CN106916226B - Application of immunoregulation medicament with CTLA-4 as target spot in treatment of tuberculosis - Google Patents

Abstract

The invention discloses application of an immunoregulation medicament taking CTLA-4 as a target spot in preparing a product for treating diseases caused by mycobacterium tuberculosis. The invention discovers for the first time that the anti-CTLA-4 monoclonal antibody can play a role in treating tuberculosis by removing Tregs, provides a basis for the anti-CTLA-4 monoclonal antibody to be used for treating tuberculosis, and simultaneously discloses that the immunoregulation medicament taking the CTLA-4 as a target has a treatment effect on the tuberculosis.

Description

Application of immunoregulation medicament with CTLA-4 as target spot in treatment of tuberculosis

Technical Field

The invention belongs to the field of biological medicine, relates to application of an immunoregulation medicament taking CTLA-4 as a target spot in preparation of a product for treating diseases caused by mycobacterium tuberculosis, and particularly relates to application of an anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) monoclonal antibody in preparation of a product for treating tuberculosis.

Background

Tuberculosis is a chronic infectious disease caused by Mycobacterium Tuberculosis (MTB), which can invade many organs, and pulmonary tuberculosis infection is the most common. Tuberculosis is caused by chronic and persistent antigen stimulation in vivo due to the fact that the immune response of the body can only inhibit mycobacterium tuberculosis but cannot completely eliminate the mycobacterium tuberculosis. There are a number of immune mechanisms in the body responsible for the clearance of mycobacterium tuberculosis, but the most prominent of these are mycobacterium tuberculosis specific type 1 CD4 positive or CD8 positive T cells, which are capable of secreting interferon-gamma (IFN- γ). An important reason why Mycobacterium tuberculosis cannot be effectively eliminated in tuberculosis patients is that immune regulatory mechanisms in vivo inhibit the function of Mycobacterium tuberculosis specific type 1 CD4 positive or CD8 positive T cells and the production of MTB specific IFN-gamma. Immune regulation mechanisms in vivo include regulatory cytokine IL-10 and transforming growth factor beta 1 (TGF-. beta.1), etc., as well as CD4CD25 double positive regulatory T cells (Tregs). Studies have found that Treg in peripheral blood and tuberculosis sites of tuberculosis patients are obviously increased^[1]Suggesting Treg-mediated immunosuppressionMay be one of the important mechanisms for the pathogenesis of tuberculosis.

Tuberculosis is still an important disease threatening human health, with about several million patients worldwide. BCG vaccine is the main means for preventing tuberculosis, but the treatment effect on adult pulmonary tuberculosis has a plurality of uncertainties^[2]. The adult pulmonary tuberculosis treatment method is few, the treatment course is long, and the drug resistance and the recurrence are easy to generate, so the development of a new tuberculosis treatment drug has important significance.

Cytotoxic T lymphocyte-associated antigen 4(CTLA-4 or CD152) belongs to one of the CD28 family members and is capable of competing with CD28 for binding to CD80 and CD86 molecules on the surface of antigen presenting cells^[3]. CTLA-4 is expressed on the surface of CD4CD25 double-positive regulatory T cells, and the expression level of the CTLA-4 is closely related to the immunosuppressive function of CD4CD25 double-positive regulatory T cells.

Disclosure of Invention

The invention aims to provide application of an immunomodulatory drug taking CTLA-4 as a target in preparing a product for treating diseases caused by mycobacterium tuberculosis.

In the above application, the disease is tuberculosis, preferably pulmonary tuberculosis.

The invention also aims to provide the application of the immune regulation medicament taking CTLA-4 as a target spot in preparing any one of the following products:

(1) a product that eliminates the tregs of CD4CD25 double positive regulatory T cells; and/or

(2) A product that antagonizes the immunosuppressive effects of CD4CD25 double positive regulatory T cell tregs.

In any of the above applications, the immunomodulatory drug is an anti-CTLA-4 antibody.

In the application, the anti-CTLA-4 antibody is an anti-CTLA-4 monoclonal antibody.

In the application, the anti-CTLA-4 monoclonal antibody is a fully human anti-CTLA-4 monoclonal antibody.

In the above applications, the sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the fully human anti-CTLA-4 monoclonal antibody are shown as SEQ ID No.1 from position 46 to 57, 73 to 79 and 112 to 120, respectively, and the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID No.2 from position 45 to 51, 70 to 76 and 118 to 126, respectively.

In any of the above applications, the encoding gene sequences of the CDR1, CDR2, and CDR3 of the light chain variable region of the fully human anti-CTLA-4 monoclonal antibody are shown as 136 th to 171 th, 217 th to 237 th, and 334 th to 360 th positions of SEQ ID No.3, respectively, and the encoding gene sequences of the CDR1, CDR2, and CDR3 of the heavy chain variable region are shown as 133 th to 153 th, 208 th to 228 th, and 352 nd to 378 th positions of SEQ ID No.4, respectively.

In any of the above applications, the amino acid sequence of the light chain variable region of the fully human anti-CTLA-4 monoclonal antibody is shown as SEQ ID No.1 from position 23 to position 130, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.2 from position 20 to position 137.

In the above application, the encoding gene sequence of the light chain variable region is shown as 67 th to 390 th positions of SEQ ID No.3, and the encoding gene sequence of the heavy chain variable region is shown as 58 th to 411 th positions of SEQ ID No. 4.

In any of the above applications, the fully human anti-CTLA-4 monoclonal antibody is IBI310, where IBI310 is composed of 2 light chains and 2 heavy chains, each light chain and each heavy chain are connected by a disulfide bond, and each heavy chain are connected by a disulfide bond; wherein, the amino acid sequence of the light chain is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 2.

In the application, the coding gene sequence of the light chain is shown as SEQ ID No.3, and the coding gene sequence of the heavy chain is shown as SEQ ID No. 4.

The invention mainly researches the therapeutic action of the anti-CTLA-4 monoclonal antibody (IBI310) on the pulmonary tuberculosis through a mouse pulmonary tuberculosis model induced by intravenous injection of the mycobacterium tuberculosis and explores the action mechanism of the anti-CTLA-4 monoclonal antibody. The research result of the invention shows that the anti-CTLA-4 monoclonal antibody (IBI310) can generate the effect of resisting the pulmonary tuberculosis of the mice by eliminating CD4CD25 double-positive regulatory T cells (Tregs) in a pulmonary tuberculosis model of the mice, inhibiting transcription expression of Foxp3 and IL-10 in the Tregs, promoting expression of IL-2 and IFN-gamma, relieving pulmonary lesion induced by mycobacterium tuberculosis and reducing formation of pulmonary granuloma, and the IBI310 is a tuberculosis treatment drug with great prospect.

The invention discovers for the first time that the anti-CTLA-4 monoclonal antibody can play a role in treating tuberculosis by removing Tregs, provides a basis for the CTLA-4 monoclonal antibody to be used for treating tuberculosis, and simultaneously prompts that immunoregulation medicaments taking the CTLA-4 as a target spot are potential tuberculosis treatment medicaments.

Drawings

FIG. 1 shows SDS-PAGE of a Protein of interest after affinity purification of Protein A.

FIG. 2 shows the CEX-HPLC detection result of the target Protein after affinity purification of Protein A.

FIG. 3 shows the SEC-HPLC detection result of the Protein of interest after Protein A affinity purification.

FIG. 4 shows the result of affinity assay of ipilimumab.

FIG. 5 shows the result of affinity detection of the target Protein obtained by Protein A affinity purification of the harvest of 56B3-29F 6.

FIG. 6 shows the result of the splicing of the IBI310 heavy chain sequencing fragment by DNASTAR-Seqman software.

FIG. 7 shows the result of the IBI310 light chain sequencing fragment spliced by DNASTAR-Seqman software.

FIG. 8 shows the effect of IBI310 on the proportion of Treg cells in peripheral blood of mice infected with Mycobacterium tuberculosis (n 10).

FIG. 9 shows the effect of IBI310 on the expression of IL-10, TGF-. beta.1 and FoxP3 in Treg cells in peripheral blood of M.tuberculosis-infected mice (n 10).

FIG. 10 shows the effect of IBI310 on IL-2 and IFN- γ expression in splenocytes from M.tuberculosis-infected mice (n 10).

FIG. 11 shows the effect of IBI310 on pulmonary lesions of M.tuberculosis-infected mice (x 200).

FIG. 12 is a graph showing the effect of IBI310 on pulmonary granuloma index in M.tuberculosis-infected mice.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The reduced SDS-PAGE method used in the following examples was as follows: according to appendix IV C of pharmacopoeia of the people's republic of China (2010 edition, three parts), the purity is detected by adopting reduced SDS-PAGE gel electrophoresis, and the purity of the sample is calculated by an area normalization method.

The charge variant detection (CEX-HPLC) method used in the following examples is as follows: the determination is carried out according to appendix III B of pharmacopoeia of the people's republic of China (2010 edition, three parts), a sample is detected by using a weak cation analytical column, and the purity of acid, alkali and main components of the sample is calculated by an area normalization method.

The size exclusion chromatography (SEC-HPLC) method used in the following examples was as follows: the determination was carried out according to appendix III B of the pharmacopoeia of the people's republic of China (2010 edition, three parts), the samples were tested with a hydrophilic silica gel size exclusion chromatography column, and the purity of the samples was calculated with an area normalization method.

The primary drug for the ipilimumab prescription formulation in the following examples was YERVOY (ipilimumab) Injection, Bristol-Myers Squibb, Application No. 125377; 3/25/2011, wherein the ipilimumab adopted in the following examples is the original grinding medicine; the main component of the imitation drug is recombinant human anti-CTLA-4 monoclonal antibody injection (IBI310 injection for short), the product of the Xindao biopharmaceutical company Limited, the IBI310 injection comprises solute and solvent, the solute is IBI310, sodium citrate, mannitol, arginine, sodium chloride, EDTA-2Na (disodium edetate) and polysorbate 80, and the solvent is water for injection; the concentration of the anti-CTLA-4 monoclonal antibody (IBI310) in the IBI310 injection is 5mg/ml, the concentration of sodium citrate is 5.88mg/ml, the concentration of mannitol is 10.02mg/ml, the concentration of arginine is 4.36mg/ml, the concentration of sodium chloride is 5.84mg/ml, the concentration of EDTA-2Na is 0.037mg/ml, and the concentration of polysorbate 80 is 0.7 mg/ml; the pH was 6.0.

The Chinese hamster ovary cell line subtype S (CHO-S) is Invitrogen, Cat.A 13696-1.

CD Forti CHO Invitrogen, catalog number A11483-01.

Anti-caking Agent is available from Invitrogen under the catalog number 0010057 AE.

MTX (methotrexate) is a product from Calbiochem under the catalog number A6770.

Puro (puromycin) is an Invitrogen company under catalog number A11138-03.

Mycobacterium tuberculosis (Mycobacterium tuberculosis) is ATCC product with the trade name of ATCC 25618.

The human CTLA-4 transgenic mouse (C57BL/6 background, female, 6-8 weeks old, 18-22 g) is a product of Nanjing Yinhe biomedicine GmbH.

Isoniazid is a product of Shanghai Xin Yihe yellow river pharmaceutical Co Ltd, and is made by the national standard H31020495 and the batch 068131001.

Rifampicin is a product of Guangdong south China pharmaceutical industry group, national medicine standard H44020771, and batch number 151001.

The recombinant Heat Shock Protein65 (Heat Shock Protein65 Mycobacterium tuberculosis sRecombinant) is a product of PROSPEC company, and has a catalog number of HSP-065.

Statistical analysis in the following examples single-factor anova with t-test using the sps 13.0for windows software, p <0.05 was considered statistically different.

Example 1 preparation of anti-CTLA-4 monoclonal antibody (IBI310)

The amino acid sequence of IBI310 is identical to that of ipilimumab (ipilimumab).

First, vector construction

According to Freedom^TMCHO-S^TMkit instructions for insertion of heavy chain gene (SEQ ID No.4) and light chain gene (SEQ ID No.3) of IBI310 into expression vector Freedom, respectively^TMpCHO1.0(Freedom^TMCHO-S^TMkit, GIBCO product, catalog number A13696-01) and a second gene expression cassette from the same plasmid to construct the IBI310 antibody expression plasmid pCHO1.0-IHEKR.

Second, expression plasmid transfection of host cells

According to Free Style^TMpMAX Reagent instructions, by Invitrogen chemical transfection Reagent (FreeStyle)^TMpMAX Reagent, cat No.: 16447-100) the transfection was accomplished by transferring the IBI310 antibody expression plasmid pCHO1.0-IHEKR into CHO-S host cells.

Thirdly, pressure screening after transfection

6 bottles of cell suspension after 48 hours of transfection were filtered and separately dispensed into two square bottles. The cells were cultured in an incubator by static culture using a selection medium (Table 1) containing Puro and MTX, and the cell viability was measured after 7 days, and the first and second-stage pressure selection was carried out based on the cell viability value.

TABLE 1 media Components Table

Fourth, cell population yield detection

And (3) screening the high-yield cell populations by adopting a 6-well plate 5-day static culture method for the 12 cell populations obtained in the first stage and the second stage respectively. The expression level of the antibody in the supernatant was determined by Fortebio assay. The method comprises the steps of detecting standard samples with different concentrations through a Protein A sensor, establishing a standard curve to calculate the concentration of a sample to be detected, and selecting a cell population YY 310-210/100-50/1000 with the highest antibody expression quantity.

Five, positive monoclonal screening

The selected cell population YY 310-210/100-50/1000 was monocloned using limiting dilution. The screening of high-producing clones was performed in two stages, 96-well plate and 6-well plate, using cloning medium (Table 1). According to the screening result of the 6-well plate, the 12 clones with the highest antibody expression level are amplified and cultured and subjected to sugar supplement experiments, and the cell strain 56B3 with relatively high antibody expression level is obtained.

Sixth, screening of subcloned cell lines

To ensure the monoclonality of the cells used to construct the master cell bank, cell line 56B3, which had good yield and stability within the IBI310 primary clone cell bank, was subcloned. The subcloning method is also limited dilution method, and 3 high-yield subclones are screened through two stages of 96-well plate and 6-well plate: 56B3-25D11, 56B3-26D 5and 56B3-29F6, an original subclone cell bank consisting of the 3 subclones was established.

Seventhly, determining cell strains for final production

The target Protein is obtained by affinity purification of Protein A from harvest liquid of three subclones 56B3-25D11, 56B3-26D 5and 56B3-29F 6. The results of detection by SDS-PAGE using ipilimumab as a control are shown in FIG. 1.

In FIG. 1, M is protein Marker (NEB product, cat # P7703); ST is ipilimumab; 25D11, 26D 5and 29F6 represent target proteins obtained by affinity purification of Protein A from harvest liquid of 56B3-25D11, 56B3-26D 5and 56B3-29F6, respectively.

FIG. 1 shows that the subcloned cell lines 56B3-25D11, 56B3-26D 5and 56B3-29F6 express proteins with the same relative molecular weight and purity as those of ipilimumab.

Using ipilimumab as a control, charge isomerism of the target Protein obtained by Protein A affinity purification of the harvest of each subclone was detected by CEX-HPLC, and the results are shown in FIG. 2.

In FIG. 2, subclones 25D11, 26D5, and 29F6 represent proteins of interest obtained by affinity purification of Protein A from the harvest of 56B3-25D11, 56B3-26D5, and 56B3-29F6, respectively.

FIG. 2 shows that the target proteins expressed by the subcloned cell strains 56B3-25D11, 56B3-26D 5and 56B3-29F6 have consistent main peaks compared with ipilimumab.

The SEC-HPLC method with ipilimumab as a control was used to detect the purity of the target Protein obtained by Protein A affinity purification of the harvest of each subclone, and the results are shown in FIG. 3.

In FIG. 3, subclones 25D11, 26D5, and 29F6 represent proteins of interest obtained by affinity purification of Protein A from the harvest of 56B3-25D11, 56B3-26D5, and 56B3-29F6, respectively.

FIG. 3 shows that the retention time of the target protein expressed by the subcloned cell strains 56B3-25D11, 56B3-26D 5and 56B3-29F6 is consistent with that of ipilimumab, and the purity is higher.

The results of affinity detection of the target Protein obtained by Protein A affinity purification of the harvest solution of 56B3-29F6 using ipilimumab as a control are shown in FIGS. 4 and 5.

FIGS. 4 and 5 show that subclone 56B3-29F6 expresses a protein of interest with an affinity similar to that of ipilimumab.

The stability test of 3 subcloned cell strains 56B3-25D11, 56B3-26D 5and 56B3-29F6 for 60 days shows that: the antibody yield of the 56B3-29F6 cell line is not reduced, and the antibody yield of the two cell lines of 56B3-25D11 and 56B3-26D5 is reduced. Finally 56B3-29F6 was selected as the IBI310 subclone.

Eighth, identification of original cell lines

Sequencing the target gene in the genome of the original cell strain 56B3-29F6, identifying the expression product, and determining the molecular weight by mass spectrum. The method of sequencing the PCR products was used for both heavy and light chains.

After the IBI310 heavy chain sequencing fragment is spliced by DNASTAR-Seqman software (shown in figure 6), a gene sequence HC Seqman is obtained, and the sequence is subjected to Blast comparison with a theoretical sequence of an ipilimumab heavy chain target gene, and the result shows that the two sequences are completely consistent.

After the IBI310 light chain sequencing fragment is spliced by DNASTAR-Seqman software (shown in FIG. 7), a gene sequence LC Seqman is obtained, and the sequence is subjected to Blast comparison with a theoretical sequence of an ipilimumab light chain target gene, and the result shows that the sequence and the theoretical sequence are completely consistent.

Ninth, identification of cell expression products

Cell line 56B3-29F6 was fed-batch cultured in a shake flask for 14 days. After the harvest liquid is subjected to Protein A affinity purification, the molecular weight of a sample is detected by liquid chromatography-mass spectrometry, and the molecular weight of IBI310 is completely the same as that of ipilimumab, as shown in Table 2.

TABLE 2 molecular weight comparison of ipilimumab to IBI310

The anti-CTLA-4 monoclonal antibody (IBI310) consists of 2 light chains and 2 heavy chains, wherein one light chain and one heavy chain are connected through a disulfide bond, and the heavy chain are connected through a disulfide bond; the amino acid sequence of the light chain is shown as SEQ ID No.1, and the amino acid sequence of the heavy chain is shown as SEQ ID No. 2; the amino acid sequence of the light chain variable region is shown as 23 rd to 130 th positions of SEQ ID No.1, and the amino acid sequence of the heavy chain variable region is shown as 20 th to 137 th positions of SEQ ID No. 2; the CDR1, CDR2 and CDR3 sequences of the light chain variable region are shown in the 46 th to 57 th, 73 th to 79 th and 112 th to 120 th positions of SEQ ID No.1, respectively, and the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region are shown in the 45 th to 51 th, 70 th to 76 th and 118 th to 126 th positions of SEQ ID No.2, respectively.

The coding gene sequence of the light chain of the monoclonal antibody is shown as SEQ ID No.3, and the coding gene sequence of the heavy chain is shown as SEQ ID No. 4; the encoding gene sequence of the light chain variable region is shown as 67 th to 390 th sites of SEQ ID No.3, and the encoding gene sequence of the heavy chain variable region is shown as 58 th to 411 th sites of SEQ ID No. 4; the encoding gene sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as positions 136 to 171, 217 to 237 and 334 to 360 of SEQ ID No.3, respectively, and the encoding gene sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as positions 133 to 153, 208 to 228 and 352 to 378 of SEQ ID No.4, respectively.

Example 2 therapeutic Effect of IBI310 on M.tuberculosis-induced pulmonary tuberculosis models in mice

Firstly, dividing a human CTLA-4 transgenic mouse (C57BL/6 mouse background, the extracellular end of CTLA-4 protein is replaced by an amino acid sequence of a human) into an IBI31010mg/kg group, a model control group and a positive control group at random, establishing a normal control group of the human CTLA-4 transgenic mouse, and treating each group with 10 mice (SPF grade, 6-8 weeks old, 18-22 g) as follows:

IBI31010mg/kg group: mice were given Mycobacterium tuberculosis (Mycobacterium tuberculosis) 5X 10 by intravenous injection⁵Colony Forming Units (CFU)/one mouse is used for establishing a mouse pulmonary tuberculosis model, and the model success can be judged by in vitro culture of lung tissue homogenate with the growth of mycobacterium tuberculosis and lung pathology with the formation of inflammatory infiltration and granuloma. The mice were treated by intraperitoneal injection of IBI310 injection (IBI310 injection is diluted to 1mg/ml with 0.9% physiological saline and used on the day of preparation) starting from the day of infection at a dose of 10mg/kg for 1 time per week for 10 consecutive weeks.

Normal control group: human CTLA-4 transgenic mice were administered without M.tuberculosis in the same manner as IBI31010mg/kg, except that the IBI310 injection was replaced with an equal volume of saline.

Model control group: the establishment and identification of the mouse pulmonary tuberculosis model are the same as those of the IBI31010mg/kg group. The administration method was the same as that of the group IBI31010mg/kg, except that the IBI310 injection solution was replaced with physiological saline.

Positive control group (isoniazid + rifampin group): the establishment and identification of the mouse pulmonary tuberculosis model are the same as those of the IBI31010mg/kg group. The mice are treated by gavage administration of 60mg/kg of isoniazid and 120mg/kg of rifampicin from the same day of contamination, the administration volumes are respectively 10ml/kg, the administration is carried out 1 time per day, and the administration is continuously carried out for 10 weeks.

Second, the Effect of IBI310 on the proportion of Treg cells in peripheral blood

Peripheral blood of mice of IBI31010mg/kg group, normal control group, model control group and positive control group are respectively collected after the last administration, and Treg cells are separated. The general process is as follows: double positive Treg separating reagent (Dynal CD4CD 25)

FlowComp Mouse CD4+ CD25+ Treg Cells, Invitrogen, Cat. 11463D) isolated and enriched Tregs from peripheral blood mononuclear Cells, CD 4-positive T Cells isolated from peripheral blood mononuclear Cells using anti-CD 14, anti-CD 56, anti-CD 19, anti-CD 8 and anti-CD 235 α antibody cocktails and depleted beads, purified CD 4-positive T Cells incubated with CD25 beads to enrich Tregs in the CellsAnd collecting the Tregs after elution on the surface of the magnetic beads (see the use instruction for the detailed process). The proportion of CD4CD25 double positive regulatory T cells (tregs), which is (number of Treg cells/number of CD4 positive T cells) × 100%, was analyzed by flow cytometry.

The results are shown in FIG. 8. In FIG. 8, # p <0.05, compared to the normal control group; p <0.05, compared to model control group.

The result shows that the proportion of Treg cells in peripheral blood of a mouse infected by mycobacterium tuberculosis is obviously increased after 10 weeks, and the proportion of Treg cells in peripheral blood of a mouse treated by IBI310 (namely a mouse in IBI31010mg/kg group) is obviously lower than that of a model control group, which indicates that the IBI310 can eliminate Tregs. The positive control drug isoniazid combined with rifampicin has no obvious influence on Treg in peripheral blood.

Third, the effect of IBI310 on the expression of IL-10, TGF-beta 1 and FoxP3 in Tregs in peripheral blood of mice infected with Mycobacterium tuberculosis

And (I) separating and purifying groups of Tregs in the same step II.

(II) measurement of expression of IL-10, TGF-. beta.1 and FoxP3 by Q-RT-PCR

1. Using RNA extraction and reverse transcription kit (Power)

Green Cells-to-CT^TMKit, thermo fisher, catalog No. 4402953) extracted RNA from each of the collected sets of tregs, and the extracted RNA samples were further treated with rnase-free dnase i to eliminate DNA contamination.

2. In reverse transcription buffer, RNA is reverse transcribed with random primers to obtain cDNA.

3. And (3) carrying out PCR amplification by taking each cDNA as a template and respectively taking an IL-10F primer pair and an IL-10R primer pair, a TGF-beta 1F primer pair and a TGF-beta 1R primer pair and a FoxP3F primer pair and a FoxP3R primer pair to respectively obtain IL-10, TGF-beta 1 and FoxP3 specific amplification products. Meanwhile, the reference gene GAPDH is subjected to PCR amplification by taking GAPDHF and GAPDHR as primer pairs. The amounts of the amplification products of IL-10, TGF-. beta.1 and FoxP3 were corrected by the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and then the expression amount of the normal control group was defined as 100%, and the relative expression amounts of each cytokine were calculated as: the relative expression amount of factor X was [ each group except the normal control group (factor X signal value/GAPDH signal value)/normal control group (factor X signal value/GAPDH signal value) ], and finally statistical analysis was performed.

Specific primers are shown in table 3.

TABLE 3

The results are shown in FIG. 9. In FIG. 9, # p <0.05, # p <0.01, compared to the normal control group; p <0.05, compared to model control group.

The results show that mRNA of IL-10 and FoxP3 in Treg in peripheral blood of mice infected with Mycobacterium tuberculosis is obviously increased after 10 weeks, and mRNA of TGF-beta 1 is also increased to a certain extent, but has no statistical significance difference compared with a normal control group. IBI310 was able to significantly reduce IL-10 and FoxP3 mRNA levels after 10 weeks of treatment, but had no significant effect on TGF-. beta.1 mRNA levels. The positive control drug isoniazid combined with rifampicin had no significant effect on the mRNA levels of IL-10, TGF- β 1 and FoxP3 of tregs in peripheral blood.

Fourth, the effect of IBI310 on IL-2 and IFN-gamma expression in splenocytes from mice infected with Mycobacterium tuberculosis

Spleens were isolated immediately after sacrifice of each group of animals following the last dose, and single cell suspensions were prepared and plated in 96-well cell culture plates containing 5X 10 cells per well⁶The cells were simultaneously added with recombinant heat shock protein65 to a final concentration of 20. mu.g/ml, 3 duplicate wells were set for each animal sample, and after 48 hours of the action, supernatants were collected and assayed for IL-2 and IFN-. gamma.levels by ELISA (Mouse IL-2ELISA Kit (Interleukin-2) Abcam product, cat # ab 100706; Mouse Interferon gamma ELISA Kit (IFNG) Abcam product, cat # ab100690) according to the protocol of the instructions.

The IL-2 was roughly detected as follows: (1) coating a 96-well plate with a mouse IL-2 specific antibody, and standing overnight at 4 ℃; (2) adding a sample to be detected and a standard substance, and incubating overnight at 4 ℃; (3) after full washing, adding a biotinylated anti-mouse IL-2 antibody, and incubating for 2h at room temperature; (4) after full washing, adding horseradish peroxidase coupled streptavidin, and incubating for 1h at room temperature; (5) after full washing, adding a TMB substrate, and reading an absorbance value at 450nm by using an enzyme-linked immunosorbent assay (ELISA) reader after developing; (6) the IL-2 concentration was calculated from a standard curve.

IFN-gamma was detected by the following approximate method: (1) coating a 96-well plate with a mouse IFN-gamma specific antibody, and standing overnight at 4 ℃; (2) adding a sample to be detected and a standard substance, and incubating overnight at 4 ℃; (3) after full washing, adding a biotinylated anti-mouse IFN-gamma antibody, and incubating for 2h at room temperature; (4) after full washing, adding horseradish peroxidase coupled streptavidin, and incubating for 1h at room temperature; (5) after full washing, adding a TMB substrate, and reading an absorbance value at 450nm by using an enzyme-linked immunosorbent assay (ELISA) reader after developing; (6) IFN-. gamma.concentrations were calculated according to the standard curve.

The results are shown in FIG. 10. In fig. 10, p <0.001, compared to the model control group.

The result shows that the splenocytes of the mice in the normal control group can express a small amount of IL-2 and IFN-gamma under the stimulation of the heat shock protein 65; although the spleen cells of the mice infected with mycobacterium tuberculosis are subjected to double stimulation by mycobacterium tuberculosis and heat shock protein65 in vivo and in vitro respectively, the expression level of IL-2 and IFN-gamma is still not high probably due to the influence of Treg; however, after the mycobacterium tuberculosis infected mice receive IBI310 treatment for 10 weeks, the expression level of IL-2 and IFN-gamma is obviously increased, and the IBI310 is suggested to promote the reaction of organisms to foreign antigens by eliminating the inhibition effect of Tregs. The positive control drug isoniazid in combination with rifampicin had no significant effect on the levels of IL-2 and IFN- γ in splenocytes.

Fifth, the effect of IBI310 on pulmonary lesions and granuloma index of mice infected with Mycobacterium tuberculosis

The lungs of each group of mice were isolated after the last administration, weighed, fixed and sectioned, HE stained, microscopically observed and calculated for the lung granuloma index (lung weight/animal weight). times.250 (HE stained see Yoshida S, Tanaka T, Kita Y, et al. DNA variation using a synergistic differentiation virus of Japan-lipid encapsulation synthesis of Mycobacterium platelet protein65and interleukin-12 binding protein expression. vaccine,24: 1191-.

HE staining results are shown in fig. 11.

The results of the granuloma index are shown in fig. 12. In FIG. 12, # # p <0.01, compared to the normal control group; p <0.01, compared to model control group.

The pathological detection results show that the lung tissue structure of a normal mouse is regular, the cell arrangement is regular, the layers are clear, and inflammatory cell infiltration is not seen; the pulmonary structure of the mycobacterium tuberculosis infected mouse is disordered, the cell arrangement is irregular, the hierarchical sense is avoided, and a large amount of inflammatory cell infiltration, wide interstitial injury and granuloma formation exist; after the infected mice are treated by IBI310 for 10 weeks, the pathological injury of the lung is obviously reduced, the infiltration of inflammatory cells is obviously reduced, and the formation of granuloma is obviously reduced; after the positive control drug isoniazid is combined with rifampicin to treat, pathological damage of the lung is obviously reduced, inflammatory cell infiltration is obviously reduced, granuloma formation is obviously reduced, and the action effect is similar to that of IBI 310.

The research result of the invention shows that the anti-CTLA-4 monoclonal antibody (IBI310) can generate the effect of resisting the pulmonary tuberculosis of the mice by removing CD4CD25 double-positive regulatory T cells (Treg) in a pulmonary tuberculosis model of the mice, inhibiting Foxp3 and IL-10 transcription expression in the Treg, promoting IL-2 and IFN-gamma expression, relieving pulmonary lesion induced by mycobacterium tuberculosis and reducing pulmonary granuloma formation, thereby prompting that the IBI310 is a very promising tuberculosis treatment drug, and simultaneously prompting that an immunoregulation drug taking the CTLA-4 as a target spot is a potential tuberculosis treatment drug.

Reference documents:

1、Guyot-Revol V,Innes JA,Hackforth S,Hinks T,Lalvani A.Regulatory Tcells are expanded in blood and disease sites in patients withtuberculosis.Am J Respir Crit Care Med,173:803-810,2006.

2、Sugawara I,Sun L,Mizuno S,Taniyama T.Protective efficacy ofrecombinant BCG Tokyo(Ag85A)in rhesus monkeys(Macaca mulatta)infectedintratracheally with H37Rv Mycobacterium tuberculosis.Tuberculosis,89:62-67,2009.

3、Wu,L.,Z.Yun,T.Tagawa,K.Rey-McIntyre and M.d.Perrot.CTLA-4 BlockadeExpands Infiltrating T Cells and Inhibits Cancer Cell Repopulation during theIntervals of Chemotherapy in Murine Mesothelioma.Mol Cancer Ther,11(8):1809-1819,2012。

sequence listing

<110> Xinda biopharmaceuticals (Suzhou) Limited

<120> application of immunoregulation medicine using CTLA-4 as target point in tuberculosis treatment

<160>12

<210>1

<211>237

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>1

Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

20 25 30

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

35 40 45

Gln Ser Val Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly

65 70 75 80

Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

100 105 110

Gln Tyr Gly Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

115 120 125

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210>2

<211>467

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile

100 105 110

Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210>3

<211>717

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

atggacttcc aggtgcagat catctccttc ctgctgatca gcgcctccgt gattatgagc 60

cggggcgaga tcgtgctgac ccaaagccct ggcacactca gcctgtcccc tggagagcgg 120

gctacactgt cctgcagggc cagccagtcc gtgggatcct cctacctggc ctggtaccag 180

cagaaacccg gccaggctcc caggctgctg atctatggcg ctttctccag ggccaccgga 240

atccccgata ggttcagcgg ctccggaagc ggaaccgact tcaccctgac catcagcagg 300

ctggagcccg aggactttgc cgtgtattac tgccagcagt acggatcctc cccctggaca360

ttcggccagg gcacaaaggt ggagatcaag aggaccgtcg ccgccccctc cgtctttatc 420

ttccccccct ccgacgagca actgaagagc ggcacagcct ccgtggtctg cctcctgaac 480

aacttctacc ccagggaggc caaggtccag tggaaagtgg acaacgccct gcagtccggc 540

aactcccagg aaagcgtcac cgagcaggac tccaaggact ccacatacag cctgtccagc 600

accctgaccc tcagcaaggc cgattacgag aagcacaagg tgtacgcctg cgaggtcaca 660

caccagggcc tgtcctcccc cgtcaccaag agctttaacc ggggcgagtg ctgatga 717

<210>4

<211>1407

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>4

atgggctggt ccctgatcct cctcttcctg gtggctgtcg ccacaagggt cctgagccag 60

gtgcagctcg tcgaatccgg aggaggagtc gtgcagcctg gcaggtccct caggctgagc 120

tgtgctgcct ccggcttcac cttcagctcc tacaccatgc attgggtgcg gcaggctcct 180

ggcaaaggcc tcgaatgggt caccttcatc agctacgatg gcaacaacaa gtattacgcc 240

gacagcgtga agggcaggtt caccatctcc cgggacaaca gcaagaacac cctctacctc 300

cagatgaaca gcctgagggc tgaggacacc gccatttatt actgcgctcg gaccggatgg 360

ctcggccctt ttgattactg gggccaaggc acactggtga ccgtgagcag cgcctccacc 420

aagggaccca gcgtgttccc tctggctccc agctccaagt ccacaagcgg cggaacagct 480

gctctgggat gcctggtcaa ggactatttc cctgagcccg tgacagtgag ctggaactcc 540

ggagccctga ccagcggagt gcatacattc cccgccgtcc tccagagctc cggactctac 600

tccctgtcct ccgtggtgac cgtgcctagc agctccctcg gcacacagac ctatatctgt 660

aacgtgaacc acaagccctc caacaccaag gtggacaaaa gagtcgagcc caagagctgc 720

gacaagaccc acacctgccc tccctgtcct gctcctgaac tgctgggcgg acccagcgtc 780

ttcctgttcc ctcccaaacc caaggatacc ctgatgatct ccaggacccc tgaggtgacc 840

tgcgtggtcg tggacgtgtc ccacgaggat cccgaagtga agttcaactg gtacgtggac 900

ggagtggaag tccataacgc caagaccaag ccccgggagg agcagtacaa ctccacatat 960

agggtcgtgt ccgtgctcac cgtcctgcat caggactggc tcaacggcaa ggagtacaaa 1020

tgcaaggtca gcaacaaagc tctccctgcc cccatcgaga agaccatcag caaggctaaa 1080

ggccagcccc gggaacctca agtctacacc ctgccccctt ccagggatga gctgaccaag 1140

aaccaggtca gcctcacctg tctggtgaag ggcttctacc cttccgacat cgctgtggag 1200

tgggagtcca acggccagcc cgagaacaac tataagacca caccccccgt gctggactcc 1260

gatggcagct tcttcctgta ctccaagctg acagtggata agtccaggtg gcagcagggc 1320

aacgtgttct cctgcagcgt gatgcacgag gccctccaca atcactatac ccaaaagtcc 1380

ctctccctga gccctggcaa atgatga 1407

<210>5

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

ctggacaaca tactgctaac cgac 24

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>6

ttcattcatg gccttgtaga cacc 24

<210>7

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>7

agacggaata cagggctttc gattca 26

<210>8

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>8

cttgggcttg cgacccacgt agta 24

<210>9

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>9

gaaacagcac attcccagag ttc 23

<210>10

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>10

atggcccagc ggatgag 17

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>11

ccacatcgct cagacaccat 20

<210>12

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>12

ggcaacaata tccactttac cagagt 26

Claims (4)

1. The application of the fully human anti-CTLA-4 monoclonal antibody in preparing products for treating diseases caused by mycobacterium tuberculosis;

the light chain amino acid sequence of the fully human anti-CTLA-4 monoclonal antibody is shown as SEQ ID No.1, and the heavy chain amino acid sequence is shown as SEQ ID No. 2.

2. Use according to claim 1, characterized in that: the disease is tuberculosis.

3. Use according to claim 2, characterized in that: the tuberculosis is pulmonary tuberculosis.

4. Use of a fully human anti-CTLA-4 monoclonal antibody in the manufacture of a product for eliminating CD4CD25 double positive regulatory T cell Treg and/or antagonizing the immunosuppressive effects of CD4CD25 double positive regulatory T cell Treg;

the light chain amino acid sequence of the fully human anti-CTLA-4 monoclonal antibody is shown as SEQ ID No.1, and the heavy chain amino acid sequence is shown as SEQ ID No. 2.

Images