CN116173200A - TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines - Google Patents
TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines Download PDFInfo
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Abstract
The invention discloses a TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines. The TNF-alpha nano antibody has the following amino acid sequence: (1) A protein consisting of the amino acid sequence shown in SEQ ID No.1 or 2 or 3; or (2) an amino acid sequence encoding the same functional protein having 80% to 100% homology with the amino acid sequence defined by the sequence SEQ ID No.1 or 2 or 3; or (3) the protein derived from (1) with equivalent activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown as SEQ ID No.1 or 2 or 3. The TNF-alpha nano antibody of the invention is expressed in escherichia coli, separated and purified and applied to preparation of ulcerative colitis medicines.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines.
Background
Ulcerative colitis (ulcerative colitis, UC) is a recurrent autoimmune disease, which is one of the inflammatory bowel diseases (Inflammatory bowel disease, IBD). The typical clinical symptoms of ulcerative colitis are bloody diarrhea, nocturnal bowel movement, emergency and tenesmus, and after the initial occult period, it is characterized by recurrent episodes and remission of mucosal inflammation. The pathogenesis of IBD is not yet defined, and genetic, immunological and environmental factors such as diet, stress, smoking and free radicals are involved in its development.
The anti-TNF-alpha nano antibody medicine is developed in the later 90 s of the 20 th century, and has achieved great success in treating autoimmune diseases such as inflammatory bowel disease, ankylosing spondylitis, psoriasis and rheumatoid arthritis. Currently common TNF- α nanobody drugs include infliximab, adalimumab, golimumab, cetuzumab, and etanercept.
Because the commercial TNF-alpha antibody has large molecular weight and complex structure, and can only be expressed by mammalian cells, the medicine of the TNF-alpha antibody has high price and always has high ranking rate of various medicines sold in the world. Development of high-activity, low-cost TNF-alpha antibody drugs, making them "affordable to the average world" has been the goal of global pharmaceutical industry efforts.
At present, a TNF-alpha nano antibody suitable for being prepared in escherichia coli and application of the TNF-alpha nano antibody in preparation of ulcerative colitis medicines are lacking.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a high-activity TNF-alpha nano antibody which is suitable for being expressed and prepared in escherichia coli and application thereof in preparing inflammatory medicaments, in particular ulcerative colitis medicaments.
In order to achieve the above object, the present invention provides the following technical solutions: the TNF-alpha nano antibody provided by the invention has the following amino acid sequence:
(1) A protein consisting of the amino acid sequence shown in SEQ ID No.1 or 2 or 3; or (b)
(2) Amino acid sequence which encodes the same functional protein with 80% to 100% homology to the amino acid sequence defined by the sequence SEQ ID No.1 or 2 or 3; or (b)
(3) The protein derived from (1) with equivalent activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No.1 or 2 or 3.
Further, the TNF-alpha nano antibody is a TNF-alpha nano antibody V19 or a TNF-alpha nano antibody V7 or a TNF-alpha nano antibody V1, the TNF-alpha nano antibody V19 is a protein composed of amino acid sequences shown in SEQ ID No.1, the TNF-alpha nano antibody V7 is a protein composed of amino acid sequences shown in SEQ ID No.2, and the TNF-alpha nano antibody V1 is a protein composed of amino acid sequences shown in SEQ ID No. 3.
A nucleic acid molecule of the invention encoding said TNF- α nanobody.
The invention relates to an application of TNF-alpha nano antibody in preparing an inflammation medicament.
The TNF-alpha nano antibody of the invention is applied in combination with other enteritis treatment medicines to prepare medicines for treating inflammation.
The invention relates to an application of TNF-alpha nano antibody in preparing a medicament for treating ulcerative colitis.
The TNF-alpha nano antibody disclosed by the invention is applied to the preparation of ulcerative colitis medicaments in combination with other enteritis treatment medicaments.
The invention relates to a combined medicine, which consists of a TNF-alpha nano antibody and annexin ANX 1.
The preparation method of the TNF-alpha nano antibody, disclosed by the invention, comprises the following steps of:
(1) Introducing a TNF-alpha nano antibody gene into a pET28a vector through enzyme digestion and enzyme linked reaction to construct an expression plasmid;
(2) Transferring the target plasmid into BL21 (DE 3) competent cells, and after transformation, taking a proper amount of bacterial liquid to spread on an LB plate containing kanamycin for overnight culture at 37 ℃;
(3) Positive clones were picked and expanded in LB medium containing kanamycin, and induced by adding 5mM IPTG at 15℃for 20h; collecting cell sediment by centrifugation, adding protein buffer solution into the system, crushing cells by using a homogenizer, and collecting supernatant after centrifugation; separating and purifying by column chromatography to obtain TNF-alpha nanometer antibody.
(4) Removing the fusion tag by protease, and purifying again to obtain the TNF-alpha nano antibody without the fusion tag; molecular weight and purity of TNF- α nanobodies were verified by 12% SDS-PAGE.
Further, in the step (1), a TNF-alpha nanobody gene shown as SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 is introduced into a pET28a vector through enzyme-linked reaction to construct a plasmid;
in the step (2), transferring the target plasmid into BL21 (DE 3) competent cells, slightly and uniformly mixing, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 45s, standing on ice for 2min, adding 600 μl of antibiotic-free LB culture medium, shaking at 37 ℃ for 1h, centrifuging, and discarding the supernatant to leave a small amount of culture medium re-suspension bacteria liquid; after transformation, a proper amount of bacterial liquid is coated on an LB plate containing kanamycin for overnight culture at 37 ℃;
in the step (3), positive clones are selected and subjected to expansion culture in LB medium containing kanamycin, and 5mM IPTG is added to induce 20h at 15 ℃; the expressed TNF-alpha nanobody protein comprises a His tag;
collecting cell sediment by centrifugation, adding protein buffer solution into the system, crushing cells by using a homogenizer, collecting supernatant after centrifugation, and allowing the expressed TNF-alpha nano antibody to exist in the supernatant in a soluble form;
after equilibrating the column containing the Ni-NTA resin with a protein buffer, the supernatant was slowly passed through Ni-NTA to bind the TNF- α nanobody of interest to Ni-NTA, followed by elution with a protein eluent; the protein buffer consists of the following components: 300mM NaCl,50mM Tris-HCl,0.5mM beta-mercaptoethanol, pH 7.8; the protein eluent consists of the following components: 80mM imidazole,300mM NaCl,50mM Tris-HCl,0.5mM beta-mercaptoethanol, pH 7.8;
in step (4), cleaving the His tag on the TNF- α nanobody with HRV3C enzyme at 4 ℃ for 12h; re-passing the solution through Ni-NTA to obtain TNF- α nanobodies without His-tag, dialyzing overnight with PBS buffer, concentrating and preserving; preparing TNF-alpha nano antibody; molecular weight and purity of TNF- α nanobodies were verified by 12% sds-PAGE.
Further, in the step (3), the speed of the first centrifugation is 2500rpm, and the centrifugation time is 5min; in the step (3), the speed of the second centrifugation is 4000rpm, the centrifugation time is 20min, and the centrifugation temperature is 4 ℃; the speed of the third centrifugation is 12000rpm, the centrifugation time is 30min, and the centrifugation temperature is 4 ℃.
Further, in step (3), the protein buffer consisted of 300mM NaCl,50mM Tris-HCl,0.5mM beta-mercaptoethanol, and the pH of the protein buffer was 7.8.
Further, in step (3), the protein eluent consists of 80mM imidazole,300mM NaCl,50mM Tris-HCl and 0.5mM beta-mercaptoethanol, and the pH of the protein eluent is 7.8; in step (4), the molecular weight and purity of the TNF-. Alpha.nanobody were confirmed by 12% SDS-PAGE.
The beneficial effects are that: the invention obtains the TNF-alpha nano antibody with high activity, which is suitable for expression and preparation in escherichia coli, wherein the affinity of the nano antibody is as high as 6.5pM, which is better than the affinity of the TNF-alpha antibody drug on the market at present. The TNF-alpha nanobody can be expressed and separated and purified in escherichia coli.
Compared with the prior art, the invention has the following advantages:
(1) Compared with the existing TNF-alpha antibody medicines, the TNF-alpha nano antibody not only has superior affinity and remarkable activity for treating inflammatory diseases including ulcerative colitis.
(2) The TNF- α nanobody of the invention can be expressed in a soluble form in e.coli, present in the cell lysis supernatant.
(3) The TNF-alpha nano antibody can be expressed in a soluble form in escherichia coli, separated and purified, so that complex and expensive mammalian cell expression and complicated and high-cost separation and preparation thereof are avoided; obviously reduces the production cost.
Drawings
FIG. 1 is a schematic representation of the purification and characterization of TNF- α nanobodies of the invention; a: expressing and purifying the TNF-alpha nanobody by affinity chromatography; b: the affinity of TNF-alpha nanobodies to murine s-TNF-alpha was detected by MST.
FIG. 2 shows the effect of different doses of TNF- α nanobody of the invention on DSS mice body weight and colon length. A: TNF-a nanobodies treat a range of body weight changes during DSS-induced colitis; b: representative colon length comparisons for each group on day 8.
FIG. 3 shows that TNF- α nanobodies of the invention resulted in increased Th17 cells in the colon of a mouse DSS mouse. A: flow cytometry analysis of intestinal Th17 cells from mice of the group of DSS and TNF-alpha nanobodies (10 mg/kg); b: stream type fineAnalysis of intestinal CD11c by cytometry + BFP fluorescence in DCs.
FIG. 4 is a plot of the amplification of Th17 in the mesenteric lymph nodes of WT mice by CPR of the TNF-alpha nanobody-TNF-alpha antigen/antibody complex of the invention; flow cytometry analysis Th17 cells in mesenteric lymph nodes treated with CPR-1 or CPR-2.
FIG. 5 is a graph of ANX1 in the colon significantly upregulated by CPR-2 of the TNF-alpha nanobody-TNF-alpha antigen/antibody complex of the invention; a: by qRT-PCR analysis, expression of ANX1 in ΔCIITA-CPR-2 was down-regulated compared to TNF- α nanobody-TNF- α antigen/antibody complex CPR-2; b: flow cytometry analysis mLN and expression of ANX1 binding receptors in the colon.
FIG. 6 is a graph showing CPR-induced Th17 cell differentiation of the ANX1 inhibited TNF-alpha nanobody-TNF-alpha antigen/antibody complex of the invention; in CPR-2 (10) 5 Cells), ANX1 (1 μg/ml) and WRW4 (10 μg/ml), flow cytometry analysis was performed on differentiated Th17 cells in colon and mLN.
FIG. 7 is a graph showing the combined treatment of DSS-induced colitis with TNF- α nanobody V7 and ANX1 of the invention; a: comparing the weight change trend of each group of mice; b: colon length comparison for each group of mice; c: representative anal bleeding for each group on day 7; d: comparison of disease activity index for each group.
FIG. 8 is a graph showing the amplification of Th17 in a DSS mouse treated with ANX1 (500. Mu.g/kg) reduced TNF-. Alpha.nanobody V7 (10 mg/kg) antibodies of the invention; a: mLN and Th17 cells in colon LPL were compared for each treatment group of mice by flow cytometry; b: the proportion of Th17 cells in each treatment group was counted.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
The TNF-alpha nano antibody provided by the invention has the following amino acid sequence:
(1) A protein consisting of the amino acid sequence shown in SEQ ID No.1 or 2 or 3; or (b)
(2) Amino acid sequence which encodes the same functional protein with 80% to 100% homology to the amino acid sequence defined by the sequence SEQ ID No.1 or 2 or 3; or (b)
(3) The protein derived from (1) with equivalent activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No.1 or 2 or 3.
The TNF-alpha nano antibody is a TNF-alpha nano antibody V19 or a TNF-alpha nano antibody V7 or a TNF-alpha nano antibody V1, the TNF-alpha nano antibody V19 is a protein formed by an amino acid sequence shown in SEQ ID No.1, the TNF-alpha nano antibody V7 is a protein formed by an amino acid sequence shown in SEQ ID No.2, and the TNF-alpha nano antibody V1 is a protein formed by an amino acid sequence shown in SEQ ID No. 3;
in the present invention, 3 different TNF- α nanobodies are used, the 3 TNF- α nanobodies having different affinities, e.g., the antibody of SEQ ID No.1 has about 100 times higher affinity than the antibody of SEQ ID No. 2. The results obtained for the 3 TNF-alpha nanobodies were essentially identical. For this reason, the present invention only takes the TNF-alpha nanobody as shown in SEQ ID No.2, which has poor affinity, as an example, and specific data are provided. The TNF-alpha nano antibody shown in SEQ ID No.2 has a code number of V7 in the research process.
The invention relates to an application of TNF-alpha nano antibody in preparing a medicament for treating inflammation including ulcerative colitis.
The TNF-alpha nano antibody disclosed by the invention is applied to the preparation of medicines for treating inflammation by being combined with other medicines for treating enteritis.
The TNF-alpha nano antibody disclosed by the invention is applied to the preparation of ulcerative colitis medicaments in combination with other enteritis treatment medicaments.
The invention relates to a combined medicine, which consists of a TNF-alpha nano antibody and annexin ANX 1.
The three TNF-alpha nano antibodies of the invention can be expressed and separated and purified in escherichia coli in a soluble way, have high anti-inflammatory biological activity, and have similar effects in spite of some difference of pharmacodynamics results. Therefore, only TNF- α nanobody V7 is presented as an example in the present invention.
Example 1
As shown in fig. 1 to 8, the invention firstly prokaryotic expresses TNF- α nanobody V7 in escherichia coli, and comprises the following specific steps: the TNF-alpha nano antibody V7 gene shown in SEQ ID No.2 is introduced into a pET28a vector through enzyme digestion and enzyme linked reaction, so as to construct an expression plasmid.
Transferring the target plasmid into BL21 (DE 3) competent cells, slightly mixing, ice-bathing for 30min, thermally shocking at 42 ℃ for 45s, standing on ice for 2min, adding 600 μl of antibiotic-free LB culture medium, shaking at 37 ℃ for 1h, centrifuging (2500 rpm,5 min), and discarding the supernatant to leave a small amount of culture medium re-suspension bacteria liquid.
After transformation, an appropriate amount of bacterial liquid was spread on LB plates containing kanamycin and cultured overnight at 37 ℃.
Positive clones were picked and grown in LB medium containing kanamycin and induced by addition of 5mM IPTG at 15℃for 20h. The expressed TNF-alpha nanobody V7 protein contains a His tag.
Cell pellet was collected by centrifugation (4000 rpm,20min,4 ℃) and protein buffer was added to the system, cells were broken up using a refiner, supernatant was collected after centrifugation (12000 rpm,30min,4 ℃) and expressed TNF- α nanobody was present in the supernatant in a soluble form.
Protein buffer formulation: (1) 300mM NaCl, (2) 50mM Tris-HCl, (3) 0.5mM beta-mercaptoethanol. (4) pH 7.8.
After equilibration of the column containing the Ni-NTA resin with the protein buffer, the supernatant was passed slowly through the Ni-NTA to bind the TNF- α nanobody V7 of interest to the Ni-NTA, followed by elution with the eluent.
The formula of the protein eluent comprises: (1) 80mM imidazole, (2) 300mM NaCl, (3) 50mM Tris-HCl, (4) 0.5mM beta-mercaptoethanol, (5) pH 7.8.
The His tag on the TNF- α nanobody V7 protein was cleaved using HRV3C enzyme at 4℃for 12h.
The solution was passed through Ni-NTA again to give TNF- α nanobody V7 without His-tag, and concentrated and stored (-80 ℃) after overnight dialysis using PBS buffer.
Molecular weight and purity of TNF- α nanobody V7 were verified by 12% SDS-PAGE.
As shown in FIG. 1A, the protein of TNF-alpha nanobody V7 after His tag removal after purification was about 15kDa.
Example 2
The invention detects the affinity of purified TNF-alpha nano antibody V7 to soluble TNF-alpha (s-TNF-alpha) through MST experiment, which comprises the following specific steps:
a100 nM solution of TNF-alpha nanobody V7 was formulated using MST buffer (50mM HEPES,100mM KCl,0.05% Tween-20).
10. Mu.M solutions of TNF-. Alpha.of human or murine origin were prepared using PBST buffer.
The highest ligand concentration of the TNF-. Alpha.solution was 5. Mu.M and diluted according to a 16-step gradient dilution method, and the final volume of each dilution series was 10. Mu.l.
Mu.l of 100nM TNF-alpha nanobody V7 was mixed with each concentration gradient of TNF-alpha sample and shaken well, after 5min incubation at room temperature the sample was drawn into a capillary and the capillary was placed in a Monolith NT.115 instrument for detection.
As shown in FIG. 1B, the dissociation constants of the TNF-alpha nanobody V7, the TNF-alpha nanobody V7 and the murine s-TNF-alpha reach 146nM, which indicates that the TNF-alpha nanobody prepared in the escherichia coli has high bioactivity, namely, has a complete and correct spatial folding protein structure. TNF-alpha nanobody V19 nanobody affinity can be as high as 6.5pM.
Example 3
The invention proves that the TNF-alpha nano antibody V7 has obvious treatment effect on 3% Dextran Sodium Sulfate (DSS) induced acute colitis mice at the dosage of 10mg/kg, and the specific steps are as follows:
model induction was performed in 7-9 week C57BL/6 female mice (body weight 18-20 g). Mice in the blank control group were fed normal drinking water daily, and mice in the DSS manufacturing module and each dosing treatment group were free to drink drinking water containing 3% DSS for a total of 8 days for induction.
Starting from the second day of molding, mice were intraperitoneally injected with 1mg/kg,5mg/kg,10mg/kg of TNF- α nanobody V7, and the administration period was once every three days.
The body weight of the mice and fecal occult blood were recorded daily.
As shown in fig. 2A, the weights of mice in the 10mg/kg (18.40 g±0.9959g, p=0.0304) and 5mg/kg (18.15 g±0.5949g, p=0.014) groups were significantly higher than those in the DSS group (17.03 g±2.443 g), while the weights of mice in the 1mg/kg (18.01 g±1.674g, p= 0.3397) group were not significantly changed. As shown in FIG. 2B, after treatment, the colon lesions were less in the 10mg/kg and 5mg/kg groups compared to the DSS group. This indicates that at a dose of 10mg/kg, TNF- α nanobody V7 has good therapeutic effect on DSS mice, being the optimal therapeutic dose of TNF- α nanobody V7.
Example 4
The invention proves that TNF-alpha nano antibody V7 causes the increase of DSS mouse colon Th17 cells, and the specific steps are as follows:
the mice were sacrificed by cervical removal and the abdominal cavity was dissected and the colon was taken.
The residual stool in the colon was purged with a cotton swab and the colon was rinsed several times with PBS solution.
The colon was dissected longitudinally and the colon was inverted with the mucosal surface facing outwards in a conical flask containing 20ml (D-Hank's solution containing 1mmol/L DTT and 1mmol/L EDTA) and incubated for 1h at 37 ℃.
The digested colon was minced and placed in a conical flask containing 20ml collagenase digest (RPMI 1640 medium+1 mg/ml collagenase I) and incubated at 37℃for 1h.
The digestions were filtered through a 300 mesh nylon screen, centrifuged (300 g,10min,4 ℃), the supernatant discarded and washed twice with RPMI1640 medium.
The cells were resuspended in 5ml of 40% Percoll, 4ml of 70% Percoll was gently added to the bottom of the centrifuge tube, the cell resuspension was gently added to form a clear liquid surface between the two layers of liquid, a Percoll gradient separation solution was prepared, and centrifuged (600 g,20min,25 ℃) to collect the lamina propria lymphocytes between the 40% Percoll and 70% Percoll interface.
Cells were resuspended in PBS with 1% BSA for use by washing 3 times with RPMI1640 medium.
The cells were centrifuged (250 g,4min,4 ℃), the supernatant discarded and the cells resuspended in PBS with 1% BSA. The labeled Th17 cells were incubated at 4℃with CD4, IL-17A antibody, and analyzed using a flow cytometer.
As shown in FIG. 3A, the intestinal Th17 cells of mice were significantly increased (31.11% + -2.787%) after TNF-. Alpha.nanobody V7 (10 mg/kg) treatment compared to DSS mice (19.82% + -2.06%).
Example 5
The invention proves that the increase of DSS mouse Th17 in TNF-alpha nano antibody V7 treatment is related to the phagocytosis of CD11c+DC cells, and the specific steps are as follows:
3% DSS model 5 days mice, intraperitoneal injection of TNF-alpha nanobody V7-BFP (Blue Fluorescent Protein) mg/kg.
After 2 hours, mice colon lamina propria lymphocytes (Lamina propria lymphocytes, LPL) were extracted, cd11c+, MHCII antibodies were added and labeled for 4 ℃ incubation and analyzed using a flow cytometer.
As shown in fig. 3B, cd11c+ DCs from the colon of treated mice showed significant BFP fluorescence, and were accompanied by upregulation of mhc ii, compared to PBS group. This suggests that TNF- α nanobody V7 was phagocytosed into cd11c+ DC cells during V7 treatment.
Example 6
The invention proves that the TNF-alpha nano antibody V7-TNF-alpha antigen/antibody complex CPR enables Th17 in the mesenteric lymph node of a WT mouse to be amplified, and the specific steps are as follows:
WT mice were taken from mesenteric lymph nodes (Mesenteric lymphocyte nodes, mLN), ground with a frosted slide and collected by rinsing with PBS.
Excised mLN was collected after passing through a 300 mesh nylon mesh.
Cells were resuspended in PBS with 1% BSA for use by washing 3 times with RPMI1640 medium.
In RAW264.7 (10) 5 Cells), CPR-1 (10 5 Cells) and CPR-2 (10 5 Cells) were stimulated in vitro for 2 hours.
After incubation of CD4, IL-17A antibody on ice for 30min, detection was performed using a flow cytometer.
As shown in fig. 4, TNF- α nanobody V7-TNF- α antigen/antibody complex CPR-2 still induced a significant increase in Th17 cell number compared to RAW264.7 (CPR-2, 35.18% ± 3.419%; RAW264.7, 19.78% ± 3.582%). However, no significant change was found by comparing CPR-1 (CPR-1, 22.02% + -2.314%) to RAW 264.7-induced Th17 cell numbers. RAW264.7 did not significantly induce Th17 cell differentiation compared to PBS (13.29% + -3.065%). This suggests that in vitro CPR can also induce Th17 cells in mLN, but not exactly the same as induction of Th17 cell production in intestinal LPL.
Example 7
The invention proves that the TNF-alpha nano antibody V7-TNF-alpha antigen/antibody complex CPR-2 significantly up-regulates ANX1 in colon, and the specific steps are as follows:
LPL from WT mice was treated with DeltaCIITA-CPR-2 (10 5 Cells) and CPR-2 (10 5 Cells) were stimulated in vitro for 2h.
The expression of ANX1 was verified by qRT-PCR analysis.
As shown in fig. 5A, CPR-2 significantly upregulates ANX1 in the colon compared to aciita-CPR-2, whereas no significant changes were found compared to RAW264.7 and PBS treatment. Furthermore, there was no significant change in the expression of ANX1 in ΔCIITA-CPR-2 compared to PBS. This trend is consistent with intestinal Th17 cells stimulated with CPR-2, indicating a correlation between ANX1 and Th17 cells.
Colon LPL and MLN of WT mice were taken and CPR-2 (10 5 Cells) for 2 hours.
The mixture was fixed with 1% paraformaldehyde for 1 hour.
Cells were fixed with an ANX1-mCherry marker and analyzed by flow cytometry.
As shown in FIG. 5B, cells from both colon and mLN were able to bind positively to ANX1 after stimulation with the TNF-alpha nanobody V7-TNF-alpha antigen/antibody complex CPR-2. This indicates that ANX1 binding to the receptor is up-regulated following CPR-2 treatment.
Example 8
The invention proves that ANX1 inhibits the CPR induced Th17 cell differentiation of TNF-alpha nanobody V7-TNF-alpha antigen/antibody complex through FPR2, and the specific steps are as follows:
colon LPL and MLN were taken from WT mice.
CPR-2 with TNF-alpha nanobody V7-TNF-alpha antigen/antibody complexes (10) 5 Cells), ANX1 (1. Mu.g/ml), WRW4 (10. Mu.g/ml) was stimulated for 2 hours.
After incubation of CD4, IL-17A antibody on ice for 30min, detection was performed using a flow cytometer.
As shown in fig. 6, ANX1 significantly inhibited CPR-2 induced intestinal Th17 cell differentiation (CPR-2, 32.46% ± 2.622%; CPR-2+anx1, 16.72% ± 1.425%); whereas in mLN differentiated Th17 cells induced by CPR-2 were also inhibited by ANX1 (CPR-2, 30.43% + -1.218%; CPR-2+ANX1,9.22% + -1.255%). In addition, WRW4 (colon: CPR-2+ANX1+WRW4, 37.49% + -7.253%; mLN: CPR-2+ANX1+WRW4, 22.31% + -0.8600%. These results indicate that ANX1 can activate FPR2 and inhibit CPR-induced Th17 cell differentiation.
Test example 1
The invention proves that the combination of the TNF-alpha nano antibody V7 (10 mg/kg) and the ANX1 (500 mug/kg) can improve the treatment effect of the TNF-alpha nano antibody V7 on the DSS mouse colonitis, and the specific steps are as follows:
model induction was performed in 7-9 week C57BL/6 female mice (body weight 18-20 g). Mice in the blank control group were fed normal drinking water daily, and mice in the DSS manufacturing module and each dosing treatment group were free to drink drinking water containing 3% DSS for 11 days total induction.
The period of administration of ANX1 (500. Mu.g/kg) and TNF-. Alpha.nanobody V7 (10 mg/kg) was once three days from the second day of molding.
The body weight of the mice and fecal occult blood were recorded daily.
As shown in fig. 7A, on day 11, the body weight of mice in the combination treatment group was significantly higher than that of DSS mice and TNF- α nanobody V7 treatment group mice (TNF- α nanobody v7+anx1, 19.62g±0.4729g; DSS group, 15.12g±1.254 g, p=0.0012; TNF- α nanobody V7, 18.77g±1.038g, p= 0.0328); while there was no significant change between ANX1 treated mice (ANX 1, 18.59g±1.274g, p= 0.05024).
As shown in fig. 7B, for macroscopic analysis of colon length, the ANX1/TNF- α nanobody V7 treated colon was significantly longer than DSS mice and ANX1 treated mice (TNF- α nanobody v7+anx1,6.2cm±0.5cm compared to DSS group, p=0.0057; ANX1,5.367cm±0.1155cm, p= 0.0482); there was no significant change between TNF- α nanobody V7 treated mice (V7, 5.3cm±0.4359cm, p=0.0785).
As shown in fig. 7C, on day 7, significant anal bleeding was observed in DSS, TNF- α nanobody V7, and ANX1, whereas no one was observed in ANX1/V7 treated mice.
As shown in FIG. 7D, the Disease Activity Index (DAI) was calculated in combination with the percent weight loss, fecal blood and fecal viscosity [35]. The DAI of ANX1/V7 was significantly lower than that of DSS, TNF-alpha nanobody V7 and ANX1 treated mice (ANX 1/V7, 1.175+ -0.1667 compared to DSS, 4.00+ -0.00, P <0.0001; TNF-alpha nanobody V7,2.583 + -0.631, P=0.032, ANX1, 2.083+ -0.1667, P=0.046). This suggests that TNF- α nanobody V7 in combination with ANX1 may have significantly improved therapeutic effects on DSS-induced colitis compared to V7 or ANX1 alone.
Test example 2
The invention demonstrates that ANX1 (500 mug/kg) reduces the amplification of Th17 when TNF-alpha nanobody V7 (10 mg/kg) antibodies are used to treat DSS mice, comprising the following steps:
model induction was performed in 7-9 week C57BL/6 female mice (body weight 18-20 g). Mice in the blank control group were fed normal drinking water daily, and mice in the DSS manufacturing module and each dosing treatment group were free to drink drinking water containing 3% DSS for 11 days total induction.
The period of administration of ANX1 (500. Mu.g/kg) and TNF-. Alpha.nanobody V7 (10 mg/kg) was once three days from the second day of molding.
LPL and mLN of mice were taken.
After incubation of CD4, IL-17A antibody on ice for 30min, detection was performed using a flow cytometer.
As shown in fig. 8, in colon LPL and mLN, TNF- α nanobody V7 treated mice had significantly lower Th17 cells than ANX1/TNF- α nanobody V7 treated mice (mLN: TNF- α nanobody V7/ANX1,5.93% ± 0.2883% compared to TNF- α nanobody V7, 8.923% ± 1.25%, p=0.0156; colon: TNF- α nanobody V7/ANX1,4.963% ± 0.9839% compared to TNF- α nanobody V7, 11.87% ± 1.344%, p=0.0002). This indicates that the combination ANX1 treatment has better efficacy on Th17 cell differentiation than anti-TNF monotherapy.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the invention or exceeding the scope of the invention as defined in the accompanying claims.
Claims (8)
1. A TNF- α nanobody, characterized by: the TNF-alpha nano antibody has the following amino acid sequence:
(1) A protein consisting of the amino acid sequence shown in SEQ ID No.1 or 2 or 3; or (b)
(2) Amino acid sequence which encodes the same functional protein with 80% to 100% homology to the amino acid sequence defined by the sequence SEQ ID No.1 or 2 or 3; or (b)
(3) The protein derived from (1) with equivalent activity by adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID No.1 or 2 or 3.
2. The TNF- α nanobody of claim 1, wherein: the TNF-alpha nano antibody is a TNF-alpha nano antibody V19 or a TNF-alpha nano antibody V7 or a TNF-alpha nano antibody V1, the TNF-alpha nano antibody V19 is a protein consisting of an amino acid sequence shown in SEQ ID No.1, the TNF-alpha nano antibody V7 is a protein consisting of an amino acid sequence shown in SEQ ID No.2, and the TNF-alpha nano antibody V1 is a protein consisting of an amino acid sequence shown in SEQ ID No. 3.
3. A nucleic acid molecule encoding the TNF- α nanobody of claim 1.
4. Use of TNF- α nanobodies of claim 1 in the preparation of a medicament for inflammation.
5. Use of TNF- α nanobodies of claim 1 in combination with other therapeutic agents for enteritis in the manufacture of a medicament for the treatment of inflammation.
6. The use of TNF- α nanobodies of claim 1 in the preparation of a medicament for the treatment of ulcerative colitis.
7. The use of TNF- α nanobody of claim 1 in combination with other enteritis treatment drugs in the preparation of ulcerative colitis drugs.
8. A combination, characterized in that: the combination drug consists of the TNF-alpha nano antibody and annexin ANX1 as claimed in claim 1.
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| CN202211308624.0A Pending CN117018185A (en) | 2022-03-18 | 2022-10-25 | Application of TNF-alpha nano antibody and PGE2 in preparation of ulcerative colitis medicines |
| CN202211499337.2A Pending CN117257933A (en) | 2022-03-18 | 2022-11-28 | Preparation method and application of TNF-alpha nano antibody |
| CN202211499338.7A Pending CN116173200A (en) | 2022-03-18 | 2022-11-28 | TNF-alpha nano antibody and application thereof in preparation of ulcerative colitis medicines |
| CN202211508681.3A Pending CN115779080A (en) | 2022-03-18 | 2022-11-29 | Application of annexin ANX1 and TNF-alpha nano antibody combination in preparation of ulcerative colitis medicine |
| CN202211508684.7A Pending CN116059352A (en) | 2022-03-18 | 2022-11-29 | Application of human annexin ANX1 in preparation of inflammatory disease medicines |
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| CN202211499337.2A Pending CN117257933A (en) | 2022-03-18 | 2022-11-28 | Preparation method and application of TNF-alpha nano antibody |
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| CN202211508684.7A Pending CN116059352A (en) | 2022-03-18 | 2022-11-29 | Application of human annexin ANX1 in preparation of inflammatory disease medicines |
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| CN115779080A (en) | 2023-03-14 |
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