A recovering intestinal barrier function antibody for treating ulcerative colitis and use thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses an antibody for treating ulcerative colitis and recovering intestinal barrier function and application thereof. The antibody is a VHH3 nanobody targeting Janus kinase 1 (JAK1), and the amino acid sequence is shown as SEQ ID NO: 4. The antibody can significantly inhibit the expression of proinflammatory factors TNF-alpha and IL-6 and promote the distribution of Claudin-1 protein of colon epithelium by specifically combining with a JAK1 antigen epitope (SEQ ID NO: 3), so as to recover the intestinal barrier function. Animal experiments show that intraperitoneal injection of 0.2-1.8 mg / kg VHH3 can effectively reduce a disease activity index (DAI), and the curative effect is dose-dependent. Compared with the prior art, the antibody has the advantages of 1.23nM affinity, small molecular weight, strong penetration and low immunogenicity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nanobody (VHH3) targeting Janus kinase 1 (JAK1) and application thereof in treating ulcerative colitis to restore intestinal barrier function, and especially relates to a screening method of the antibody, binding characteristics and anti-inflammatory mechanism in vivo. BACKGROUND

[0002] Ulcerative colitis (UC) is an autoimmune disease characterized by chronic inflammation of the colonic mucosa, and is clinically manifested as repeated abdominal pain, mucopurulent bloody stool, diarrhea, and intestinal barrier damage with or without other systemic symptoms and extraintestinal manifestations. Existing biological agents (such as anti-TNF-α monoclonal antibodies, representative drugs infliximab and adalimumab) are only effective for 40-60% of patients, and long-term use can easily cause infection, lymphoma and other risks (Danese et al., Gut 2020). Traditional small molecule drugs (such as 5-aminosalicylic acid, representative drug mesalamine) have limited efficacy for moderate to severe UC, and there is an urgent need to develop new targeted therapies with high efficiency and low toxicity.

[0003] Janus kinase 1 (JAK1) is a core protein that mediates UC inflammatory signal transduction, and its abnormal activation can drive the release of pro-inflammatory factors such as IL-6 and TNF-α (Shea et al., Annu Rev Med 2015). Although JAK inhibitors (representative drugs tofacitinib and upadacitinib) have been approved for UC treatment, their broad-spectrum inhibition of JAK1 / JAK2 / JAK3 can lead to anemia, infection, herpes zoster and other systemic side effects (Sandborn et al., NEJM 2017). Therefore, the development of inhibitors that specifically target JAK1 and avoid off-target effects is crucial to break through the current treatment bottleneck.

[0004] Nanobodies (VHH) are derived from heavy chain antibodies of camels, and have the characteristics of small molecular weight (about 15 kDa), strong penetration and low immunogenicity (Hamers-Casterman et al., Nature 1993). However, existing nanobodies are mostly focused on the field of tumors or infections, and there is no high-affinity nanobody targeting JAK1 for UC treatment. In particular, nanobodies targeting specific domains of JAK1 still face technical challenges such as complex epitope design and insufficient in vivo functional verification. SUMMARY

[0005] The present application aims to solve the three defects in the treatment of ulcerative colitis (UC): 1. The existing JAK inhibitors (such as tofacitinib, upadacitinib) cause serious systemic side effects due to broad-spectrum inhibition of the JAK family; 2. Traditional monoclonal antibodies (such as anti-TNF-alpha monoclonal antibodies, representative drugs infliximab, adalimumab) have large molecular weights, poor penetration, and limited effect on intestinal mucosal barrier repair; 3. Lack of high-affinity nanobodies targeting specific domains of JAK1.

[0006] The present application first provides a nanobody for treating ulcerative colitis to restore intestinal barrier function, which is a VHH3 antibody, and the amino acid sequences of the complementarity determining regions CDR1-3 are respectively as shown in SEQ ID NO: 6, 8, and 10.

[0007] In some embodiments, the amino acid sequence of the nanobody is as shown in SEQ ID NO: 4.

[0008] The present application also designs an enhanced antigen epitope JK1 (SEQ ID NO: 3) based on the characteristics of the JAK1 target point: the structural composition of the antigen epitope is the functional domain FERM_C_JAK1 (SEQ ID NO: 1) and PTK_Jak1_rpt1 (SEQ ID NO: 2) of JAK1 in series, connected by a flexible linker (GGGGS); this epitope covers the key catalytic region (583-846 aa) and the regulatory region (282-426 aa) of JAK1, significantly improving the specificity of antibody screening.

[0009] The present application also provides a pharmaceutical composition comprising the nanobody and a pharmaceutically acceptable carrier.

[0010] The present application finally provides an application, which is the application of the nanobody or its pharmaceutical composition in the preparation of a drug for treating ulcerative colitis to restore intestinal barrier function.

[0011] Compared with the prior art, the present application has at least the following beneficial effects:

[0012] The present application first provides the core characteristics of the VHH3 nanobody, which has the following molecular characteristics: molecular weight 12.03 kDa, purity 99.3% ( Figure 4VHH3 can be efficiently expressed in prokaryotes; its affinity for the JK1 antigen reaches 1.23 nM; VHH3 treats UC by precisely blocking JAK1 signaling: significantly reducing serum TNF-α and IL-6 levels in UC mice; intraperitoneal injection of 0.2-1.8 mg / kg shows an increasing anti-inflammatory effect with increasing dose; the core evidence for VHH3's repair of the intestinal mucosal barrier lies in the upregulation of tight junction proteins, namely promoting the expression and distribution of Claudin-1 in the colon, with the proportion of positive areas in the high-dose group (1.8 mg / kg) returning to more than 90% of the normal level; at the same time, it improves clinical indicators, namely achieving a dose-dependent reduction in the disease activity index (DAI); the small molecule properties of VHH3 nanobody enhance intestinal tissue penetration, and its immunogenicity is lower than that of traditional monoclonal antibodies. Attached Figure Description

[0013] Figure 1 Schematic diagram of the structure of the JK1 antigen fragment.

[0014] Figure 2 SDS-PAGE identification of JK1 antigen fragments.

[0015] Figure 3 Positive clones were detected by ELISA.

[0016] Figure 4 SDS-PAGE identification of VHH3 nanobodies.

[0017] Figure 5 Affinity detection of VHH3 nanobodies binding to antigen proteins.

[0018] Figure 6 Disease activity index (DAI) detection in ulcerative colitis model mice.

[0019] Figure 7 Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of TNF-α and IL-6 in mouse serum.

[0020] Figure 8 The distribution of Claudin-1 expression in the colon of mice with ulcerative colitis. Detailed Implementation

[0021] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0022] Example 1: Preparation of Janus kinase 1 (JAK1) antigen

[0023] Domain from Janus kinase 1 (a protein tyrosine kinase) [Homo sapiens], GenBank: AAI32730.1 according to NCBI as epitope sequence:

[0024] FERM_C_JAK1 (282..426):

[0025] GAEIFETSMLLISSENEMNWFHSNDGGNVLYYEVMVTGNLGIQWRHKPNV

[0026] VSVEKEKNKLKRKKLENKHKKDEEKNKIREEWNNFSYFPEITHIVIKESVVS INKQDNKKMELKLSSHEEALSFVSLVDGYFRLTADAHHYLCTD (SEQ ID NO: 1);

[0027] PTK_Jak1_rpt1 (583..846):

[0028] LVQGEHLGRGTRTHIYSGTLMDYKDDEGTSEEKKIKVILKVLDPSHRDISLA

[0029] FFEAASMMRQVSHKHIVYLYGVCVRDVENIMVEEFVEGGPLDLFMHRKSD

[0030] VLTTPWKFKVAKQLASALSYLEDKDLVHGNVCTKNLLLAREGIDSECGPFI

[0031] KLSDPGIPITVLSRQECIERIPWIAPECVEDSKNLSVAADKWSFGTTLWEICY

[0032] NGEIPLKDKTLIEKERFYESRCRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDIN (SEQ ID NO: 2);

[0033] The enhanced JAK1-C epitope fragment is prepared from the linker (GGGGS) in series with FERM_C_JAK1 and PTK_Jak1_rpt1:

[0034] GAEIFETSMLLISSENEMNWFHSNDGGNVLYYEVMVTGNLGIQWRHKPNVVSVEKEKNKLKRKKLENKHKKDEEKNKIREEWNNFSYFPEITHIVIKESVVSINKQDNKKMELKLSSHEEALSFVSLVDGYFRLTADAHHYLCTDGGGGSGGGGSGGGGSGGGGSLVQGEHLGRGTRTHIYSGTLMDYKDDEGTSEEKKIKVILKVLDPSHRDISLAFFEAASMMRQVSHKHIVYLYGVCVRDVENIMVEEFVEGGPLDLFMHRKSDVLTTPWKFKVAKQLASALSYLEDKDLVHGNVCTKNLLLAREGIDSECGPFIKLSDPGIPITVLSRQECIERIPWIAPECVEDSKNLSVAADKWSFGTTLWEICYNGEIPLKDKTLIEKERFYESRCRPVTPSCKELADLMTRCMNYDPNQRPFFRAIMRDIN (SEQ ID NO: 3);

[0035] The Nde I and Xba I were introduced at the beginning and the end of the fragment, respectively, and the fragment was named JK1 Figure 1 ), which was synthesized by Nanjing Kings River Biotech Co., Ltd.

[0036] The synthesized JK1 protein antigen epitope fragment was cloned into the expression vector pCZN1, and the ligation product was transformed into Arctic-Express BL21 (DE3) competent cells and plated on LB (containing Amp) plates for culture. Then a single colony was picked and inoculated into LB liquid medium, which was cultured at 37°C, 120 r / min constant temperature shaker until the D600 value was 0.6-0.8. Then IPTG was used for further induction, and the bacteria were collected by centrifugation at 5000 r / min for 10 min, and the supernatant was discarded. After ultrasonic lysis and disruption, the bacteria were collected by centrifugation at 8000 r / min for 10 min, and the supernatant containing the target protein of JK1 was collected and purified using a His-tag protein purification kit (Ni-NTA) (Shanghai Genechem Co., Ltd.). Finally, the protein expression was identified by SDS-PAGE, as shown in Figure 2 .

[0037] Figure 2 The results showed that the molecular weight of the JK1 antigen fragment was 48.50 kDa, and the purity was 99.4%.

[0038] Example 2, Screening, Expression and Identification of Anti-Janus Kinase 1 (JAK1) Nanobody

[0039] After obtaining sufficient antigen, the camel was immunized. 1 mg of JK1 antigen protein was mixed with Freund's adjuvant at a ratio of 1:1. The first immunization used Freund's complete adjuvant, and the subsequent 5 immunizations used Freund's incomplete adjuvant. A total of 6 immunizations were performed, with an interval of 1 week between each immunization. Peripheral blood (100 mL) was collected 1 week before immunization and 1 week after the last immunization. The upper serum was taken for antibody titer detection. Peripheral blood lymphocytes were isolated, total RNA of peripheral blood lymphocytes was extracted using an RNA extraction kit, and RNA was reverse transcribed into cDNA using a reverse transcription kit. The nucleic acid sequence VHH of Nbs was obtained by nested PCR. The nucleic acid sequence VHH of Nbs and the vector pMECS were subjected to restriction endonuclease PstI and NotI digestion, respectively, and then connected using T4 DNA ligase. The JK1 phage library was constructed by electrotransformation into E. coli TG1.

[0040] The purified JK1 protein (diluted to 10 mg / L) was coated on a 96-well plate, 100 μL was added to each well, and a negative control well (coated with PBS) was set up. After overnight incubation at 4°C, the coating solution was discarded, and the wells were washed 3 times with 0.05% PBST. 200 μL of 5% skimmed milk was added, and the plate was incubated at 37°C for 2 h. After washing 3 times with 0.05% PBST, 100 μL of the JK1 phage library prepared above (adjusted to a concentration of 1.2×10 9 PFU / mL) was added, and the plate was incubated at 37°C for 2 h. The phage sample was discarded, and the wells were washed 3 times with 0.05% PBST. 100 μL of 0.1 mol / L triethylamine solution was added to each well, and the plate was allowed to stand at room temperature for 10 min. The same volume of 1 mol / L Tris-HCl (pH 7.4) was added to neutralize the solution. One round of eluate was obtained, and the eluted recombinant phage was subjected to the first round of titer determination, which was the first round of panning. The above operations were repeated for a total of 3 rounds of panning to effectively enrich the positive phage.

[0041] The phage obtained by the third round of panning was used to infect TG1 bacteria, and the bacterial solution was spread on 2YTAG solid culture plates. Ninety-eight single clones were randomly selected and transferred to culture solution, which was incubated at 37°C. The solution was then transferred to a 96-well deep well plate, and KM13 helper phage was added. The plate was incubated overnight to release the phage into the supernatant. The supernatant was collected and added to the JK1 antigen pre-coated ELISA plate. After overnight reaction, an anti-M13 phage antibody coupled with HRP was added as a secondary antibody. The positive clones were detected by ELISA, and the D450 value was measured. The D450 value of the sample well was set as P, and the D450 value of the negative control well was set as N. Clones with P / N≥3 were considered positive, and the strain with the largest ratio was selected for sequencing, as shown in Table 1. Figure 3 .

[0042] Figure 3The results showed that the clones with P / N≥3 were determined as positive, and a total of 4 positive clones could specifically bind to JK1 antigen, wherein the OD value of VHH3 was significantly higher than that of other positive clones. Therefore, VHH3 was subjected to subsequent sequencing analysis, expression and purification.

[0043] The TG1 strain corresponding to the screened VHH3 positive clone was resuscitated and cultured, and the phage plasmid DNA carried thereby was extracted. The universal sequencing primer on both sides of the phagemid vector was used to perform bidirectional sequencing by Sanger dideoxy chain termination method. The obtained sequencing peak map was spliced, translated and subjected to sequence analysis to confirm the open reading frame integrity, especially the sequence of CDR1-3 region, and finally the accurate gene sequence and deduced amino acid sequence of VHH3 nanobody were obtained, as shown in Table 1.

[0044] Table 1, sequencing results of VHH3 nanobody

[0045]

[0046] The sequenced VHH3 was inserted into the pCZN1 vector with HIS tag to construct the pCZN1-VHH3 recombinant plasmid, which was then transformed into Arctic-Express competent cells for expression at 37°C overnight. Positive clones were picked and inoculated into LB liquid medium for overnight culture at 37°C with 2000 r / min shaking. VHH3 was further purified by Ni column affinity chromatography, and the size and purity of the nanobody were identified by SDS-PAGE, as shown in Figure 4 .

[0047] Figure 4 The results showed that the size of VHH3 nanobody with HIS tag was 12.03 kDa, which was consistent with the expectation, and the purity was 99.3%.

[0048] JK1 antigen was diluted to 1 μg / mL, 100 μL / well, added to the high adsorption 96-well plate, and incubated at 4°C overnight; the plate was washed with 200 μL / well of PBST buffer for 5 times, and then dried immediately, 300 μL of blocking solution was added, and incubated at room temperature for 1 h. The plate was washed with 300 μL / well of PBST buffer for 3 times, and then dried, and then VHH3 nanobodies were added at a starting concentration of 100 μg / mL and then serially diluted, 100 μL / well was added, and incubated at room temperature for 90 min. The plate was washed with 200 μL / well of PBST buffer for 5 times, and then dried, and then 100 μL / well of HRP-labeled anti-His tag secondary antibody diluted at 1:2500 was added, and incubated at room temperature for 60 min, and then the plate was washed with 200 μL / well of PBST buffer for 5 times, and then dried. TMB solution was added to the reaction plate at 100 μL / well, and whether color development occurred was observed; after the reaction was completed, 60 μL / well of 2M sulfuric acid was added to terminate the reaction, the plate edge was gently tapped until the color in the well was uniform, and then the plate was immediately read to measure the OD450 value, see Figure 5 .

[0049] Figure 5 The results show that the affinity of the VHH3 nanobodies to the antigen protein is 1.23 nM, which provides a basis for subsequent candidate antibodies to be studied in experimental animals.

[0050] Example 3, Application of VHH3 Nanobodies in the Treatment of Ulcerative Colitis Mouse Models

[0051] Forty SPF level Balb / c mice (20-25 g, 8 weeks old) were randomly divided into 5 groups (n=8): a blank control group, a model group (DSS), a VHH3 low-dose group (0.2 mg / kg), a VHH3 medium-dose group (0.6 mg / kg), and a VHH3 high-dose group (1.8 mg / kg). Except for the blank control group, the mice in the other groups were given 3% DSS drinking water for 7 consecutive days to induce acute ulcerative colitis. From the day of DSS induction, the mice in the VHH3 administration groups were intraperitoneally injected with the corresponding dose of VHH3 nanobodies (dissolved in PBS, injection volume 10 μL / g body weight) every day, and the mice in the model group were injected with the same volume of PBS. The body weight, fecal characteristics, and blood in the stool were recorded daily, and the disease activity index (DAI) was calculated, wherein DAI score=(body weight change score+fecal form score+blood in stool score) / 3, see Figure 6 .

[0052] Table 2 Disease Activity Index

[0053] Evaluation Criteria Scoring Details Score 1. Body weight change Weight gain or no change 0 Weight loss 1-5% (relative to initial body weight) 1 Weight loss 6-10% 2 Weight loss 11-15% 3 Weight loss >15% 4 2. Stool consistency Normal formed pellets 0 Soft stool (slightly sticky, can maintain shape) 1 Loose stool (semi-solid, slightly sticky to anus) 2 Watery stool (liquid, no solid component, easily sticky to anus) 3 Watery stool (completely liquid, automatically flows out) 4 3. Degree of blood in stool No blood in stool (normal stool color, negative occult blood test) 0 Positive occult blood (not visible to the naked eye, confirmed by benzidine method / paper test) 1 Slight visible blood stool (a small amount of blood or light pink on the surface of the stool) 2 Obvious blood stool (stool is red or dark red) 3 Severe blood stool / rectal bleeding (anal dripping blood or blood clots) 4

[0054] Figure 6The results showed that the DAI scores of all mice drinking DSS increased, indicating that the UC mouse model was successfully established. Compared with the model group, the DAI scores of each VHH3 dose group were significantly reduced (P<0.05), indicating that the increase in DAI scores of UC mice was improved, and the improvement was more obvious with the increase of VHH3 concentration.

[0055] The enzyme-linked immunosorbent assay (ELISA) kit (NeoBioscience, Beijing) was used to detect the expression levels of TNF-α and IL-6 in mouse serum, as shown in Figure 7 .

[0056] Figure 7 The results showed that the contents of pro-inflammatory factors TNF-α and IL-6 in the serum of the model group mice were significantly higher than those of the blank control group (P<0.05), which indicated that the UC mouse model was successfully established, that is, DSS could cause oxidative stress reaction by activating TNF-α and IL-6 pro-inflammatory factors; however, after VHH3 nanobody intervention, the contents of TNF-α and IL-6 were significantly lower than those of the model group (P<0.01); this indicated that VHH3 nanobody could inhibit the overexpression of pro-inflammatory factors TNF-α and IL-6, reduce inflammatory reaction, repair intestinal barrier damage, and achieve the purpose of treating ulcerative colitis.

[0057] The colon tissues of the mice in each group were fixed with 4% paraformaldehyde, embedded in paraffin, and then 4 μm thick sections were prepared, followed by deparaffinization, hydration, antigen heat repair (pH 6.0 sodium citrate buffer), and 3% H2O2 blocking endogenous peroxidase. The sections were added with rabbit anti-Claudin-1 primary antibody (1:200 dilution) and incubated at 4°C overnight, washed with PBS, and then incubated with HRP-labeled goat anti-rabbit secondary antibody (1:500) at room temperature for 1 hour, stained with DAB color developing agent, stained with hematoxylin for nuclei, and mounted with neutral resin. Image-Pro Plus 6.0 software was used to randomly select 5 non-overlapping fields (200x) to determine the percentage of brown positive area, and the data were expressed as the percentage of positive area (%), and the comparison between groups was performed by one-way ANOVA (ANOVA), as shown in Figure 8 .

[0058] Figure 8The results show that, compared with the blank control group, the positive expression area of Claudin-1 protein in the colon tissue of the model group mice is significantly reduced (P<0.05). In the VHH3 low, medium and high dose groups, the positive area of Claudin-1 protein is significantly increased compared with the model group (P<0.05), especially in the VHH3 high dose group, the positive area is increased more significantly. This shows that after the intervention of VHH3 nanobody, the expression and distribution of Claudin-1 protein can be promoted, which is close to the blank control group, and the effect of treating ulcerative colitis is realized by maintaining the intestinal epithelial barrier function.

[0059] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A nanobody for treating ulcerative colitis to restore intestinal barrier function, characterized in that, The nanobody is a VHH3 antibody, the amino acid sequences of the complementarity determining regions CDR1-3 thereof are shown in SEQ ID NO: 6, 8, 10 respectively.

2. The Nanobody according to claim 1, characterized in that, The nanobody specifically binds to a JAK1 antigen epitope, the amino acid sequence of the antigen epitope is shown in SEQ ID NO:

3.

3. The Nanobody according to any one of claims 1-2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

4.

4. The Nanobody according to any one of claims 1-2, characterized in that, The nanobody has an affinity of 1.23 nM to the JAK1 antigen, and a molecular weight of 12.03 kDa.

5. A pharmaceutical composition comprising the nanobody of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. Use of the nanobody of any one of claims 1-4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating ulcerative colitis.

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