A nanobody targeting the ectodomain of dr3

Abstract

The present application relates to the technical field of biopharmaceuticals, and particularly relates to a nano-antibody targeting DR3 extracellular domain. The present application immunizes a llama with DR3 extracellular domain of an insect expression system, extracts total RNA from peripheral blood lymphocytes, reversely transcribes and amplifies nano-antibody sequences, and finally separates and obtains a nano-antibody, named A6. SPR results show that the nano-antibody exhibits high affinity binding with human DR3 extracellular domain, and the nano-antibody provided by the present application is expected to provide experimental fact basis for the treatment idea of DR3 related diseases.

Description

[0001] This application is a divisional application of application No. 202310649138.3, titled "Nanobody targeting extracellular domain of DR3 and application thereof", filed on June 2, 2023. TECHNICAL FIELD

[0002] The present application relates to the technical field of biopharmaceuticals, and particularly relates to a nanobody targeting extracellular domain of DR3. TECHNICAL BACKGROUND

[0003] 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, costimulation of lymphocytes, and determination of lymphocyte fate. Among the TNFRSF family, the death receptor 3 (DR3) has the highest amino acid sequence homology with TNFR1. DR3 is detected to have a large amount of expression in thymus and spleen lymphocytes and peripheral blood lymphocytes, and plays a certain pro-apoptotic role in the development process of thymus cells, while in lymphocytes, it mainly plays a costimulatory role with its only ligand TL1A (TNFSF15). When the ligand TL1A activates the signal pathway, the intracellular region of DR3 can recruit its downstream adaptor molecule TRADD (TNFR-associated death domain), which can 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), and studies have shown that the DR3-TL1A signaling pathway can promote Th1 and Th17 immune responses and thus play a pathogenic role in inflammatory bowel disease, such as TL1A / DR3 can play an inflammatory role in the intestine by activating ILC3 (Activation of DR3 signaling causes loss of ILC3s and exacerbates intestinal inflammation).

[0004] In view of the key role of TL1A and DR3 signaling pathway in autoimmune and inflammatory diseases, studies have shown that neutralizing TL1A with soluble DR3 protein can effectively alleviate α-CD40 and DSS-induced intestinal inflammation, which indicates that inhibiting TL1A-DR3 interaction may be an effective treatment 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 shown an upward trend, but the efficacy of traditional drug treatment such as 5-aminosalicylic acid is uncertain and may have a high recurrence rate, and it is ineffective for patients with moderate to severe IBD. Targeting ligand and receptor drugs related to the signaling pathway have become the most promising target for the development of anti-IBD drugs. Developing an antibody targeting the extracellular segment of DR3 to prevent the activation of the signaling pathway with the ligand TL1A will have great clinical significance. SUMMARY

[0005] To solve the problems in the prior art, one of the purposes of the present application is to provide a nanobody targeting the extracellular domain of DR3, the nanobody being any one of A2, A6 and H10, the A2, A6 and H10 each comprising three antigenic complementarity determining regions CDR1, CDR2 and CDR3, wherein:

[0006] the three antigenic complementarity determining regions of the A2 have amino acid sequences that are greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;

[0007] the three antigenic complementarity determining regions of the A6 have amino acid sequences that are greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6;

[0008] the three antigenic complementarity determining regions of the H10 have amino acid sequences that are greater than or equal to 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to 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 substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

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

[0011] The amino acid sequence of A2 is:

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

[0013] The amino acid sequence of A6 is:

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

[0015] The amino acid sequence of H10 is:

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

[0017] The three antigenic complementarity determining region amino acid sequences CDR1, CDR2 and CDR3 of the above-mentioned nanobody are shown in bold line portions, respectively, as follows:

[0018] The antigenic complementarity determining region amino acid sequences of A2 are:

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

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

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

[0022] The three antigenic complementarity determining region amino acid sequences of A6 are:

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

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

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

[0026] Three antigenic complementarity determining region amino acid sequences 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 application also provides an antibody targeting the extracellular domain of DR3, which has any one of the nanobodies A2, A6, H10 and an Fc domain as described above.

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

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

[0033] The present application 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 exogenous proteins, such as bacteria, yeast, insect cells, mammalian cells.

[0034] The present application provides a pharmaceutical composition containing the nanobodies A2, A6 or H10 as described above, or containing 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 nanobody / antibody provided by the application can be used for preparing a treatment and / or diagnosis of diseases related to DR3, such as Crohn's disease, ulcerative colitis, rheumatoid arthritis.

[0037] The application also provides the use of the nanobody A2, A6 or H10 or the antibody with Fc domain as described above in the preparation of a kit for the treatment and / or diagnosis of diseases related to DR3, such as Crohn's disease, ulcerative colitis or rheumatoid arthritis.

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

[0039] The application has the following beneficial effects:

[0040] 1. The llama is immunized three times with the DR3 extracellular domain of an insect expression system, and then peripheral blood lymphocytes (PBMCs) are separated by blood drawing, and total RNA extraction is performed on the separated PBMCs, which is then immediately reverse transcribed into cDNA, and the cDNA is used as a template to amplify the nanobody sequence, and finally three nanobodies are isolated and obtained, which are named A2, A6 and H10 respectively.

[0041] The nanobody (VHH) is derived from natural camel heavy chain antibodies, which has the advantages of 1) simple structure, small molecular weight, which is beneficial to expression and use; 2) convenient to express in E. coli and various eukaryotic systems; 3) as a single domain antibody, it has only one binding site, which has better permeability, specificity and detection linearity as a diagnostic reagent; 4) it is easy to couple with various fusion proteins or be labeled with various markers; 5) it is easier to prepare bifunctional antibodies, which is more conducive to the development of targeted drugs and cell target directional transportation; 6) as a drug development, it has little immunogenicity to human and is not easy to produce immune rejection.

[0042] 2. The three nanobodies provided by the application have different antigenic complementarity determining regions, the antibody binding to the human DR3 extracellular domain is expressed and secreted by a mammalian cell (HEK293F), the antibody is fused with human IgG1 Fc, and is cloned into a mammalian expression vector pTT5, and 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 by a Protein A column, and the yield of the three nanobodies is greater than 50 mg / L.

[0043] The ELISA results show that the three nanobodies all show high affinity binding to the human DR3 extracellular domain, and the EC 50 at 0.072±0.021nM, the EC of A6-IgG1 Fc is50 At 0.146±0.041 nM, the EC of H10-IgG1 Fc 50 At 0.063±0.017 nM. The nanobody provided by this invention holds promise for providing experimental evidence for treatment strategies related to DR3-related diseases. Attached Figure Description

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

[0045] Figure 2 Figure 1 shows the results of antigen and antibody purification and SDS-PAGE analysis. Figure A shows the 24 mL molecular sieve result and SDS-PAGE result of the extracellular region of human DR3 antigen; Figure B shows the elution diagram and SDS-PAGE result of nanobody A2 purified by Protein A column; Figure C shows the elution diagram and SDS-PAGE result of nanobody A6 purified by Protein A column; Figure D shows the elution diagram and SDS-PAGE result of nanobody H10 purified by Protein A column.

[0046] Figure 3 Results of competitive ELISA analysis of the binding of nanobodies to the extracellular region of human DR3; ECGs of three nanobodies 50 The values ​​are A2 and EC respectively. 50 =0.072±0.021nM; A6: EC 50 =0.146±0.041nM; G10: EC 50 =0.063±0.017nM. Detailed Implementation

[0047] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to embodiments:

[0048] Example 1

[0049] Purified extracellular domains of human DR3 were used for immunization and screening of alpacas.

[0050] 1) By constructing expression plasmid, it is transformed into DH10Bac competent containing Bacmid and helper plasmid, the target gene is transposed to Bacmid under the action of transposase; through the resistance screening of kanamycin, gentamicin and tetracycline and blue-white spot screening, the clone containing recombinant transposon is obtained. After further identification by PCR, high-purity and high-concentration recombinant Bacmid is purified and transfected into the corresponding insect cells to obtain recombinant baculovirus containing the target gene. After three generations of amplification of the recombinant baculovirus using SF9 cells, the virus in the supernatant is collected, which 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 extracellular region of human DR3 protein 26-199 residues is purified by affinity chromatography and size exclusion chromatography, and the sequence is shown in SEQ ID NO: 17.

[0051] For the immunization of alpaca, the first immunization uses Freund's complete adjuvant, and the last two times use Freund's incomplete adjuvant. Each time uses an antigen dose of 200 μg / time, and the antigen and adjuvant are mixed at a volume ratio of 1:1. The first two injections are subcutaneous injections, and the last injection is intramuscular injection. The alpaca (Vicugna pacos) is immunized 3 times with an interval of 3 weeks.

[0052] 2) At the 11th week, venous blood is collected and peripheral blood lymphocytes are separated using Ficoll of Solerbio.

[0053] 3) Total RNA is extracted using the RNA extraction kit of Omega Biotek, and genomic DNA is removed. The RNA is reversely transcribed into cDNA using the PrimeScript TM II First strand cDNA synthesis kit.

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

[0055] 5) E. coli T1 (MRC Laboratory of Molecular Biology) competent cells are prepared using 10% glycerol washing method. The activated TG1 is cultured in 250 mL 2xYT (formula 1L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) medium to OD 600About 0.6-0.8, 5000g centrifugation for 15 min, then washed with 250 mL, 250 mL, 100 mL of pre-cooled 10% glycerol three times, finally resuspended with 1 mL 10% glycerol and aliquoted to 500 μL per tube.

[0056] 6) Transformation of E. coli TG1 competent cells using BTX ECM 399 electroporator. Gibson assembled products were mixed with 500 μL of TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by 2.5KV electroporation. The electroporation products were resuspended in 20 mL of 2x medium and incubated at 37°C, 220 rpm for 1 h. 10 μL of the bacterial solution was diluted into 990 μL of 2x YT and 40 μL of the bacterial solution was diluted into 160 μL of 2x YT, followed by plating 100 μL and incubation at 37°C overnight. The next day, the library size was calculated (library size = number of colonies x 10 5 ). The bacteria were spread on 5 150 mm 2x YT plates supplemented with 100 μg / mL ampicillin and 2% glucose to amplify the phage library and incubated at 37°C overnight. Next, the transformed colonies were scraped from the plates, mixed thoroughly with 25% glycerol to a final concentration, frozen in 1 mL aliquots in liquid nitrogen and stored at -80°C, and the size of the phage library was calculated.

[0057] 7) Amplification of the Nanobody phage display library: To amplify the phage library of Nanobodies, 0.2 ml of the frozen library was thawed on ice, diluted into 200 mL of 2x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose and incubated at 37°C, 220 rpm. Next, 1 x 1010 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 min. The cell pellet was separated by high speed centrifugation and resuspended in 200 mL of 2x YT medium supplemented with 0.1% glucose, 50 μg / mL kanamycin and 100 μg / mL ampicillin. 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 the phage particles. The precipitated phage particles were resolubilized in 1x 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.

[0058] 8) Screening: The purified extracellular part of human DR3 was diluted in PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plates) and one well of the ELISA plate was left out as a negative control. After washing 3 times with 1 x PBS, 300 μL MPBS (1 x PBS containing 5% skimmed milk) was added to each well of the ELISA plate and incubated for 2 hours at room temperature to block unbound sites. Next, the plate was washed 3 times with 1 x PBS and 1 x 10 11 pfu (diluted in 100 μL MPBS) phage library was added to each well and after one hour of incubation at room temperature, washed 3 times with PBST (1 x PBS containing 0.1 % Tween 20). The phage displaying the nanobody specific for the extracellular part of human DR3 were eluted by incubation with trypsin at a final concentration of 0.5 mg / ml for one hour at room temperature. 10 μL of the eluted phage was added to 1 mL of E. coli TG1 competent cells and incubated at 37 °C (water bath) for 45 minutes to allow infection, after which the bacterial culture was plated on 2 x YT supplemented with 100 μg / mL ampicillin and 2% glucose at 37 °C overnight.

[0059] 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 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). This was incubated at 37 °C, 180 rpm for 12 hours. Next, 5 μL of this culture was inoculated into a new 96-well round bottom culture dish containing 200 μL of 2 x YT medium supplemented with 100 μg / mL ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37 °C, 250 rpm for 1.5 hours until the OD260was about 0.5. 50 μL of 2 x YT medium containing 4 x 10 8 pfu of KM13 helper phage was added to each well of the plate and incubated at 37 °C for 45 minutes without shaking to allow infection. After infection, 150 μL of supernatant was discarded and the remaining volume was centrifuged at 3500 g for 15 minutes, the supernatant was discarded and the bacterial pellet was resuspended in 200 mL of 2 x YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1 % glucose (w / v) and incubated at 25 °C, 250 rpm overnight for about 14-16 hours. The next day, the culture was centrifuged at 3500 g for 30 minutes, after which 150 μL of supernatant was transferred to a new 96-well plate and stored at 4 °C for screening of the nanobodies.

[0060] 10) Phage ELISA test. 96-well immunological ELISA plates were coated with 100 μL of human DR3 extracellular segment at a final concentration of 0.2 μg / mL by dilution using 1x PBS and incubated at 4°C for 16 hours. Next, the ELISA plates were washed 3 times with 1x PBS. The plates were blocked with MPBS (1x PBS containing 5% skim milk) for 2 hours at room temperature. After completion, the wells were washed 4 times with PBS-0.1% Tween 20. 100 μL of 1x 10 11 pfu of phage was added to each well and incubated at room temperature for 1 hour. Then the ELSA plates were washed 5 times with PBS-0.1% Tween 20. 100 μL of HRP-KM13 diluted 1:8000 with MPBS was added to the ELISA plates per well, and incubated at room temperature for 1 hour. Subsequently, the wells were washed 4 times with PBS-0.1% Tween 20, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added per well, and incubated for color development for 7 minutes in the dark, immediately after which 50 μL of 1M H2SO4 was added to stop the reaction, and the OD 450nm values were measured by an enzyme label meter, and the results are shown in Figure 1

[0061] 11) Positive clones with OD 450nm values greater than 1 were picked 20 times for sequencing and sequence analysis, and finally 3 positive phage nanobodies specific to human DR3 extracellular region protein were determined, designated as 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), and the 3 nanobodies have specific CDR regions that bind to human DR3 extracellular region.

[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 about 2.5x 10 6 cells / ml) using polyethyleneimine (PEI), and the cells were cultured in Freestyle TM ​293 expression medium (purchased from Yonglian Biotechnology) was used to culture the transfected mammalian cells at 5% CO2, 150 rpm, 37℃ for 4 days. Then the mammalian cells were cultured for 5 days, and the supernatant of the mammalian cell culture was collected by centrifugation at 2000 rpm for 10 minutes. The nanobody-FC fusion protein was purified by a protein A column, eluted with 0.1 M acetic acid, and analyzed by SDS-PAGE electrophoresis as shown in Figure 2 , and a high-purity nanobody-FC fusion protein was obtained from the supernatant.

[0066] Example 3

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

[0068] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL of human DR3 extracellular segment protein at room temperature for 2 h. After washing the ELISA plates with 1 × PBS three times, 240 μL of MPBS was added to each well at room temperature for 2 h to block unbound sites. Next, the nanobody-Fc was gradient diluted (starting at 50 nM, three consecutive gradient dilutions, 12 dilution gradients) with a diluent 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 with 1 × PBST three times, and the HRP-anti-human IgG Fc (Beijing Yiqiao God) was diluted at a ratio of 1:10,000 with a diluent containing 5% milk prepared with PBST and incubated at room temperature for 1 h. The ELISA plates were washed with 1 × PBST three more times, and 100 μL of TMB was added to each well at room temperature in the dark for 7 min. Finally, 50 μL of 1 M H2SO4 was added to stop the reaction and the absorbance at 450 nm was detected.

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

[0070] The above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; even though the present application has been described in detail with reference to the foregoing embodiments, those ordinarily skilled in the art should understand that any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A nanobody targeting the extracellular domain of DR3, characterized in that, The nanobody is named A6, and A6 contains three antigen complementarity-determining regions CDR1, CDR2 and CDR3, with the amino acid sequences of the three antigen complementarity-determining regions shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

2. The nanobody targeting the extracellular domain of DR3 as described in claim 1, characterized in that, The amino acid sequence of A6 is shown in SEQ ID NO:

11.

3. An antibody targeting the extracellular domain of DR3, having the nanobody as described in claim 1 and the Fc domain.

4. The antibody as described in claim 3, characterized in that, The Fc domain is the human IgG1 Fc domain.

5. A polynucleotide encoding a nanobody as described in any one of claims 1-2, or an antibody as described in any one of claims 3-4.

6. An expression vector comprising the polynucleotide as described in claim 5.

7. A host cell comprising the expression vector as described in claim 6.

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