A nanobody targeting the extracellular domain of DR3 and its application

Abstract

The present invention relates to the field of biopharmaceutical technology, and in particular to a nano antibody targeting the extracellular domain of DR3 and its application. The present invention uses the DR3 extracellular domain of an insect expression system to immunize alpacas, separates peripheral blood lymphocytes for total RNA extraction, reverse transcribes and amplifies the nano antibody sequence, and finally separates and obtains three nano antibodies, which are named A2, A6 and H10 respectively. The three nano antibodies have different antigen complementary determining regions, and the SPR results show that the three nano antibodies all show high affinity binding to the human DR3 extracellular domain. The nano antibodies provided by the present invention are expected to provide experimental factual basis for the treatment ideas of DR3-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a nano antibody targeting the DR3 extracellular domain and an application thereof. Technical Background

[0002] The tumor necrosis factor receptor superfamily (TNFRSF) is one of the most important families in the immune system, which widely regulates various immune responses such as the development of immune organs, co-stimulation of lymphocytes, and determination of the fate of lymphocytes. In the TNFRSF family, death receptor 3 (DR3) has the highest homology with TNFR1 amino acid sequence. DR3 is detected to be expressed in large quantities in lymphocytes of the thymus and spleen and peripheral blood lymphocytes, and plays a certain role in promoting apoptosis during the development of thymocytes. 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 region of DR3 can recruit its downstream adapter molecule TRADD (TNFR-associated death domain), which can eventually activate NF-κB signaling. The most important chronic autoimmune diseases associated with 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 Th1 and Th17 immune responses. For example, TL1A / DR3 can play an inflammatory role in the intestine by activating ILC3 (Activation of DR3signaling causes loss of ILC3s and exacerbatesintestinal inflammation).

[0003] Given the key role of TL1A and DR3 signaling pathways in autoimmune and inflammatory diseases, studies have shown that neutralizing TL1A with soluble DR3 protein can effectively alleviate α-CD40 and DSS-induced enteritis, indicating that inhibiting TL1A-DR3 interaction may be an effective therapeutic strategy to improve autoimmune diseases and local inflammation of 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, but the efficacy of traditional drug treatments such as 5-aminosalicylate is uncertain and may also have a high recurrence rate and is ineffective for patients with moderate to severe IBD. Ligand and receptor drugs targeting related signaling pathways have become the most promising targets for the development of anti-IBD drugs. Developing an antibody that targets the extracellular segment of DR3 and prevents it from activating the signaling pathway with its ligand TL1A will have great clinical significance. Summary of the invention

[0004] In order to solve the problems in the prior art, one of the objects of the present invention is to provide a nanobody targeting the extracellular domain of DR3, wherein the nanobody is any one of A2, A6, and H10, wherein A2, A6, and H10 all comprise three antigen complementary determining regions CDR1, CDR2, and CDR3, wherein:

[0005] The amino acid sequences of the three antigen complementary determining regions of A2 are 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% to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;

[0006] The amino acid sequences of the three antigen complementary determining regions of A6 are 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% to the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;

[0007] The amino acid sequences of the three antigen complementary determining regions of H10 are amino acid sequences with a homology greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively, and preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0008] The above homologous sequences also include amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0009] Preferably, the amino acid sequences of A2, A6 and H10 are as follows:

[0010] Amino acid sequence of A2:

[0011]

[0012] Amino acid sequence of A6:

[0013]

[0014] Amino acid sequence of H10:

[0015]

[0016] The amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of the above-mentioned nanobody are shown in bold lines, namely:

[0017] The amino acid sequence of the antigenic complementary determining region of A2 is:

[0018] CDR1: GDTICISAM (SEQ ID NO: 1)

[0019] CDR2: LVAGITSS (SEQ ID NO: 2)

[0020] CDR3:NADLGSPGVYE(SEQ ID NO:3)

[0021] The amino acid sequences of the three antigenic complementary determining regions of A6 are:

[0022] CDR1: GDTICISGM (SEQ ID NO: 4)

[0023] CDR2: LVAAITSGGS (SEQ ID NO: 5)

[0024] CDR3: NADGSRCPGE (SEQ ID NO: 6)

[0025] The amino acid sequences of the three complementary determining regions of H10 are:

[0026] CDR1: GLTSDDYTM (SEQ ID NO: 7)

[0027] CDR2: GVSCISRVDGN (SEQ ID NO: 8)

[0028] CDR3:ATDCRLTPNSDWNGPLRFGS(SEQ ID NO:9)

[0029] 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.

[0030] Preferably, the Fc domain is a human IgG1 Fc domain, and the amino acid sequence is shown in SEQ ID NO:13.

[0031] The present invention also provides a polynucleotide, which encodes the nanoantibody 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 shown in SEQ ID NO: 14, the nucleotide sequence encoding A6 is shown in SEQ ID NO: 15, and the nucleotide sequence encoding H10 is shown in SEQ ID NO: 16.

[0032] The present invention also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector as described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, and mammalian cells.

[0033] The present invention provides a pharmaceutical composition comprising the nanobody A2, A6 or H10 as described above, or the antibody having an Fc domain as described above.

[0034] Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0035] The nanobodies / antibodies provided by the present invention can be used to prepare treatments and / or diagnose diseases associated with DR3, such as Crohn's disease, ulcerative colitis, and rheumatoid arthritis.

[0036] The present invention also provides the use of the above-mentioned nanoantibodies A2, A6 or H10, or antibodies containing the above-mentioned antibodies with Fc domains in the preparation of a kit for treating and / or diagnosing a disease associated with DR3, wherein the disease is Crohn's disease, ulcerative colitis or rheumatoid arthritis.

[0037] Preferably, the Nanobody / antibody further comprises a second antibody, which comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0038] The beneficial effects of the present invention are:

[0039] 1. Alpacas were immunized three times with the DR3 extracellular domain of the insect expression system, and then blood was drawn to isolate peripheral blood lymphocytes (PBMCs). Total RNA was extracted from the isolated PBMCs and then immediately reverse transcribed into cDNA. The cDNA was used as a template to amplify the nanoantibody sequence. Finally, three nanoantibodies were isolated and named A2, A6 and H10.

[0040] The nano antibody (VHH) is derived from natural camel heavy chain antibodies, and has the advantages of 1) simple structure and small molecular weight, which is conducive to expression and use; 2) convenient and efficient large-scale expression in Escherichia coli and various eukaryotic systems; 3) since it has only one binding site, it is a single-domain antibody, and has better permeability, specificity and detection linearity as a diagnostic reagent; 4) it is easy to couple with various fusion proteins or be more easily labeled with various markers; 5) it is easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and directional transport of cell targets; 6) as a drug development, it has the advantages of low immunogenicity to humans and is not easy to produce immune rejection.

[0041] 2. The three nanobodies provided by the present invention have different antigen complementary determining regions. The antibody binding to the extracellular domain of human DR3 is expressed and secreted by mammalian cells (HEK293F), and the antibody is fused with human IgG1 Fc and cloned into the mammalian expression vector pTT5. The vector is transfected into mammalian cells 293F, and the supernatant is collected after 4 days of culture. The fusion protein in the supernatant is purified using a Protein A column, and the yield of the three nanobodies is greater than 50 mg / L.

[0042] ELISA results showed that all three nanoantibodies showed high affinity binding to the extracellular domain of human DR3, among which the EC of A2-IgG1 Fc 50 At 0.072 ± 0.021 nM, the EC of A6-IgG1 Fc 50 The EC of H10-IgG1 Fc was 0.146±0.041 nM. 50 At 0.063±0.017 nM. The nano-antibody provided by the present invention is expected to provide experimental factual basis for the treatment of DR3-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The ELISA results of monoclonal phages that specifically bind to the extracellular domain of human DR3.

[0044] Figure 2Figure 2 is the results of antigen and antibody purification and SDS-PAGE analysis; A is the 24 mL molecular sieve result and SDS-PAGE result of the extracellular region of human DR3 antigen, B is the elution graph and SDS-PAGE result of nanobody A2 purified by Protein A column, C is the elution graph and SDS-PAGE result of nanobody A6 purified by Protein A column, and D is the elution graph and SDS-PAGE result of nanobody H10 purified by Protein A column.

[0045] Figure 3 The results of the competitive ELISA analysis of the binding of nanobodies to the extracellular region of human DR3; the EC values ​​of the three nanobodies 50 The values ​​are A2:EC 50 =0.072±0.021nM; A6:EC 50 =0.146±0.041nM; G10:EC 50 =0.063±0.017nM. DETAILED DESCRIPTION

[0046] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0047] Example 1

[0048] Purification of the extracellular domain of human DR3 for immunization and screening of alpacas

[0049] 1) By constructing an expression plasmid and transforming it into a DH10Bac competent state containing Bacmid and an auxiliary plasmid, the target gene is transposed to Bacmid under the action of transposase; clones containing recombinant transposons are obtained through resistance screening of kanamycin, gentamicin and tetracycline and blue-white screening. After further PCR identification, the high-purity and high-concentration recombinant Bacmid is purified and transfected into corresponding insect cells to obtain a recombinant baculovirus containing the target gene. The recombinant baculovirus is amplified three generations using SF9 cells, and the virus in the supernatant is collected, and it is added to Hi5 cells at a ratio of 1:1000 for virus infection and expression of the target protein. After 4 days, the cell supernatant is collected and the residues 26-199 of the extracellular region of the human DR3 protein are purified by affinity chromatography and size exclusion chromatography, and the sequence is shown in SEQ ID NO:17.

[0050] For alpaca immunization, Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the next two immunizations. Each time, 200 μg of antigen was used, and the antigen and adjuvant were mixed in a 1:1 volume ratio. The first two injections were injected subcutaneously, and the last injection was injected intramuscularly. The alpaca (Vicugna pacos) was immunized 3 times, once every 3 weeks.

[0051] 2) At week 11, venous blood was collected and peripheral blood lymphocytes were separated using Ficoll from Solebol.

[0052] 3) Total RNA was extracted using Omega Biotek's RNA extraction kit and genomic DNA was removed. PrimeScript from Takara was used TM II First Strand cDNA Synthesis Kit, reverse transcribe RNA into cDNA.

[0053] 4) Construction of nanobody phage display library: Using the above cDNA as a template, the coding sequence of nanobody was obtained by PCR amplification using specific alpaca VHH primers. The amplified nanobody sequence was inserted into the NcoI and NotI sites of phagemid pR2 by Gibson assembly. The obtained Gibson assembly product is the initial nanobody phage library, and the product is recovered.

[0054] 5) Prepare competent E. coli T1 (MRC Laboratory of Molecular Biology) cells using the 10% glycerol washing method. The activated TG1 cells were cultured in 250 mL 2×YT medium (formula 1 L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) until the OD 600 After centrifugation at about 0.6-0.8, 5000g for 15 min, wash three times with 250mL, 250mL, and 100mL of pre-cooled 10% glycerol, and finally resuspend with 1mL of 10% glycerol and dispense into 500μL per tube.

[0055] 6) Use BTX ECM 399 electroporator to transform Escherichia coli TG1 competent cells. Mix the product recovered from Gibson assembly with 500 μL TG1 competent cells and transfer to a 0.1 cm electroporation cuvette, followed by electrotransformation at 2.5 KV. Resuspend the electroporation product in 20 mL 2× culture medium and incubate at 37°C, 220 rpm for 1 h. Take 10 μL of bacterial solution, dilute to 990 μL 2×YT, mix well, then take 40 μL of bacterial solution, dilute to 160 μL 2×YT, plate 100 μL and culture overnight at 37°C. Count and calculate the library size on the second day (library size = number of counts × 10 5 ). The bacteria were spread on five 150 mm 2×YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library and cultured overnight at 37°C. Next, the transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, snap-frozen in liquid nitrogen in 1 mL aliquots and stored at -80°C, and the size of the phage library was calculated.

[0056] 7) Amplification of the Nanobody Phage Display Library: To amplify the Nanobody phage library, 0.2 ml of the frozen library was thawed on ice, diluted into 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and cultured at 37°C at 220 rpm. Next, 1×10 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37°C (water bath) for 45 minutes. The cell pellet was separated by high-speed centrifugation and resuspended in 200 mL of 2×YT medium to which 0.1% glucose, 50 μg / mL kanamycin and 100 μg / mL ampicillin were added. The cells were incubated at 25°C, 220 rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate phage particles. The precipitated phage particle pellet was dissolved 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 stored in 1 mL aliquots at -80°C in the presence of 25% glycerol.

[0057] 8) Screening: The purified extracellular segment of human DR3 was diluted with PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plate), and one well of the ELISA plate was set aside as a negative control. After washing three times with 1×PBS, 300 μL of MPBS (1×PBS containing 5% skim milk) was added to each well of the ELISA plate and incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed three times with 1×PBS and 1×10 11 pfu (diluted in 100 μL MPBS) phage library was added to each well, and after one hour of incubation at room temperature, it was washed three times with PBST (1×PBS containing 0.1% Tween 20). Phages displaying nanobodies specific for the extracellular segment of human DR3 were eluted by incubating at room temperature for one hour with trypsin at a final concentration of 0.5 mg / ml. 10 μL of eluted phage was added to 1 mL of E. coli TG1 competent cells and incubated at 37°C (water bath) for 45 minutes for infection, and then the bacterial culture was plated on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubated overnight at 37°C.

[0058] 9) Preparation of monoclonal phage: After one round of panning, 48 individual colonies were picked into a 96-well round-bottom culture dish containing 100 μL of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). Incubate at 37°C, 180 rpm for 12 hours. Next, 5 μL of the culture solution was inoculated into a new 96-well round-bottom culture dish containing 200 μL of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37°C, 250 rpm for 1.5 hours until the OD260 was about 0.5. 50 μL containing 4×10 8 2×YT medium with pfu KM13 helper phage was added to each well of the plate and incubated at 37°C for 45 minutes without shaking for infection. After infection, 150 μL of supernatant was discarded and the remaining volume was centrifuged at 3500g for 15 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1% glucose (w / v), and incubated overnight at 25°C, 250rpm for about 14-16 hours. The next day, the culture was centrifuged at 3500g for 30 minutes, and then 150 μL of supernatant was transferred to a new 96-well plate and stored at 4°C for screening of nanobodies.

[0059] 10) Phage ELISA test. Use 1×PBS for dilution, and coat a 96-well round-bottom immunoELISA plate with 100 μL of human DR3 extracellular segment at a final concentration of 0.2 μg / mL, and incubate at 4°C for 16 hours. Next, wash the ELISA plate 3 times with 1×PBS. Block the plate with MPBS (1×PBS containing 5% skim milk) at room temperature for 2 hours. After the wells are washed 4 times with PBS-0.1% Tween 20. Add 100 μL of 1×10 11 pfu of phage was added to each well and incubated at room temperature for 1 hour. The ELSA plate was then washed 5 times with PBS-0.1% Tween 20. HRP-KM13 diluted 1:8000 in MPBS was added to each well at 100 μL and incubated at room temperature for 1 hour. The wells were then washed 4 times with PBS-0.1% Tween 20, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, incubated in the dark for 7 minutes to develop color, and then 50 μL of 1M H 2 SO 4 Terminate the reaction and measure the OD using a microplate reader. 450nm Value, the result is Figure 1 shown.

[0060] 11) OD 450nm Twenty positive clones with values ​​greater than 1 were picked for sequencing and the sequence results were analyzed. Finally, three positive phage nanobodies specific for human DR3 extracellular region protein were determined, and they were named A2 (amino acid sequence as shown in SEQ ID NO: 10), A6 (amino acid sequence as shown in SEQ ID NO: 11) and H10 (amino acid sequence as shown in SEQ ID NO: 12). These three nanobodies have specific CDR regions that bind to the extracellular region of human DR3.

[0061] Example 2

[0062] Expression and purification of nanobody-FC fusion protein

[0063] The peptide gene sequence that guides 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.

[0064] The constructed vector was transfected into HEK293F cells (density about 2.5 x 10 6 cells / ml), in Freestyle TM 293 expression medium (purchased from Yonglian Biotechnology) was used to culture transfected mammalian cells in 5% CO 2 , 150 rpm, 37 ° C for 4 days, and then centrifuged at 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 using a protein A column and eluted with 0.1 M acetic acid. The eluted protein was analyzed by SDS-PAGE electrophoresis, such as Figure 2 As shown, highly pure Nanobody-Fc fusion protein was obtained from the supernatant.

[0065] Example 3

[0066] Analysis of the binding affinity of nanobodies to the extracellular segment of human DR3

[0067] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL human DR3 extracellular segment protein and allowed to stand at room temperature for 2 hours. The ELISA plate was washed three times with 1×PBS and 240 μL of MPBS was added to each well and allowed to stand at room temperature for 2 hours to block unbound sites. Next, nanoantibody-Fc was gradient diluted with a diluent containing 5% milk in PBST (starting at 50 nM, three-fold continuous gradient dilution, 12 gradient dilutions). 100 μL was added to each well and incubated at room temperature for 1 hour. After 1 hour, the ELISA plate was washed three times with 1×PBST and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) was diluted with a diluent containing 5% milk in PBST at a ratio of 1:10000 and incubated at room temperature for 1 hour. The ELISA plate was washed three times with 1×PBST and 100 μL of TMB was added to each well and reacted in the dark at room temperature for 7 minutes. Finally, 50 μL of 1M H 2 SO 4 The reaction was stopped and the absorbance at 450 nm was measured.

[0068] The results of ELISA analysis are as follows Figure 3 As shown, the EC of human DR3 extracellular segment and nanobody A2-IgG1 Fc 50 At 0.072±0.021nM, the EC of nanobody A6-IgG1 Fc 50 At 0.146±0.041nM, the EC of nanobody G10-IgG1Fc 50 At 0.063±0.017 nM, the prepared nanobody has a high affinity to the extracellular segment of human DR3.

[0069] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody targeting the extracellular domain of DR3, It is characterized in that The nanobody comprises three antigen complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequences of the three antigen complementary determining regions are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. A nanobody targeting the DR3 extracellular domain according to claim 1, It is characterized in that The amino acid sequence of the Nanobody is shown in SEQ ID NO:

10.

3. An antibody targeting the DR3 extracellular domain, comprising the nanobody as claimed in claim 1 and an Fc domain.

4. The antibody according to claim 3, It is characterized in that The Fc domain is a human IgG1 Fc domain.

5. A polynucleotide encoding the Nanobody according to any one of claims 1 to 2, or encoding the antibody according to any one of claims 3 to 4. An expression vector comprising the polynucleotide according to claim 5 . A host cell comprising the expression vector according to claim 6 .

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