQ ID NO:10)

[0013] Amino acid sequence of A6:

[0014] QVQLVESGGGLVQPGGSLRLSCAAS GDTICISGM GWYRQAPGKERE LVAAITSGGS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADGSRCPGE YDYWGQGTQVTVSS(SEQ ID NO:11)

[0015] Amino acid sequence of H10:

[0016] QVQLVESGGGLVQPGGSLRLSCTAS GLTSDDYTM GWFRQAPGKERE GVSCISRVDGN TYYADSVKGRFTISRGNAKNTVYLQMNSLKPEDTAVYYC ATDCRLTPNSDWNGPLRFGS WGQGTQVTVSS(SEQ ID NO:12)

[0017] The amino acid sequences of the three antigen - complementary determining regions CDR1, CDR2 and CDR3 of the above nanobodies are shown as the bold - underlined parts respectively, that is:

[0018] Amino acid sequence of the antigen - complementary determining region of A2:

[0019] CDR1:GDTICISAM(SEQ ID NO:1)

[0020] CDR2:LVAGITSS(SEQ ID NO:2)

[0021] CDR3:NADLGSCPGVYE(SEQ ID NO:3)

[0022] Amino acid sequences of the three antigen - complementary determining regions of A6:

[0023] CDR1: GDTICISGM (SEQ ID NO:4)

[0024] CDR2: LVAAITSGGS (SEQ ID NO:5)

[0025] CDR3: NADGSRCPGE (SEQ ID NO:6)

[0026] Amino acid sequences of three antigen - complementary determining regions of H10:

[0027] CDR1: GLTSDDYTM (SEQ ID NO:7)

[0028] CDR2: GVSCISRVDGN (SEQ ID NO:8)

[0029] CDR3: ATDCRLTPNSDWNGPLRFGS (SEQ ID NO:9)

[0030] The present invention also provides an antibody targeting the extracellular domain of DR3, which has any one of the nanobodies A2, A6, and H10 as described above and an Fc domain.

[0031] Preferably, the Fc domain is a human IgG1 Fc domain, and its amino acid sequence is as shown in SEQ ID NO:13.

[0032] The present invention also provides a polynucleotide, which encodes the nanobody A2, A6 or H10 as described above, or encodes the antibody with an Fc domain as described above; wherein the nucleotide sequence encoding A2 is as shown in SEQ ID NO:14, the nucleotide sequence encoding A6 is as shown in SEQ ID NO:15, and the nucleotide sequence encoding H10 is as shown in SEQ ID NO:16.

[0033] The present invention also provides an expression vector containing the polynucleotide as described above, and a host cell containing the expression vector as described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, mammalian cells.

[0034] The present invention provides a pharmaceutical composition, which contains the nanobody A2, A6 or H10 as described above, or contains the antibody with an Fc domain as described above.

[0035] Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0036] The nanobodies / antibodies provided by the present invention can be used for preparing medicaments for treating and / or diagnosing DR3-related diseases, such as Crohn's disease, ulcerative colitis, and rheumatoid arthritis.

[0037] The present invention also provides the use of the above-mentioned nanobody A2, A6 or H10, or an antibody containing an Fc domain as described above in the preparation of a kit for treating and / or diagnosing DR3-related diseases, which are Crohn's disease, ulcerative colitis or rheumatoid arthritis.

[0038] Preferably, the nanobody / antibody further comprises a second antibody, and the second antibody comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0039] The beneficial effects of the present invention are as follows:

[0040] 1. The extracellular domain of DR3 of an insect expression system was used to immunize alpacas three times, and then blood was drawn to isolate peripheral blood lymphocytes (PBMCs). Total RNA was extracted from the isolated PBMCs and immediately reverse transcribed into cDNA. The cDNA was used as a template to amplify the nanobody sequence, and finally three nanobodies were isolated and named A2, A6 and H10 respectively.

[0041] The nanobody (VHH) is derived from the natural camel heavy chain antibody, and has the following advantages: 1) simple structure and small molecular weight, which are beneficial to expression and use; 2) convenient for high-efficiency and large-scale expression in Escherichia coli and various eukaryotic systems; 3) since it has only one binding site and belongs to a single-domain antibody, it has better permeability, specificity and detection linearity as a diagnostic reagent; 4) convenient for coupling with various fusion proteins or easier to be labeled with various markers; 5) easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and cell target-directed transportation; 6) as a drug development, it has low human immunogenicity and is not easy to cause immune rejection.

[0042] 2. The three nanobodies provided by the present invention have different antigen complementarity determining regions. The antibody that binds to the extracellular domain of human DR3 was expressed and secreted using mammalian cells (HEK293F). The antibody was fused with human IgG1 Fc and cloned into the mammalian expression vector pTT5. The vector was transfected into mammalian cells 293F, and the supernatant was collected after culturing for 4 days. The fusion protein in the supernatant was purified using a Protein A column, and the yields of the three nanobodies were all greater than 50 mg / L.

[0043] ELISA results showed that the three nanobodies all showed high-affinity binding to the extracellular domain of human DR3, and the EC 50 of A2-IgG1 Fc was 0.072 ± 0.021 nM, and the EC50 At 0.146 ± 0.041 nM, the EC of H10-IgG1 Fc 50 is at 0.063 ± 0.017 nM. The nanobody provided by the present invention is expected to provide experimental factual basis for the treatment ideas of DR3-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 ELISA results of monoclonal phages specifically binding to the extracellular domain of human DR3.

[0045] Figure 2 Results of antigen and antibody purification and SDS-PAGE analysis; in the figure, A is the results of 24 mL molecular sieve and SDS-PAGE of the extracellular region of antigen human DR3, B is the elution diagram and SDS-PAGE results of nanobody A2 purified by Protein A column, C is the elution diagram and SDS-PAGE results of nanobody A6 purified by Protein A column, and D is the elution diagram and SDS-PAGE results of nanobody H10 purified by Protein A column.

[0046] Figure 3 Results of binding analysis of nanobodies to the extracellular region of human DR3 by competitive ELISA; the EC 50 values are respectively A2: EC 50 = 0.072 ± 0.021 nM; A6: EC 50 = 0.146 ± 0.041 nM; G10: EC 50 = 0.063 ± 0.017 nM. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] For ease of understanding, the technical solutions of the present invention will be described more specifically below in combination with embodiments:

[0048] Example 1

[0049] Purify the extracellular domain of human DR3 for immunizing alpacas and screening

[0050] 1) The expression plasmid was constructed and transformed into DH10Bac competent cells containing Bacmid and helper plasmid. The target gene was transposed onto Bacmid under the action of transposase. Through the resistance screening of kanamycin, gentamicin and tetracycline and blue-white screening, clones containing recombinant transposons were obtained. After further PCR identification, high-purity and high-concentration recombinant Bacmid was purified and transfected into corresponding insect cells to obtain recombinant baculovirus containing the target gene. The recombinant baculovirus was amplified in SF9 cells for three generations, and the virus in the supernatant was collected. It was added to Hi5 cells at a ratio of 1:1000 for virus infection and expression of the target protein. Four days later, the cell supernatant was collected and the extracellular region residues 26-199 of human DR3 protein were purified by affinity chromatography and size exclusion chromatography, and the sequence was shown as SEQ ID NO:17.

[0051] For alpaca immunization, Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the next two times. Each time, the antigen dose was 200 μg / time, and it was mixed according to the volume ratio of antigen to adjuvant of 1:1. The first two injections were given by subcutaneous injection, and the last injection was given by intramuscular injection. The alpaca (Vicugna pacos) was immunized 3 times at intervals of 3 weeks.

[0052] 2) At the 11th week, blood was collected from the vein and peripheral blood lymphocytes were isolated using Ficoll from Solarbio.

[0053] 3) Total RNA was extracted using the RNA extraction kit from Omega Biotek and genomic DNA was removed. Using the PrimeScript TM II First Strand cDNA Synthesis Kit from Takara, RNA was reverse transcribed into cDNA.

[0054] 4) Construction of the nanobody phage display library: Using the above cDNA as a template, the coding sequence of the nanobody was amplified by PCR using specific alpaca VHH primers. The amplified nanobody sequence was inserted into the NcoI and NotI sites of the phagemid pR2 by Gibson assembly. The obtained Gibson assembly product was the initial nanobody phage library, and the product was recovered.

[0055] 5) Preparation of Escherichia coli T1 (MRC Laboratory of Molecular Biology) competent cells using the 10% glycerol washing method. The activated TG1 was cultured in 250 mL of 2×YT (formula per liter: 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl) medium until OD 600Approximately 0.6 - 0.8. After centrifugation at 5000g for 15 min, wash three times with 250 mL, 250 mL, and 100 mL of pre-cooled 10% glycerol, and finally resuspend in 1 mL of 10% glycerol and aliquot into 500 μL per tube.

[0056] 6) Transform Escherichia coli TG1 competent cells using a BTX ECM 399 electroporator. Mix the product recovered from Gibson assembly with 500 μL of TG1 competent cells and transfer to a 0.1 cm electroporation cuvette, followed by electroporation at 2.5 KV. Resuspend the electroporated product in 20 mL of 2× medium and incubate at 37 °C and 220 rpm for 1 h. Take 10 μL of the bacterial solution, dilute it to 990 μL of 2× YT and mix well, then take 40 μL of the bacterial solution, dilute it to 160 μL of 2× YT, plate 100 μL, and culture overnight at 37 °C. Count the colonies the next day to calculate the library size (library size = number of counted colonies × 10 5 ). Spread the bacteria on 5 150 mm 2× YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library and culture overnight at 37 °C. Next, scrape the transformed colonies from the plates, vortex thoroughly with glycerol at a final concentration of 25%, snap-freeze in 1 mL aliquots in liquid nitrogen, and store at -80 °C, and calculate the size of the phage library.

[0057] 7) Amplification of the nanobody phage display library: To amplify the phage library of nanobodies, thaw 0.2 ml of the frozen library on ice, dilute it into 200 mL of 2× YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and culture at 37 °C and 220 rpm. Next, add 1×10 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) to the culture and incubate at 37 °C (water bath) for 45 min. Separate the cell pellet by high-speed centrifugation and resuspend in 200 mL of 2× YT medium supplemented with 0.1% glucose, 50 μg / mL kanamycin, and 100 μg / mL ampicillin. Incubate the cells at 25 °C and 220 rpm for 20 h to amplify the phage library. After centrifugation, add polyethylene glycol (PEG) to the culture supernatant to precipitate the phage particles. Dissolve the precipitated phage particle pellet in 1× PBS (formula: 10 mmol / L Na2HPO4; 1.75 mmol / L KH2PO4; 137 mmol / L NaCl; 2.65 mmol / L KCl; pH 7.2 - 7.6) and store in 1 mL aliquots at -80 °C in the presence of 25% glycerol.

[0058] 8) Screening: Dilute the extracellular domain of purified human DR3 with PBS to a final concentration of 0.1 mg / ml, and coat it into one well of a 96-well immunoplates ELISA (Nunc maxsorp plate), and leave one well of the ELISA plate as a negative control. After washing 3 times with 1×PBS, add 300 μL of MPBS (1×PBS containing 5% skim milk) to each well of the ELISA plate, and incubate at room temperature for 2 hours to block unbound sites. Next, wash the plate 3 times with 1×PBS, and add the phage library containing 1×10 11 pfu (diluted in 100 μL of MPBS) specific for the extracellular domain of human DR3 to each well. After incubating at room temperature for one hour, wash 3 times with PBST (1×PBS containing 0.1% Tween 20). Elute the phages displaying nanobodies specific for the extracellular domain of human DR3 by incubating with trypsin at a final concentration of 0.5 mg / ml at room temperature for one hour. Add 10 μL of the eluted phages to 1 mL of competent Escherichia coli TG1 cells, and incubate at 37 °C (water bath) for 45 minutes for infection, then spread the bacterial culture on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubate overnight at 37 °C.

[0059] 9) Preparation of monoclonal phages: After one round of panning, pick 48 individual colonies into a 96-well round-bottom culture dish containing 100 μL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). Incubate continuously at 37 °C, 180 rpm for 12 hours. Next, inoculate 5 μL of this culture into a new 96-well round-bottom culture dish containing 200 μL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). Incubate the freshly inoculated plate at 37 °C, 250 rpm for 1.5 hours until the OD260 is approximately 0.5. Add 50 μL of 2×YT medium containing 4×10 8 pfu of KM13 helper phages to each well of the plate, and incubate at 37 °C for 45 minutes without shaking for infection. After infection, discard 150 μL of the supernatant, centrifuge the remaining volume at 3500 g for 15 minutes, discard the supernatant, resuspend the bacterial pellet in 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1% glucose (w / v), and incubate overnight at 25 °C, 250 rpm for about 14 - 16 hours. The next day, centrifuge the culture at 3500 g for 30 minutes, then transfer 150 μL of the supernatant to a new 96-well plate and store at 4 °C for screening of nanobodies.

[0060] 10) Phage ELISA assay. Dilution was performed using 1×PBS. 100 μL of the extracellular domain of human DR3 at a final concentration of 0.2 μg / mL was used to coat a 96-well round-bottom immunological ELISA plate and incubated at 4 °C for 16 h. Next, the ELISA plate was washed 3 times with 1×PBS. The plate was blocked with MPBS (1×PBS containing 5% skim milk) at room temperature for 2 h. After that, the wells were washed 4 times with PBS-0.1% Tween 20. Then, 100 μL of phage containing 1×10 11 pfu was added to each well and incubated at room temperature for 1 h. Then, the ELISA plate was washed 5 times with PBS-0.1% Tween 20. Next, HRP-KM13 diluted 1:8000 with MPBS was added to the ELISA plate at 100 μL per well and incubated at room temperature for 1 h. Subsequently, the wells were washed 4 times with PBS-0.1% Tween 20, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well for color development. Incubation was carried out in the dark for 7 min to develop color, and then 50 μL of 1 M H2SO4 was immediately added to terminate the reaction, and the OD 450nm value was measured using an enzyme-linked immunosorbent assay reader. The results are as Figure 1 shown.

[0061] 11) Twenty positive clones with an OD 450nm value greater than 1 were picked for sequencing and sequence analysis. Finally, three positive phage nanobodies specific for the extracellular region protein of human DR3 were identified and named A2 (amino acid sequence shown in SEQ ID NO:10), A6 (amino acid sequence shown in SEQ ID NO:11), and H10 (amino acid sequence shown in SEQ ID NO:12). These three nanobodies have specific CDR regions that bind to the extracellular region of human DR3.

[0062] Example 2

[0063] Expression and purification of nanobody-Fc fusion protein

[0064] A peptide gene sequence for guiding secretion was designed and fused to the N-terminus of the nanobody gene to ensure secretion after expression. Human IgG1 Fc was fused to the C-terminus of the nanobody gene, and the nanobody gene and human IgG1 Fc were recombined and then cloned into the mammalian expression vector pTT5.

[0065] The constructed vector was transfected into HEK293F cells (density approximately 2.5x 10 6 cells / ml) using polyethyleneimine (PEI), and in Freestyle TMThe 293 expression medium (purchased from Yonglian Biology) was used to culture the transfected mammalian cells at 5% CO2, 150 rpm, and 37 °C for 4 days, and then rotated at a speed of 3000 rpm for 10 minutes. The mammalian cells were cultured for 5 days, and then the supernatant of the mammalian cell culture was collected by centrifugation at 2000 rpm for 10 minutes. The nanobody-Fc fusion protein was purified with a protein A column and eluted with 0.1 M acetic acid. The eluted protein was analyzed by SDS-PAGE electrophoresis. As Figure 2 shown, a high-purity nanobody-Fc fusion protein was obtained from the supernatant.

[0066] Example 3

[0067] Analysis of the binding affinity of nanobodies to the extracellular domain of human DR3

[0068] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL of the extracellular domain protein of human DR3 and left standing at room temperature for 2 h. After washing the ELISA plates three times with 1×PBS, 240 μL of MPBS was added to each well and left standing at room temperature for 2 h to block the unbound sites. Next, the nanobody-Fc was serially diluted (starting from 50 nM, three-fold serial dilution, diluted for 12 gradients) with a dilution solution containing 5% milk prepared with PBST. 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plates were washed three times with 1×PBST, and then HRP-anti-human IgG Fc (Beijing Proteintech) was diluted with a dilution solution containing 5% milk prepared with PBST at a ratio of 1:10000 and incubated at room temperature for 1 h. After washing the ELISA plates three times with 1×PBST again, 100 μL of TMB was added to each well and reacted in the dark at room temperature for 7 min. Finally, 50 μL of 1 M H2SO4 was added to terminate the reaction and the absorbance at 450 nm was measured.

[0069] The analysis results of ELISA are as Figure 3 shown. The EC 50 of the extracellular domain of human DR3 and nanobody A2-IgG1 Fc was 0.072 ± 0.021 nM, the EC 50 of the extracellular domain of human DR3 and nanobody A6-IgG1 Fc was 0.146 ± 0.041 nM, and the EC 50 of the extracellular domain of human DR3 and nanobody G10-IgG1 Fc was 0.063 ± 0.017 nM. The prepared nanobodies had a high affinity for the extracellular domain of human DR3.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should all be included within the protection scope of the present invention.

Claims

1. A nanobody with high affinity for the extracellular domain of human DR3, characterized in that, The nanobody is named H10, and H10 contains three antigen - complementary determining regions CDR1, CDR2, and CDR3. The amino acid sequences of the three antigen - complementary determining regions are successively shown as SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 respectively.

2. The nanobody with high affinity for the extracellular domain of human DR3 according to claim 1, characterized in that, The amino acid sequence of H10 is shown as SEQ ID NO:

12.

3. A nanobody with high affinity for the extracellular domain of human DR3, which has the nanobody as claimed in claim 1 and an Fc domain.

4. The antibody according to claim 3, wherein The Fc domain is a human IgG1 Fc domain.

5. A polynucleotide that encodes the nanobody as claimed in any one of claims 1 - 2, or encodes the antibody as claimed in any one of claims 3 - 4.

6. An expression vector that contains the polynucleotide as claimed in claim 6.

7. A host cell that contains the expression vector as claimed in claim 7.

Images