CN106046174A

CN106046174A - Tocilizumab/CH12 coupled simulated epitope

Info

Publication number: CN106046174A
Application number: CN201610431258.6A
Authority: CN
Inventors: 杨麟
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-06-17
Filing date: 2016-06-17
Publication date: 2016-10-26
Anticipated expiration: 2036-06-17
Also published as: CN106046174B

Abstract

The invention discloses tocilizumab/CH12 coupled simulated epitope, having an amino acid sequence that is formed by: connecting C terminal of tocilizumab/CH12 coupled simulated epitope (KTMSAEEFDNWL) and C terminal of CH12 coupled simulated epitope through a linker peptide (GGGSGGS-SGGGSGGG) ('='represents disulfide bond); three polypeptides can be synthesized by using a chemical synthetic process and have linear structure. A dual-specific antibody prepared using the epitope can identify IL-6R and can identify EGFRvIII, thus inhibiting the activity of IL-6 and EFG, producing tocilizumab and enabling add-up of the tocilizumab monoclonal and CH12 and overcoming the problem that there is no need for multiple passive immunity. The simulated epitope of the invention provides novel pharmaceutical means to prevent and treat rheumatoid arthritis and other diseases.

Description

Tocilizumab/CH12 coupled mimic epitope peptide

Technical Field

The invention relates to a mimic antigen peptide in the technical field of biological engineering, in particular to a Tocilizumab/CH12 coupled mimic epitope peptide.

Background

Rheumatoid Arthritis (RA) is a chronic, systemic, pleiotropic inflammatory autoimmune disease that mainly affects the small joints of the hands and feet, and is characterized most by persistent synovitis, with symptoms including joint pain, joint swelling, and progressive joint destruction that ultimately leads to disability in the patient.

At present, drugs for treating rheumatoid arthritis mainly include hormones, non-steroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs (DMARDs) and emerging biologics. Compared with the traditional medicine, the biological agent directly acts on the main inflammatory factors, can more quickly and effectively relieve symptoms and inhibit the damage of joint structures. Currently, the biological agents used for treating RA include monoclonal antibodies (mabs), cytokines and antagonists, including anti-T lymphocyte surface differentiation antigen mabs, anti-interleukin-2 receptor (IL-2R) mabs, anti-CD 54 receptor mabs, anti-Tumor Necrosis Factor (TNF) -alpha antibodies, recombinant human interleukin-1 receptor antagonists, and the like.

In 2010, tocilizumab (roactemra) antibody was FDA approved for the treatment of rheumatoid arthritis. Tocilizumab is a novel IL-6R-targeting monoclonal antibody that reduces the immunogenic response that might occur in a patient by shifting the complementarity determining regions of a murine anti-human IL-6R antibody into the framework of human IgG1 antibody, and is the same as the original murine antibody and chimeric anti-IL-6R antibody in terms of both antigen binding and inhibition of IL-6 function. Whereas humanized antibodies are less immunogenic and have a longer half-life. Tocilizumab, which is capable of specifically binding to the soluble IL-6 receptor and the membrane IL-6 receptor and inhibiting the signaling of soluble IL-6 and membrane IL-6, was approved by Japan as a drug for the treatment of Castelman's syndrome in 2005. Tocilizumab monoclonal antibody is the first clinical trial to demonstrate better efficacy than anti-TNF monoclonal antibody alone with MTX monoclonal antibody (Jones G, Sebba A, Gu J et al, company of Tocilizumab of cytotoxic monoclonal antibody with modulation to segment rheum sativum, 69: 88; 96,2010; Kremer JM, et al, Tocilizumab inhibition structure j. oil delivery genes with amino antibody reactivity genes, reaction promoter with amino antibody reactivity reagent 609. recovery reagent of receptor complex of reaction of receptor complex of receptor of strain of reaction of receptor of 3. about. At The same time, The antibody in combination with DMARDs also showed therapeutic effects over DMARDs alone (Weinblatt M, et al, safety of Tocilizumab monoterpy and TCZ plus DMARDs in a US RA publication with amino equivalent response to biologics or DMARDs: The ACT-STAR study. London: EULAR, 2011). Therefore, the application prospect of Tocilizumab is probably better than that of anti-TNF biological agent.

Although the clinical biotherapy of various monoclonal antibodies advances the treatment of autoimmune diseases such as rheumatoid arthritis or other refractory diseases, the monoclonal antibodies still have many defects in immunotherapy: firstly, in order to maintain the sufficient titer of the antibody in the antibody immunotherapy, the immunization must be carried out repeatedly; ② the use cost of the antibody is very expensive due to repeated immunization; ③ the side effects of antibody therapy also limit the application range, mainly the susceptibility to infection and drug hypersensitivity. Therefore, the induction of the body to produce the required antibodies through active immunization becomes a new choice for researchers, and the defects that passive immunization needs to be repeated and non-human parts have immunological damage to human bodies can be overcome.

To overcome these application problems of monoclonal antibodies, foreign scholars have proposed the design of mimotope peptides. The mimotope peptide, also called mirror image epitope, is a polypeptide capable of stimulating an organism to generate specific immunity, and the minimum structure of the mimotope peptide is 6-20 amino acids in length. The mimic epitope peptide can activate the immune system of an organism and generate active immune target molecules, thereby playing a role in treating diseases and avoiding the clinical problem of the monoclonal antibody. The sequence of the mimotope peptide may be different from that of the natural epitope, but the spatial structures of the mimotope peptide and the natural epitope are similar, so that the polyclonal antibody generated after the immune animal not only can be combined with the mimotope peptide, but also can be combined with the natural epitope to mediate further biological behaviors. The epitope peptide is simulated through immunity, and an organism is induced to generate an antibody targeting target molecule. It has low cost, good safety and high specificity (Riemer AB, Jensen-Jarolime. Mimotope vaccines: epipope mimics index-cancer antibodies. Immunol Lett2007Oct 31; 113(1): 1-5).

Chinese patent publication No. 104945485A (published Japanese 2015-09-30) discloses a mimotope peptide of anti-IL-6 receptor Tocilizumab. The antibody produced by the mimic epitope immune stimulator body can not only recognize the mimic epitope, but also recognize IL-6 receptor so as to inhibit the activity of IL-6, produce the same effect as Tocilizumab monoclonal antibody, and simultaneously overcome the problem of needing multiple passive immunizations.

Some rheumatoid diseases have obvious correlation with malignant tumors, such as dermatitis and various solid tumors, sicca syndrome and lymphoma, systemic sclerosis and lung adenocarcinoma, and the occurrence of tumors can be caused by long-term use of immunosuppressive agents. The condition that rheumatoid arthritis patients are combined with malignant tumors is reported at home and abroad, and the specific pathogenesis of the rheumatoid arthritis patients is unknown. Recent reports show that RA patients have certain relevance not only to blood and lymphatic system tumors such as leukemia, lymphoma and the like, but also to solid tumors such as lung cancer, breast cancer, thyroid cancer and the like. RA patients may have malignant tumor at first, and then have rheumatoid arthritis, and some patients have malignant tumor after having rheumatoid arthritis for several years, and two diseases occur simultaneously and have parallel course of disease.

The relation between RA and malignant tumor is clear, and the enhancement of the understanding and the exploration of the relation has important significance for diagnosis and treatment, however, corresponding medicines are lacked in the diagnosis and treatment at present.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a Tocilizumab/CH12 coupled mimotope peptide, wherein a polyclonal antibody obtained after an animal is immunized can target specific targets of two diseases, and inhibit signal paths and malignant phenotypes of the two targets, so that a treatment effect is achieved.

The amino acid sequence of the Tocilizumab/CH12 coupled mimotope peptide is shown in SEQ ID NO. 1, and the Tocilizumab mimotope peptide is formed by connecting the C end of the Tocilizumab mimotope peptide shown in SEQ ID NO. 2with the C end of the CH12 mimotope peptide shown in SEQ ID NO. 3 through a connecting peptide shown in SEQ ID NO. 4; wherein,

tocilizumab mimotope peptide fragment: n-terminal → C-terminal KTMSAEEFDNWL (SEQ ID NO: 2);

CH12 mimotope peptide fragment: n-terminal → C-terminal WHTEILKSYPHE (SEQ ID NO: 3);

connecting peptide: n-terminal → C-terminal GGGSGGS ═ SGGGSGGG (SEQ ID NO:4), where "═" denotes a disulfide bond.

The amino acid sequence of SEQ ID NO. 1 is as follows:

KTMSAEEFDNWLGGGSGGS is SGGGSGGGEHPYSKLIETHW, amino acid K and amino acid W on both sides are the N-terminal end of the polypeptide, amino acid L and amino acid E on both ends of the linker peptide are the C-terminal end of the polypeptide, the linker peptide is GGGSGGS-SGGG, which represents a disulfide bond.

CH12 is a still experimental monoclonal antibody against EGFRvIII that binds to EGFRvIII expressed only in tumors or some malignant diseases and to EGFR that is overexpressed (not bound to EGFR at normal expression levels), thereby inhibiting downstream signaling pathways. Monoclonal antibody CH12 can effectively inhibit the cell malignancy phenotype of tumors in animal models of various tumors, and shows good application prospects (see the literature: Xu W, et al. combination of an anti-EGFRvIII antigen CH12with Rapamycin synthetic antigens: 10.18632/oncotarget.8407; Xu W et al. synthetic antigen activity of the EGFRvIII + HER2+ bacterial antigens of the growth promoter with anti-inflammatory genes with anti-EGFRvIII antigen-induced III CH12. oncotarget.12. oncogene expression, nov 17: 38840-53; growth of cancer antigen H12. mutation of cancer cells, 12. expression of cancer cells, 12. oncogene, 12. expression of cancer cells, 17, 6. 38840-53; growth of cancer cells, and mutation H2. mutation. In view of the important pathogenesis of EGFR/EGFRvIII in malignant diseases such as rheumatoid arthritis and malignant tumors, the invention takes EGFR and a mutant thereof as a treatment target, designs a mimic epitope of a monoclonal antibody CH12 for recognizing the target, couples the mimic epitope with a Tocilizumab mimic epitope, and obtains the coupled mimic epitope peptide so as to realize the effect that the antibody can be combined with two targets for treatment.

During design: the C end of the Tocilizumab mimotope peptide is connected with the N end of the peptide, and the C end of the CH12 coupling mimotope peptide is connected with the C end of the peptide. In actual manufacturing, an optional implementation is as follows: the Tocilizumab mimotope peptide is chemically synthesized along with one half of the linker peptide (KTMSAEEFDNWLGGGSGGS), and similarly, the CH12 conjugate mimotope peptide is chemically synthesized along with the other half of the linker peptide (WHTEILKSYPHEGGGSGGGS), and the two final phases are linked by a disulfide bond.

The Tocilizumab mimotope peptide and the CH12 mimotope peptide are coupled through a linear connecting peptide, and the connecting peptide in the middle is linear and has the characteristics of higher flexibility and swinging. Such a structure activates the immune system to produce a bispecific antibody that targets both IL-6R and EGFRvIII. The antibody produced by the design can replace two different monoclonal antibodies and play the role of adding the two monoclonal antibodies. In possible clinical application in the future, the frequency of using the medicine is reduced, the use cost of the medicine is reduced, and the safety is improved.

The invention also provides a group of functional gene sequences, and the polypeptide obtained by post-transcription and translation can be used for forming the Tocilizumab/CH12 coupled mimotope peptide, which comprises a coding gene of the Tocilizumab mimotope peptide, a coding gene of the CH12 coupled mimotope peptide and a coding gene of the connecting peptide. Under the condition that the amino acid sequence of the polypeptide is known, the codon corresponding to each amino acid residue can be one or more, on the basis of the codon, different codon connection combinations can be made by adopting the prior art means according to needs, and different coding genes formed by combining a plurality of codons corresponding to each polypeptide are within the protection scope of the invention.

The gene encoding the Tocilizumab mimotope peptide is preferably 5'-aag acg atg agt gct gag gagttt gat aat tgg ctg ggt gga ggt tcg-3' (SEQ ID NO: 5).

The gene encoding the CH12 mimotope peptide is preferably 5'-tgg cat act gag att ctg aag tct tatccg cat ggg ggt gga ggt tcg-3' (SEQ ID NO: 6).

The coding gene of the Tocilizumab/CH12 coupled mimotope peptide is also in the protection scope of the invention. The coding gene can also be any one of a plurality of DNA sequences formed by different codon combinations.

The invention also provides a biological material containing the coding gene, wherein the biological material is an expression vector, a transgenic cell line or a host bacterium.

The application of the Tocilizumab/CH12 coupled mimotope peptide as an antigen in preparing a medicament for inducing the generation of an antibody is also within the protection scope of the invention.

The Tocilizumab mimotope peptide and the CH12 mimotope peptide on the Tocilizumab/CH12 coupled mimotope peptide can simultaneously exert the biological functions thereof, and can be used as immunogen to activate an immune system to generate a bispecific antibody, and the antibody can simultaneously target IL-6R and EGFRvIII.

The invention also provides a polyclonal antibody, which is prepared by the Tocilizumab/CH12 coupled mimic epitope peptide immune animal. The antibody can specifically recognize IL-6R and EGFRvIII.

The invention also provides application of the polyclonal antibody in preparation of a medicament for preventing and/or treating rheumatoid arthritis.

The invention also provides application of the polyclonal antibody in preparation of a medicament for preventing and/or treating tumors.

Preferably, the tumor is histiocytic lymphoma, cervical cancer, lung cancer, liver cancer or brain glioma.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, two different mimic epitope peptides are coupled, so that the polyclonal antibody obtained after an immune animal can target two disease-specific targets IL-6R and EGFRvIII, and inhibit signal paths and malignant phenotypes of the two targets. Therefore, the active immunity caused by the coupling mimotope peptide can generate the similar treatment effect with Tocilizumab (ROACTEMRA) and CH12 monoclonal antibody, and has the advantages of high specificity (targeting disease specific target), low cost (low price for polypeptide synthesis), safety (no infection source in the process of polypeptide synthesis), no need of repeated administration (similar vaccine, only needing 3-4 times of immunization) and small side effect (no species heterogeneity), thereby being superior to the biological treatment of the monoclonal antibody.

Drawings

FIG. 1 shows the electrophoresis results of IL-6R and EGFRvIII for detecting whether polyclonal antibody can be combined with receptor over-expression cell line or tumor cell line expression by immunoblotting experiment. The electrophoresis results with Tocilizumab on the top show binding of Tocilizumab to IL-6R, the electrophoresis results with purefined pAb on the top show binding of purified polyclonal antibody to IL-6R, the electrophoresis results with CH12 on the top show binding of monoclonal antibody CH12 to EGFR/EGFRvIII, and the electrophoresis results with purefined pAb on the top show binding of purified polyclonal antibody to EGFR/EGFRvIII.

FIG. 2 is the result of electrophoresis of the polyclonal antibody to detect whether it can bind to IL-6R and EGFRvIII expressed in synovial tissue of patients with rheumatoid arthritis or osteoarthritis by immunoblotting. Wherein, the electrophoresis diagram marked with mimo-pAb at the top shows the detection result of the binding of the polyclonal antibody generated by Tocilizumab/CH12 coupled mimotope peptide immune animals by the immunoblotting method; the electrophoresis chart marked with Tocilizumab at the top shows the combination detection result of the monoclonal antibody Tocilizumab; the electrophoresis chart with the top labeled with CH12 shows the binding detection result of monoclonal antibody CH 12; the relative position band of EGFR is about 170KD, the relative position band of EGFRvIII is about 130KD, the relative position band of IL-6R is about 72KD and 55KD, and beta-actin is internal reference protein.

FIG. 3 is the result of electrophoresis of the polyclonal antibody to detect whether it can bind to IL-6R and EGFRvIII expressed by the fibroblast synovial cells of patients with rheumatoid arthritis or osteoarthritis by immunoblotting experiments. The figures are labeled as in FIG. 2.

FIG. 4 is a photograph showing the result of detecting the internalization degradation of IL-6R and EGFRvIII expressed by the fibroblast synoviocytes of patients with rheumatoid arthritis caused by polyclonal antibody by immunofluorescence assay. In the figure, the first row is, from left to right, sequentially provided with a nuclear stain DAPI, a control group polyclonal antibody combined with IL-6R, a coupling mimic peptide group polyclonal antibody combined with IL-6R by the nuclear stain DAPI, and IL-6R; the second row is sequentially provided with a nuclear stain DAPI, a control group polyclonal antibody combined with the EGFRvIII, an EGFRvIII, a nuclear stain DAPI, a coupling mimic peptide group polyclonal antibody combined with the EGFRvIII, and an EGFRvIII from left to right; control pAb represents a control group polyclonal antibody, mimo-pAb represents a polyclonal antibody (peptidomimetic group polyclonal antibody) prepared by coupling mimotope peptide immunized animals of the invention, and DAPI represents a nuclear stain.

FIG. 5 is the result of an immunoblotting experiment for detecting the inhibition of EGF and IL-6 signal channels in the fibroblast synoviocytes of patients with rheumatoid arthritis by using a polyclonal antibody prepared by coupling a mimotope peptide.

FIG. 6 is a bar graph showing the effect of polyclonal antibody prepared by coupling mimotope peptide on proliferation of EGF and IL-6 stimulated fibroblast synoviocytes of rheumatoid arthritis patients by using a cell counting kit.

Detailed Description

The present invention will be further described with reference to specific examples to assist understanding of the invention.

The preparation and identification process of the Tocilizumab/CH12 coupled mimotope peptide basically comprises the following steps:

1. synthesizing Tocilizumab/CH12 coupled mimic epitope peptide;

2. Tocilizumab/CH12 coupled with mimotope peptide to immunize animals;

3. purifying animal serum to obtain polyclonal antibody;

4. detecting the capacity of the purified polyclonal antibody to combine with IL-6R and EGFRvIII expressed by an overexpression cell line and a tumor cell line by using an immunoblotting method;

5. detecting the capability of the purified polyclonal antibody combining IL-6R and EGFRvIII expressed by rheumatoid arthritis synovial tissue by using an immunoblotting method;

6. detecting the capacity of the purified polyclonal antibody for combining IL-6R and EGFRvIII expressed by the rheumatoid arthritis fibroblast-like synovial cells by using an immunoblotting method;

7. detecting the influence of the purified polyclonal antibody on IL-6 and EGF signal channels of rheumatoid arthritis fibroblast synoviocytes by an immunoblotting method;

8. the effect of the purified polyclonal antibody on proliferation induced by IL-6 and EGF in rheumatoid arthritis fibroblast-like synoviocytes was observed using a cell counting kit.

Sources of biological materials and reagents:

Tocilizumab/CH12 coupled mimic epitope peptide for immunizing animals

(1) Preparation of Tocilizumab/CH12 coupled mimotope peptide

The synthesis and coupling of the polypeptides was performed by the company beijing seibuten (SBS) with a purity > 98%. Polypeptides of similar length were set as control polypeptides (control peptides:

MTLHSNSTTSPLGGGSGGGS-SGGGSGGGFPNISSSWVHSP, the coupling method is the same as the invention) Tocilizumab/CH12 is coupled with a mimotope peptide (hereinafter, coupling peptide) and a control peptide as antigens for immunization, the dose of each immunization is 200 mu g of protein, and the adjuvants used are complete Freund's adjuvant (first immunization) and incomplete Freund's adjuvant (second, third and fourth immunizations).

(2) Preparation of Wistar rats

Wistar rats were provided by witnessee, inc, at a breeding site of SPF grade Wistar rats, 8 weeks old, female, from the animal center of the department of medicine, beijing university. Rats were divided into 2 groups, 1 group was a conjugated peptide group (immune Tocilizumab/CH12 conjugated mimotope peptide), and 1 group was a control peptide group (immune control polypeptide).

(3) Immunization of Wistar rats

Wistar rats were immunized by subcutaneous injection on days 1, 22, 43, 65 for 4 times, and blood was collected from the inner canthus of the eye 7 days after each immunization. Standing whole blood at 4 deg.C for 24hr, centrifuging at 4000rpm for 15min, collecting supernatant, packaging, and storing at-20 deg.C.

Purification of rat antiserum

Purification of rat polyclonal antibody purification was performed using the Montage ProSep G antibody purification kit from Millipore.

(1) Rat serum was diluted 1:1 with Buffer A provided in the kit, and after addition to the purification column, 500g was centrifuged at room temperature for 20 min.

(2) The column was washed once with 15ml Buffer A and centrifuged at 500g at room temperature for 20 min.

(3) Bound polyclonal antibody was eluted with 10ml Buffer B and centrifuged at 500g for 20min at room temperature.

(4) Buffer B was neutralized with 150. mu.l of Buffer C.

(5) The polyclonal antibody system is treated by a desalting concentration column, then diluted by PBS, subpackaged and stored at-20 ℃.

Thirdly, detecting the binding capacity of the purified polyclonal antibody with an over-expression cell line and tumor cell lines EGFRvIII and IL-6R by using an immunoblotting method (Western Blot)

(1) Extraction of proteins

Removing culture solution from HeLa, U937, Jurkat, Huh7-EGFRvIII, Huh7-IL-6R, A431, A549 and TF-1 cells which grow well in a plate or a culture bottle respectively, washing the cells by using a proper amount of precooled PBS, adding 600 mu l of PBS into the culture dish, scraping the cells by using cell scraping, collecting the cells in an Eppendorf tube, centrifuging at 8000rpm for 1min, and discarding supernatant; adding 100 mul of prepared cell lysate into the precipitate, adding protease inhibitor according to the volume ratio of 1:100, blowing and suspending the precipitate, standing on ice for 30min, centrifuging at 12000rpm for 15min, collecting supernatant, and measuring the protein concentration by using a BCA method.

(2) Preparing gel for PAGE electrophoresis:

the separating gel formula comprises:

the concentrated gel formula comprises:

the concentrated gel was placed on top of the separation gel, and the comb was inserted and left at room temperature for 15 min. After the lamination layer is solidified, the sample adding comb can be pulled out for standby.

(3) Loading: adding 6 × protein electrophoresis sample liquid into the cell extract protein, mixing, and treating with boiling water bath for 15 min. Centrifuging at 10000rpm for 5min, and collecting 20 μ g sample for protein electrophoresis.

(4) Electrophoresis: electrophoresis was performed at constant voltage. Firstly, the voltage of 80V is about 30min to ensure that the protein sample reaches the interface between the concentrated gel and the separation gel, and then the voltage is changed to 120V for about 1-1.5 hr until the protein runs to the bottom of the separation gel.

(5) Electric conversion: the proteins were transferred to a PVDF membrane previously activated with methanol by wet electrotransformation at a constant current of 250mA for about 2.5 hr.

(6) And (3) sealing: blocking with TBST 5% bovine serum albumin blocking solution at room temperature for 1hr, and shaking gently on shaking table.

(7) Incubating the primary antibody: polyclonal antibody (10. mu.g/ml) was incubated with PVDF membrane and gently shaken overnight on a shaker at 4 ℃.

(8) Rinsing: rinse primary antibody with TBST, shake rinse on shaker for 15min each time for 3 times.

(9) Incubation of secondary antibody: secondary antibodies, which were fluorescent antibodies provided by the central laboratory, were selected according to the primary antibody source at a concentration of 1:10000 and were gently shaken on a shaker at room temperature for 1 hr.

(10) Rinsing: the secondary antibody was rinsed with TBST and gently rinsed on a shaker for 15min each time 3 times.

(11) Scanning: and spreading the rinsed PVDF membrane on a western blot scanner, clicking Odysey software, scanning and storing an experimental result.

The results suggest that polyclonal antibodies generated from animals immunized with the conjugated peptide are capable of binding to EGFRvIII and IL-6R expressed by both over-expressed cell lines and tumor cell lines. Referring to FIG. 1, the electrophoresis results are shown in the top labeled Tocilizumab shows that the monoclonal antibody Tocilizumab binds to IL-6R, the top labeled purefined pAb shows that the purified polyclonal antibody binds to IL-6R, the relative positions of IL-6R are in the vicinity of 72KD and 55KD, the black bands show binding, and the darker color indicates the higher binding concentration. FIG. 1 shows the results of electrophoresis using CH12 at the top, wherein the binding of mAb CH12 to EGFR/EGFRvIII is shown, and the binding of the above purified polyclonal antibody to EGFR/EGFRvIII is shown by the results of electrophoresis using PURIFIED pAb at the top, wherein the EGFR relative position is approximately 170KD, the EGFRvIII relative position is approximately 130KD, and the dark color shows the binding, and the darker color indicates the higher binding concentration.

Fourthly, Western Blot for detecting expression of EGFRvIII or IL-6R of synovial tissue of rheumatoid arthritis

The extraction method of the synovial tissue protein is basically the same as the extraction method of the total protein of cells, and the basic method comprises the following steps: freezing-80 deg.C synovial tissue from RA and OA patients, waiting to room temperature, and cutting with scissors to obtain a block of about 5mm³Loading synovial tissue into EP tube, weighing, adding RIPA lysate containing protease inhibitor at a mass/volume ratio of 1:5, cutting tissue block with small scissors on ice, homogenizing with tissue homogenizer at rotating speed4000rpm for 3 times, 30 seconds each, until a homogeneous tissue suspension was obtained, the entire procedure was performed on ice, and then the protein concentration was determined using the BCA protein quantification kit. And the Western blot method is the same as the third step.

The results suggest that polyclonal antibodies generated from animals immunized with the conjugated peptide can detect EGFRvIII and IL-6R expressed in synovial tissues of patients with rheumatoid arthritis, as shown in FIG. 2.

Fifth, Western Blot for detecting the expression of EGFRvIII or IL-6R of rheumatoid arthritis fibroblast-like synoviocytes

The method is the same as the third method, and the used cells are rheumatoid arthritis fibroblast-like synoviocytes.

The results suggest that polyclonal antibodies generated from animals immunized with the conjugated mimotope peptide can detect EGFRvIII and IL-6R expressed in fibroblast-like synovial cells of patients with rheumatoid arthritis (see FIG. 3).

Sixth, Immunofluorescence (Immunofluorescence) method for observing the effect of polyclonal antibody on the receptor internalization of rheumatoid arthritis fibroblast-like synoviocytes

(1) The rheumatoid arthritis fibroblast-like synoviocytes were seeded into confocal dishes (1.0 × 10)⁴)，37℃5％CO₂Culturing to 70-90%.

(2) Wash 3 times with PBS for 10min each.

(3) Cells were fixed with 4% paraformaldehyde at room temperature for 15 min.

(4) Wash 3 times with PBS for 10min each.

(5) Blocking with 10% sheep serum (dissolved in PBS/pH 7.3) at room temperature for 1 hr.

(6) Wash 3 times with PBST for 10min each.

(7) Polyclonal antibody (10. mu.g/ml) was incubated at room temperature for 1 hr.

(8) Wash 3 times with PBST × 10 min.

(9) Northern lights546anti-rat IgG antibody (1:100) and DAPI (1:1000) were added, both diluted in PBS and incubated at room temperature for 30 min.

(10) Wash 3 times with PBS for 15min each.

(11) The sheets were mounted with glycerol.

(12) Observed with a common fluorescence microscope and photographed.

The results suggest that polyclonal antibodies can internalize and degrade receptors on the surface of fibroblasts in rheumatoid arthritis patients. Referring to FIG. 4, red is bound polyclonal antibody and green is IL-6R or EGFR receptor. The polyclonal antibody of the negative control polypeptide group (control-pAb) hardly binds to IL-6R and EGFR (DAPI: for nuclear staining; control-pAb is weak in red light; EGFRvIII and IL-6R are strong in green fluorescence), and the coupling peptide polyclonal antibody group shows that the binding rate of the polyclonal antibody is very high (mimo-pAb is strong in red fluorescence; EGFRvIII and IL-6R are weak in green fluorescence, and internalization degradation of the receptor is shown).

Seventhly, detecting the influence of the polyclonal antibody on the fibroblast synoviocytes signal channel of the rheumatoid arthritis patient caused by IL-6 and EGF stimulation by Western Blot

(1) A well-grown RA-FLS cell is taken out, subcultured to 96 cm culture dishes, and cultured until the whole dish is full.

(2) The serum-containing medium was decanted and starved for 48hr with serum-free medium.

(3) Serum-free medium was decanted and positive control group was added: tocilizumab and CH12 (10. mu.g/ml), polyclonal antibody (divided into 5 concentration gradients: 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml), incubated at 37 ℃ for 3 hr.

(4) EGF + IL-6(100ng/ml) was added after 3 hr. The cytokine is stimulated for 30 min.

(5) Washing cells with a proper amount of precooled PBS, adding 600 mu l of PBS into a culture dish, scraping the cells with a cell scraper, collecting the cells in an Eppendorf tube, centrifuging at 8000rpm for 1min, and discarding the supernatant; adding 100 μ l of prepared cell lysate into the precipitate, adding protease inhibitor according to the volume ratio of 1:100, blowing and beating the suspension precipitate, and standing on ice for 30 min; centrifugation was carried out at 12000rpm for 15min, and the supernatant was collected and assayed for protein concentration by BCA method. Adding 6 × protein electrophoresis sample liquid into the cell extract protein, mixing, and treating with boiling water bath for 15 min. Centrifuging at 10000rpm for 5min, and collecting 30 μ g sample for protein electrophoresis.

(6) Incubating the primary antibody: the signal path-associated antibody was diluted at a concentration of 1:1000 and incubated with PVDF membrane, and gently shaken overnight on a shaker at 4 ℃.

(7) Rinsing: rinse primary antibody with TBST, shake rinse on shaker for 15min each time for 3 times.

(8) Incubation of secondary antibody: secondary antibodies, which were fluorescent antibodies provided by the central laboratory, were selected according to the primary antibody source at a concentration of 1:10000 and were gently shaken on a shaker at room temperature for 1 hr.

(9) Rinsing: the secondary antibody was rinsed with TBST and gently rinsed on a shaker for 15min each time 3 times.

(10) Scanning: and spreading the rinsed PVDF membrane on a western blot scanner, clicking Odyssey software, scanning and storing the experimental result.

The results are shown in FIG. 5, with the signaling pathway proteins and sizes shown on the left, and the cytokines and antibodies added annotated on the top. Column 1 without any cytokine or antibody; column 2with 100ng/ml EGF and IL-6; column 3, 100ng/ml EGF and IL-6, and 10. mu.g/ml Tocilizumab; column 4, EGF and IL-6 at 100ng/ml, and CH12 mAb at 10. mu.g/ml; columns 5-9, EGF and IL-6 were added at 100ng/ml, as well as polyclonal antibodies at concentrations corresponding to 10-0.001. mu.g/ml, respectively. It is suggested that polyclonal antibody generated by Tocilizumab/CH12 coupled with mimotope peptide immunization animals can inhibit activation of a fibroblast-like synovial cell signaling pathway of rheumatoid arthritis patients caused by IL-6 and EGF.

Eighthly, the CCK-8 method detects the influence of the polyclonal antibody on the proliferation of the fibroblast synoviocytes of the rheumatoid arthritis patient caused by IL-6 and EGF

The RA-FLS cells with good growth state are inoculated on a 96-well plate, and the cell number of each well is 5.0 × 10³Each processing group is provided with 5 complex holes which are divided into the following groups:

serum-free group: DMEM medium (negative control);

seropositive group: DMEM medium plus 10% FBS serum (positive control);

IL-6 group: IL-6 alone (at a concentration of 100 ng/ml);

EGF group: EGF alone (concentration 100ng/ml)

IL-6+ EGF group: adding IL-6 and EGF (concentration 100ng/ml)

IL-6+ EGF + CH12 group: adding IL-6, EGF and CH12 monoclonal antibody (IL-6 and EGF concentration is 100ng/ml, CH12 monoclonal antibody concentration is 10 mug/ml)

IL-6+ EGF + Tocilizumab group: adding IL-6, EGF and Tocilizumab (IL-6 and EGF concentration is 100ng/ml, Tocilizumab concentration is 10 mug/ml)

IL-6+ EGF + polyclonal antibody (5 concentrations) groups: IL-6, EGF and polyclonal antibody (IL-6 and EGF at 100ng/ml, polyclonal antibody at 10. mu.g/ml, 1. mu.g/ml, 0.1. mu.g/ml, 0.01. mu.g/ml, 0.001. mu.g/ml) were added.

Treating RA-FLS cells under different conditions for 72hr, removing culture medium, washing each well with serum-free medium, adding DMEM medium 100 μ l into each well, adding CCK8 cell proliferation detection solution 20 μ l into each well, and adding 5% CO₂Culturing in a cell culture box at 37 deg.C for 2hr, measuring absorbance at 490nm with enzyme labeling instrument, and zeroing with blank cell hole for color comparison.

The percent cell viability (percent of cell viability) was calculated by the following formula:

percent cell survival rate ═ a_{Mean value of experimental wells}÷A_{Mean values of seropositive groups})×100％。

Results referring to fig. 6, the absorbance values are plotted on the ordinate, representing the cell proliferation coefficient, and the group on the abscissa. From left to right, the group is serum-free, seropositive, IL-6, EGF, IL-6+ EGF + CH12, IL-6+ EGF + Tocilizumab, and IL-6+ EGF + polyclonal antibody (5 concentrations). The results suggest that polyclonal antibodies generated by animals immunized with the conjugated mimotope peptide can inhibit the proliferation of fibroblast-like synovial cells of patients with rheumatoid arthritis caused by IL-6 and EGF.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

Tocilizumab/CH12 coupled mimotope peptide, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 1, and the Tocilizumab mimotope peptide is formed by connecting the C end of the Tocilizumab mimotope peptide shown as SEQ ID NO. 2with the C end of the CH12 mimotope peptide shown as SEQ ID NO. 3 through a connecting peptide shown as SEQ ID NO. 4; wherein,

tocilizumab mimotope peptide: n-basal end → C-distal end KTMSAEEFDNWL;

CH 12-coupled mimotope peptides: n-basal end → C-distal end WHTEILKSYPHE;

connecting peptide: n-terminal → C-terminal GGGSGGS ═ SGGGSGGG, where "═" denotes a disulfide bond.
2. A set of functional gene sequences, wherein the polypeptides obtained by posttranscriptional translation can be used for constituting the Tocilizumab/CH12 coupled mimotope peptide as claimed in claim 1, which comprises a coding gene of the Tocilizumab mimotope peptide, a coding gene of the CH12 coupled mimotope peptide and a coding gene of the connecting peptide.
3. The gene encoding Tocilizumab/CH 12-conjugated mimotope peptide of claim 1.
4. Biological material comprising the coding gene of claim 3, said biological material being an expression vector, a transgenic cell line or a host bacterium.
5. Use of the Tocilizumab/CH 12-conjugated mimotope peptide of claim 1 as an antigen in the preparation of a medicament for inducing antibody production.
6. A polyclonal antibody prepared from an animal immunized with the Tocilizumab/CH12 conjugated mimotope peptide of claim 1.
7. Use of a polyclonal antibody as defined in claim 6 for the preparation of a medicament for the prevention and/or treatment of rheumatoid arthritis.
8. Use of a polyclonal antibody according to claim 6 for the preparation of a medicament for the prevention and/or treatment of a tumor.
9. The use of claim 8, wherein the tumor is a histiocytic lymphoma, cervical cancer, lung cancer, liver cancer or brain glioma.