CN106916226A - Immunoregulation medicament with CTLA 4 as target spot treats application lungy - Google Patents

Abstract

The invention discloses application of the immunoregulation medicament with CTLA 4 as target spot in the product for preparing the disease that treatment is caused by mycobacterium tuberculosis.Present invention firstly discovers that the monoclonal antibodies of anti-CTLA 4 can play the phthisical effect for the treatment of by removing Treg, foundation is provided for the monoclonal antibodies of anti-CTLA 4 are used for treatment lungy, while the immunoregulation medicament disclosed with CTLA 4 as target spot has therapeutic action to tuberculosis.

Description

Immunoregulation medicament with CTLA-4 as target spot treats application lungy

Technical field

The invention belongs to biomedicine field, it is related to the immunoregulation medicament with CTLA-4 as target spot preparing treatment by tying Application in the product of the disease that core mycobacteria causes, and in particular to anti-cell Cytotoxic T lymphocytes related antigen 4 (CTLA- 4) application of the monoclonal antibody in treatment product lungy is prepared.

Background technology

Tuberculosis is the chronic infectious disease caused by mycobacterium tuberculosis (MTB), can be invaded and many internal organs, with pulmonary Mycobacterium Infection is most commonly seen.Tuberculosis be because the immune response of body can only suppress mycobacterium tuberculosis, but but can not be by it It is fully erased, cause to there is chronic, lasting antigenic stimulus in vivo and cause.There is panimmunity mechanism is responsible for removing in vivo Mycobacterium tuberculosis, but wherein it is most importantly 1 type CD4 of the mycobacterium tuberculosis specificity positives or CD8 positive T cells, This kind of cell can secrete interferon-γ (IFN-γ).What the mycobacterium tuberculosis in tuberculosis patient body can not be removed efficiently One major reason is that internal immune regulation mechanism inhibits 1 type CD4 of mycobacterium tuberculosis specificity positive or CD8 is positive The generation of function and MTB the specificity IFN-γ of T cell.Internal immune regulation mechanism includes regulatory cell factor IL-10 With transforminggrowthfactor-β1 (TGF-β 1) etc. and the double positive regulatory T cells (Treg) of CD4CD25.Research has been found that tuberculosis The peripheral blood of patient and the Treg at tuberculosis position substantially increase^[1], the immunosupress for pointing out Treg mediations is probably Tuberculosis One of important mechanisms.

Tuberculosis is still at present the class important diseases for threatening human health, the about millions of patient in the whole world. BCG vaccine is prevention Main Means lungy, but is also existed to the phthisical therapeutic effect of adult many uncertain Property^[2].Adult pulmonary tuberculosis treatment method is less, and the course for the treatment of is long, easily produces drug resistance and recurrence, therefore develops new tuberculosis and controls Treat medicine significant.

Cytotoxic t lymphocyte-associated antigen 4 (CTLA-4 or CD152) belongs to one of CD28 family members, can With CD80 the and CD86 molecules on CD28 competitive binding antigen presenting cells surface^[3].It is double positive that CTLA-4 is expressed in CD4CD25 Regulatory T cells surface, its expression is closely related with the immune suppression function of the double positive regulatory T cells of CD4CD25.

The content of the invention

The immunoregulation medicament that an object of the present invention is to provide with CTLA-4 as target spot is preparing treatment by tuberculosis point Application in the product of the branch microbial disease of bar.

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

The immunoregulation medicament that the second object of the present invention is to provide with CTLA-4 as target spot is following any shown in preparation Product in application：

(1) product of the double positive regulatory T cells Treg of CD4CD25 is removed；And/or

(2) product of the immunosuppressive action of the double positive regulatory T cells Treg of antagonism CD4CD25.

In any of the above-described described application, the immunoregulation medicament is anti-CTLA-4 antibody.

In above-mentioned application, the anti-CTLA-4 antibody is anti-CTLA-4 monoclonal antibodies.

In above-mentioned application, the anti-CTLA-4 monoclonal antibodies are the anti-CTLA-4 monoclonal antibodies in full people source.

In above-mentioned application, CDR1, CDR2, CDR3 sequence of the light chain variable district of the complete anti-CTLA-4 monoclonal antibodies in people source Row respectively such as SEQ ID No.1 the 46th to the 57th, the 73rd to the 79th, shown in the 112nd to the 120th, heavy chain CDR1, CDR2, CDR3 of variable region respectively as the 45th to the 51st of SEQ ID No.2, the 70th to the 76th, the 118th Position is to shown in the 126th.

In any of the above-described described application, the CDR1 of the light chain variable district of the complete anti-CTLA-4 monoclonal antibodies in people source, The coding gene sequence of CDR2, CDR3 respectively as the 136th to the 171st of SEQ ID No.3, the 217th to the 237th, Shown in 334th to the 360th, the coding gene sequence of CDR1, CDR2, CDR3 of weight chain variable district is respectively such as SEQ ID The 133rd to the 153rd of No.4, the 208th to the 228th, shown in the 352nd to the 378th.

In any of the above-described described application, the amino acid of the light chain variable district of the complete anti-CTLA-4 monoclonal antibodies in people source Sequence as shown in the 23rd to the 130th of SEQ ID No.1, the amino acid sequence such as SEQ ID No.2 of weight chain variable district Shown in 20th to the 137th.

In above-mentioned application, the 67th to the 390th of the coding gene sequence such as SEQ ID No.3 of the light chain variable district Shown, the coding gene sequence of the weight chain variable district is as shown in the 58th to the 411st of SEQ ID No.4.

In any of the above-described described application, the complete anti-CTLA-4 monoclonal antibodies in people source are IBI310, and IBI310 is by 2 Light chain and 2 heavy chain compositions, by disulfide bond between a light chain and a heavy chain, pass through two between heavy chain and heavy chain Sulfide linkage is connected；Wherein, the amino acid sequence of light chain is as shown in SEQ ID No.1, the amino acid sequence such as SEQ ID No.2 of heavy chain It is shown.

In above-mentioned application, the coding gene sequence of the light chain as shown in SEQ ID No.3, the encoding gene of the heavy chain Sequence is as shown in SEQ ID No.4.

The main mouse lung tuberculosis model induced by being injected intravenously mycobacterium tuberculosis of the invention has investigated anti-CTLA- Therapeutic action of 4 monoclonal antibodies (IBI310) to pulmonary tuberculosis, and explore its mechanism of action.Result of study table of the invention Bright, anti-CTLA-4 monoclonal antibodies (IBI310) can be by removing the double positive tune of the CD4CD25 in mouse lung tuberculosis model Section property T cell (Treg), the transcriptional expression for suppressing Foxp3 and IL-10 in Treg, promotion IL-2 and IFN-γ expression, mitigation knot The lung lesion of core mycobacteria induction produces the effect of anti-mouse pulmonary tuberculosis with lungs Granuloma formation is reduced, and points out IBI310 is very promising tuberculotherapy medicine.

Present invention firstly discovers that anti-CTLA-4 monoclonal antibodies can play the phthisical work for the treatment of by removing Treg With for CTLA-4 monoclonal antibodies provide foundation for treatment lungy, while immune tune of the prompting with CTLA-4 as target spot Section medicine is the potential tuberculotherapy medicine of a class.

Brief description of the drawings

Fig. 1 is the SDS-PAGE testing results of destination protein after Protein A affinity purifications.

Fig. 2 is the CEX-HPLC testing results of destination protein after Protein A affinity purifications.

Fig. 3 is the SEC-HPLC testing results of destination protein after Protein A affinity purifications.

Fig. 4 is the affinity testing result of her monoclonal antibody.

The affinity detection knot of the destination protein that Fig. 5 is obtained for the harvest liquid of 56B3-29F6 through Protein A affinity purifications Really.

Fig. 6 is the result that IBI310 heavy chains sequencing fragment is spliced through DNASTAR-Seqman softwares.

Fig. 7 is the result that IBI310 light chains sequencing fragment is spliced through DNASTAR-Seqman softwares.

Fig. 8 is influences (n=10) of the IBI310 to mycobacterium tuberculosis exposed Mice peripheral blood T reg cell proportions.

Fig. 9 is IBI310 to IL-10, the and of TGF-β 1 in mycobacterium tuberculosis exposed Mice peripheral blood T reg cells The influence (n=10) of FoxP3 expression.

Figure 10 is the influence (n=that IBI310 is expressed IL-2 in mycobacterium tuberculosis exposed Mice splenocyte and IFN-γ 10)。

Figure 11 is influences (× 200) of the IBI310 to mycobacterium tuberculosis exposed Mice lung lesion.

Figure 12 is influences of the IBI310 to mycobacterium tuberculosis exposed Mice lungs granuloma index.

Specific embodiment

Experimental technique used in following embodiments is conventional method unless otherwise specified.

Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.

Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate this Invention is not for restriction the scope of the present invention.

Reduced form SDS-PAGE methods employed in following embodiments are as follows：Press《Pharmacopoeia of People's Republic of China》 (version in 2010, three) C of annex IV, using reduced form PAGE gel electrophoresis detection purity, area normalization method calculates sample Product purity.

It is as follows that (CEX-HPLC) method is surveyed in charge alterations physical examination employed in following embodiments：According to《The Chinese people are total to With state's pharmacopeia》(version in 2010, three) B of annex III is measured, and detects that area is returned to sample with weak cation analytical column One changes method calculates sample acid, alkali and principal component purity.

Molecular-exclusion chromatography (SEC-HPLC) method employed in following embodiments is as follows：According to《Chinese people's republicanism State's pharmacopeia》(version in 2010, three) B of annex III is measured, and sample is detected with hydrophilic silica gels size exclusion chromatograph post, Sample purity is calculated with area normalization method.

The original of square preparation grinds medicine for YERVOY (ipilimumab) Injection at her monoclonal antibody in following embodiments, Bristol-Myers Squibb companies, Application No.:125377；Approval Date:3/25/2011, it is following Her monoclonal antibody used in embodiment is for the original grinds medicine；Its imitation medicine is the complete anti-CTLA-4 monoclonal antibody injections liquid in people source of restructuring (abbreviation IBI310 parenteral solutions), Innovent Biologics's product, its main component is IBI310, the IBI310 Parenteral solution is made up of solute and solvent, and solute is IBI310, sodium citrate, mannitol, arginine, sodium chloride, EDTA-2Na (second Edetate disodium) and polyoxyethylene sorbitan monoleate, solvent is water for injection；Anti- CTLA-4 monoclonal antibodies (IBI310) are at this Concentration in IBI310 parenteral solutions is 5mg/ml, and sodium citrate is 5.88mg/ml, and mannitol is 10.02mg/ml, and arginine is 4.36mg/ml, sodium chloride is 5.84mg/ml, and EDTA-2Na is 0.037mg/ml, and polyoxyethylene sorbitan monoleate is 0.7mg/ml；PH is 6.0。

Chinese hamster ovary line S hypotypes (CHO-S) is Invitrogen Products, and catalog number is A13696-1。

CD Forti CHO are Invitrogen Products, and catalog number is A11483-01.

Anti-Clumping Agent (anticaking agent) are Invitrogen Products, and catalog number is 0010057AE。

MTX (methotrexate) is Calbiochem Products, and catalog number is A6770.

Puro (puromycin) is Invitrogen Products, and catalog number is A11138-03.

Mycobacterium tuberculosis (Mycobacterium tuberculosis) is ATCC products, and article No. is ATCC 25618.

People CTLA-4 transgenic mices (C57BL/6 backgrounds, female, 6~8 week old, 18~22g) are the biological doctor of the Nanjing milky way Medicine Co., Ltd product.

Isoniazid is upper Hisense's friendship the Yellow River pharmaceutical Co. Ltd product, Chinese medicines quasi-word H31020495, lot number 068131001.

Rifampin is GuangDong HuaNan Pharmacy Group Co., Ltd's product, Chinese medicines quasi-word H44020771, lot number 151001.

Recombinant Heat shock protein 65kD (Heat Shock Protein 65Mycobacterium Tuberculosis Recombinant it is) PROSPEC Products, catalog number (Cat.No.) is HSP-065.

Statistical analysis in following embodiments carries out one-way analysis of variance using spss 13.0for windows softwares With reference to t inspections, p<0.05 thinks with significant difference.

The preparation of embodiment 1, anti-CTLA-4 monoclonal antibodies (IBI310)

The amino acid sequence of IBI310 is identical with the amino acid sequence of her monoclonal antibody (ipilimumab).

First, vector construction

According to Freedom^TMCHO-S^TMKit specifications, respectively by the heavy chain gene (SEQ ID No.4) of IBI310 and gently Chain gene (SEQ ID No.3) insertion expression vector Freedom^TM pCHO1.0(Freedom^TMCHO-S^TMKit, GIBCO are produced Product, catalog number is A13696-01) first and second destination gene expression framework, be built into IBI310 antibody tables Up to plasmid pCHO1.0-IHEKR.

2nd, expression plasmid transfection host cell

According to Free Style^TMPMAX Reagent specifications, by Invitrogen chemical transfection reagents (Free Style^TMPMAX Reagent, article No.：IBI310 antibody expressing plasmids pCHO1.0-IHEKR 16447-100) is transferred to CHO-S In host cell, transfection is completed.

3rd, pressurizeed after transfecting and screened

Dispensed respectively into two square vases after 6 bottles of cell suspensions filtering after transfecting 48 hours.Using containing Puro, The screening and culturing medium (table 1) of MTX, the quiescent culture in incubator detects Cell viability after 7 days, complete according to Cell viability numerical value Pressurizeed into first and second stage and screened.

The nutrient media components table of table 1

4th, cell mass yield detection

12 cell masses respectively obtained to the first and second stages carry out height using 6 orifice plate, 5 days methods of quiescent culture Produce the screening of cell mass.Supernatant antibody expression amount is detected with Fortebio sizing techniques.The method passes through Protein A sensor The standard sample of various concentrations is detected, sets up standard curve to calculate the concentration of testing sample, choose antibody expression amount highest Cell mass YY 310-2 10/100-50/1000.

5th, positive monoclonal screening

Monoclonal is carried out to the cell mass YY 310-2 10/100-50/1000 for choosing using limiting dilution assay.With clone Culture medium (table 1), through 96 orifice plates, two stages of 6 orifice plate, carries out the screening of High producing clones.According to 6 orifice plate the selection results, will be anti- The 12 clonal expansion cultures of body expression quantity highest simultaneously carry out mending sugared experiment, obtain the of a relatively high cell line of antibody expression amount 56B3。

6th, the screening of subcloned cells strain

To ensure to build the monoclonicity of cell used by master cell bank, to the yield in IBI310 original clone cell banks and The good cell line 56B3 of stability is subcloned.Subcloning procedures used are all limiting dilution assay, through 96 orifice plates, 6 holes Two stages of plate filter out 3 high yield subclones：56B3-25D11,56B3-26D5 and 56B3-29F6, establish by 3 Asias Clone the original subcloned cells storehouse for constituting.

7th, final production cell line is determined

Tri- harvest liquids of subclone of 56B3-25D11,56B3-26D5 and 56B3-29F6 are through Protein A affinity purifications Obtain destination protein.It is control with her monoclonal antibody, is detected using SDS-PAGE methods, as a result as shown in Figure 1.

In Fig. 1, M is albumen Marker (NEB products, article No. is P7703)；ST:Her monoclonal antibody；25D11,26D5 and 29F6 The purpose that the harvest liquid of 56B3-25D11,56B3-26D5 and 56B3-29F6 is obtained through Protein A affinity purifications is represented respectively Albumen.

Fig. 1 shows, the destination protein expressed by the strain of 56B3-25D11,56B3-26D5 and 56B3-29F6 subcloned cells Relative molecular weight size and purity are consistent with her monoclonal antibody.

It is control with her monoclonal antibody, CEX-HPLC methods detect that the harvest liquid of each subclone is obtained through Protein A affinity purifications The electric charge isomery of the destination protein for arriving, as a result as shown in Figure 2.

In Fig. 2, subclone 25D11,26D5 and 29F6 represent 56B3-25D11,56B3-26D5 and 56B3-29F6 respectively The destination protein that harvest liquid is obtained through Protein A affinity purifications.

Fig. 2 shows, the destination protein expressed by the strain of 56B3-25D11,56B3-26D5 and 56B3-29F6 subcloned cells with She compares a monoclonal antibody, and main peak is consistent.

It is control with her monoclonal antibody, SEC-HPLC methods detect that the harvest liquid of each subclone is obtained through Protein A affinity purifications The purity of the destination protein for arriving, as a result as shown in Figure 3.

In Fig. 3, subclone 25D11,26D5 and 29F6 represent 56B3-25D11,56B3-26D5 and 56B3-29F6 respectively The destination protein that harvest liquid is obtained through Protein A affinity purifications.

Fig. 3 shows, the destination protein expressed by the strain of 56B3-25D11,56B3-26D5 and 56B3-29F6 subcloned cells Retention time is consistent with her monoclonal antibody, and purity is higher.

It is control, the destination protein obtained through Protein A affinity purifications to the harvest liquid of 56B3-29F6 with her monoclonal antibody Affinity detection is carried out, as a result as shown in Figure 4 and Figure 5.

Fig. 4 and Fig. 5 show that the affinity of the destination protein of subclone 56B3-29F6 expression is close with her monoclonal antibody.

To 56B3-25D11,56B3-26D5 and 56B3-29F6, this 3 subcloned cells strains carry out 60 days Detection of Stability, Result shows：56B3-29F6 cell lines antibody production is without reduction, two antibody of cell line of 56B3-25D11 and 56B3-26D5 Yield decreases.Finally selected 56B3-29F6 is IBI310 subclones.

8th, the identification of initial cell strain

Genes of interest sequencing in genome is carried out to initial cell strain 56B3-29F6, expression product identification, mass spectrum determines to divide Son amount.The method being sequenced using PCR primer to heavy chain and light chain.

IBI310 heavy chains are sequenced (as shown in Figure 6) after fragment is spliced through DNASTAR-Seqman softwares, obtain gene order HC Seqman, the sequence carries out Blast contrasts with her theoretical sequence of a monoclonal antibody heavy chain genes of interest, as a result shows that the two is complete It is complete consistent.

IBI310 light chains are sequenced (as shown in Figure 7) after fragment is spliced through DNASTAR-Seqman softwares, obtain gene order LC Seqman, the sequence carries out Blast contrasts with her theoretical sequence of a monoclonal antibody light chain genes of interest, as a result shows that the two is complete It is complete consistent.

9th, the identification of cell expression product

Cell line 56B3-29F6 carries out shaking flask stream and adds culture 14 days.Harvest liquid is led to after Protein A affinity purifications The molecular weight that liquid chromatograph mass spectrography detects sample is crossed, the molecular weight of IBI310 is identical with the molecular weight of her monoclonal antibody, As shown in table 2.

Her monoclonal antibody of table 2 is contrasted with IBI310 molecular weight

Anti- CTLA-4 monoclonal antibodies (IBI310) are made up of 2 light chains and 2 heavy chains, a light chain and heavy chain it Between by disulfide bond, disulfide bond is passed through between heavy chain and heavy chain；The amino acid sequence of light chain such as SEQ ID No.1 Shown, the amino acid sequence of heavy chain is as shown in SEQ ID No.2；The amino acid sequence of light chain variable district such as SEQ ID No.1 Shown in 23rd to the 130th, the amino acid sequence of weight chain variable district is as shown in the 20th to the 137th of SEQ ID No.2； CDR1, CDR2, CDR3 sequence of light chain variable district are respectively such as the 46th to the 57th, the 73rd to the 79th of SEQ ID No.1 Position, shown in the 112nd to the 120th, CDR1, CDR2, CDR3 of weight chain variable district respectively such as the 45th of SEQ ID No.2 extremely 51st, the 70th to the 76th, shown in the 118th to the 126th.

The coding gene sequence of the light chain of the monoclonal antibody as shown in SEQ ID No.3, the coding gene sequence of heavy chain As shown in SEQ ID No.4；The coding gene sequence of light chain variable district as shown in the 67th to the 390th of SEQ ID No.3, The coding gene sequence of weight chain variable district is as shown in the 58th to the 411st of SEQ ID No.4；The CDR1 of light chain variable district, The coding gene sequence of CDR2, CDR3 respectively as the 136th to the 171st of SEQ ID No.3, the 217th to the 237th, Shown in 334th to the 360th, the coding gene sequence of CDR1, CDR2, CDR3 of weight chain variable district is respectively such as SEQ ID The 133rd to the 153rd of No.4, the 208th to the 228th, shown in the 352nd to the 378th.

The therapeutic action of the mouse lung tuberculosis model that embodiment 2, IBI310 is induced mycobacterium tuberculosis

First, by people CTLA-4 transgenic mices, (C57BL/6 mouse backgrounds, it is people's that the extracellular end of CTLA-4 albumen is replaced Amino acid sequence) IBI310 10mg/kg groups, model control group and positive controls are randomly divided into, and setter CTLA-4 turns base Because of mouse Normal group, every group 10 (SPF ranks, 6~8 week old, 18~22g) are handled as follows to each group：

IBI310 10mg/kg groups：Mouse mainline gives mycobacterium tuberculosis (Mycobacterium tuberculosis)5×10⁵CFU (CFU)/only, set up mouse lung tuberculosis model, lung tissue homogenate liquid The visible M. tuberculosis growth of the outer culture and visible inflammatory infiltration of lungs pathology and Granuloma formation can determine whether to be model success. IBI310 was given to mouse peritoneal injection IBI310 parenteral solutions (by 0.9% physiology of IBI310 parenteral solutions since the same day of contaminating Salt solution is used after being diluted to 1mg/ml, and the preparation same day on the same day uses) to be treated, dosage is 10mg/kg, Per-Hop behavior 1 It is secondary, continuously give 10 weeks.

Normal group：People's CTLA-4 transgenic mices, do not inject mycobacterium tuberculosis, the same IBI310 of medication 10mg/kg groups, only replace with isometric physiological saline by IBI310 parenteral solutions.

Model control group：The foundation and identification of mouse lung tuberculosis model are with IBI310 10mg/kg groups.Medication is same IBI310 10mg/kg groups, only replace with physiological saline by IBI310 parenteral solutions.

Positive controls (isoniazid+rifampin group)：The foundation of mouse lung tuberculosis model and the same IBI310 of identification 10mg/kg groups.Isoniazid 60mg/kg joints rifampin 120mg/kg was given to mouse stomach since the same day of contaminating to control Treat, administered volume is respectively 10ml/kg, is administered once daily, continuously gives 10 weeks.

2nd, in IBI310 human peripheral bloods Treg cell proportions influence

IBI310 10mg/kg groups, Normal group, model control group and positive controls are gathered after last dose respectively The peripheral blood of mouse, separates Treg cells.Substantially process is as follows：Using the double positive Treg separation agents of Dynal CD4CD25 (FlowComp Mouse CD4+CD25+Treg Cells, Invitrogen Products, catalog number (Cat.No.) 11463D) separated from PMBC and be enriched with Treg, with anti-CD14, anti-CD56, anti-CD19, anti-CD8 and anti- CD235 Alpha antibodies cocktail and removal magnetic bead separate CD4 positive T cells from PMBC, and the CD4 positives T of purifying is thin Born of the same parents make Treg be enriched in magnetic bead surfaces after being incubated with CD25 magnetic beads, and by collecting Treg after wash-out, (detailed process is said referring to use Bright book).With the ratio of the double positive regulatory T cells (Treg) of flow cytometry analysis CD4CD25, Treg cell proportions= (Treg cell numbers/CD4 positive T cells number) × 100%.

Result is as shown in Figure 8.In Fig. 8, #p<0.05, compare with Normal group；*p<0.05, with model control group ratio Compared with.

Result shows, mycobacterium tuberculosis exposed Mice after 10 weeks peripheral blood T reg cell proportions substantially increase, give The mouse (i.e. IBI310 10mg/kg groups mouse) of IBI310 treatments, peripheral blood T reg cell proportions are significantly lower than model comparison Group, points out IBI310 to remove Treg.Treg has no significant effect in positive control drug isoniazid joint rifampin human peripheral blood.

3rd, IBI310 is to IL-10, TGF-β 1 and FoxP3 expression in mycobacterium tuberculosis exposed Mice peripheral blood T reg Influence

(1) each group Treg separates and purifies same step 2.

(2) Q-RT-PCR methods determine the expression of IL-10, TGF-β 1 and FoxP3

1st, extracted and Reverse Transcriptase kit (Power using RNAGreen Cells-to-CT^TMKit, ThermoFisher Products, catalog number (Cat.No.) 4402953) RNA is extracted from each group Treg for collecting, the RNA samples of extraction enter One step is processed to eliminate the pollution of DNA with the DNA enzymatic I without RNase.

2nd, in RT Buffer, with random primer by RNA reverse transcriptions, cDNA is obtained.

3rd, with each cDNA as template, respectively with IL-10F and IL-10R primer pairs, TGF-β 1F and TGF-β 1R primer pairs and FoxP3F and FoxP3R primer pairs enter performing PCR amplification, respectively obtain IL-10, TGF-β 1 and FoxP3 specific amplification products.Together When with GAPDHF and GAPDHR as primer pair, reference gene GAPDH is entered performing PCR amplification.IL-10, TGF-β 1 and FoxP3 amplification The amount of the amount glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of product is corrected, and then defines the expression quantity of Normal group It is 100%, calculating each cell factor relative expression quantity computational methods is：Factor X relative expression quantities=[in addition to Normal group Each group (factor X signal value/GAPDH signal values)/Normal group (factor X signal value/GAPDH signal values)], finally carry out Statistical analysis.

Specific primer is as shown in table 3.

Table 3

Result is as shown in Figure 9.In Fig. 9, #p<0.05、##p<0.01, compare with Normal group；*p<0.05, with model Control group compares.

Result shows, the IL-10's and FoxP3 in Treg of the mycobacterium tuberculosis exposed Mice after 10 weeks in peripheral blood MRNA substantially increases, although increasing to a certain degree also occurs in the mRNA of TGF-β 1, compared with Normal group, without system Meter learns significant difference.After IBI310 is treated 10 weeks, the mRNA level in-site of IL-10 and FoxP3 can be substantially reduced, but to TGF- The mRNA level in-site of β 1 has not significant impact.IL-10, TGF- of Treg in positive control drug isoniazid joint rifampin human peripheral blood The mRNA level in-site of β 1 and FoxP3 has no significant effect.

4th, the influence that IBI310 is expressed mycobacterium tuberculosis exposed Mice splenocyte IL-2 and IFN-γ

Spleen will be after death immediately disconnected at each group animal after last dose, prepares single cell suspension, be inoculated in 96 hole cells Culture plate, 5 × 10 are contained per hole⁶Individual cell, while adding Recombinant Heat shock protein 65kD, final concentration of 20 μ g/ml, each animal sample 3 multiple holes of this setting, effect takes supernatant after 48 hours, operation to specifications, and IL-2 and IFN- is detected with ELISA method (Mouse IL-2ELISA Kit (Interleukin-2) are Abcam products to the level of γ, and catalog number (Cat.No.) is ab100706；Mouse Interferon gamma ELISA Kit (IFNG) is Abcam products, and catalog number (Cat.No.) is ab100690).

IL-2 substantially detection methods are as follows：(1) mouse IL-2 specific antibodies are coated with 96 orifice plates, and 4 DEG C overnight；(2) add Testing sample and standard items, 4 DEG C of overnight incubations；(3) fully after washing, biontnylated anti-mouse IL-2 antibody is added, room temperature is incubated Educate 2h；(4) fully after washing, horseradish peroxidase Streptavidin is added, is incubated at room temperature 1h；(5) fully after washing, Tmb substrate is added, absorbance is read at 450nm with ELIASA after colour developing；(6) the dense of IL-2 is calculated according to standard curve Degree.

IFN-γ substantially detection method is as follows：(1) mouse IFN-γ specific antibody is coated with 96 orifice plates, and 4 DEG C overnight；(2) Add testing sample and standard items, 4 DEG C of overnight incubations；(3) fully after washing, biontnylated anti-mouse IFN-γ antibody is added, Incubation at room temperature 2h；(4) fully after washing, horseradish peroxidase Streptavidin is added, is incubated at room temperature 1h；(5) fully After washing, tmb substrate is added, absorbance is read at 450nm with ELIASA after colour developing；(6) calculated according to standard curve The concentration of IFN-γ.

Result is as shown in Figure 10.In Figure 10, * * * p<0.001, compare with model control group.

Result shows that Normal group mouse boosting cell can express a small amount of IL-2 under the stimulation of HSP65 And IFN-γ；Although the splenocyte of mycobacterium tuberculosis exposed Mice in vivo and it is external receive respectively mycobacterium tuberculosis and The double stimuli of HSP65, but may be due to the influence of Treg, and the expression quantity of IL-2 and IFN-γ is not still high；But It is that the expression quantity of IL-2 and IFN-γ substantially increases, and carries after the IBI310 treatments that mycobacterium tuberculosis exposed Mice receives 10 weeks Show that IBI310 may promote reaction of the body to exogenous antigen by eliminating the inhibitory action of Treg.Positive control drug isoniazid Joint rifampin has no significant effect to the level of IL-2 in splenocyte and IFN-γ.

5th, influences of the IBI310 to mycobacterium tuberculosis exposed Mice lung lesion and granuloma index

The lungs of each group mouse are separated after last dose, weighed, fixed and cut into slices, HE dyeing, micro- sem observation is simultaneously calculated Lungs granuloma index, granuloma index=(lung weight/the weight of animals) × 250 (HE is dyeed referring to document Yoshida S, Tanaka T,Kita Y,et al.DNA vaccine using hemagglutinating virus of Japan- liposome encapsulating combination encoding mycobacterial heat shock protein 65and interleukin-12confers protection against Mycobacterium tuberculosis by T cell activation.Vaccine,24:1191-1204,2006；Granuloma exponential experiment referring to document Moore VL, Myrvik QN,Leake ES.Specificity of a BCG-induced pulmonary granulomatous response in rabbits.Infection and Immunity,7:743-746,1973)。

HE coloration results are as shown in figure 11.

The result of granuloma index is as shown in figure 12.In Figure 12, ##p<0.01, compare with Normal group；**p<0.01, Compare with model control group.

Above pathology testing result shows that the lung tissue tactical rule of normal mouse, cell arrangement is regular, level Clearly, inflammatory cell infiltration is had no；Mycobacterium tuberculosis exposed Mice lung structure is chaotic, and cell arrangement is irregular, without level Sense, and there is substantial amounts of inflammatory cell infiltration and the damage of extensive interstitial and Granuloma formation；Exposed Mice gives After IBI310 is treated 10 weeks, lungs pathology damage substantially mitigates, and inflammatory cell infiltration is significantly reduced, and Granuloma formation substantially subtracts It is few；Lungs pathology damage substantially mitigates after positive control drug isoniazid joint rifampicin treatment, and inflammatory cell infiltration is significantly reduced, Granuloma formation is significantly reduced, and action effect is close with IBI310.

Result of study of the present invention shows that anti-CTLA-4 monoclonal antibodies (IBI310) can be by removing mouse pulmonary tuberculosis Foxp3 and IL-10 transcriptional expressions, promotion IL- in the double positive regulatory T cells (Treg) of CD4CD25, suppression Treg in model 2 and IFN-γ expression, mitigate mycobacterium tuberculosis induction lung lesion and reduce lungs Granuloma formation and produce anti-mouse The effect of pulmonary tuberculosis, it is very promising tuberculotherapy medicine to point out IBI310, while prompting is with CTLA-4 as target spot Immunoregulation medicament be the potential tuberculotherapy medicine of a class.

Bibliography：

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

2、Sugawara I,Sun L,Mizuno S,Taniyama T.Protective efficacy of recombinant BCG Tokyo(Ag85A)in rhesus monkeys(Macaca mulatta)infected intratracheally 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 Blockade Expands Infiltrating T Cells and Inhibits Cancer Cell Repopulation during the Intervals of Chemotherapy in Murine Mesothelioma.Mol Cancer Ther,11(8):1809- 1819,2012。

Sequence table

<110>Xinda bio-pharmaceuticals（Suzhou）Co., Ltd

<120>Immunoregulation medicament with CTLA-4 as target spot treats application lungy

<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 cccctggaca 360

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 (10)

1. the immunoregulation medicament with CTLA-4 as target spot is in the product for preparing the disease that treatment is caused by mycobacterium tuberculosis Application.

2. application according to claim 1, it is characterised in that：The disease is tuberculosis, preferably pulmonary tuberculosis.

3. application of the immunoregulation medicament with CTLA-4 as target spot in following any shown product is prepared：

(1) product of the double positive regulatory T cells Treg of CD4CD25 is removed；And/or

(2) product of the immunosuppressive action of the double positive regulatory T cells Treg of antagonism CD4CD25.

4. the application according to claim any one of 1-3, it is characterised in that：The immunoregulation medicament is anti-for anti-CTLA-4 Body.

5. application according to claim 4, it is characterised in that：The anti-CTLA-4 antibody is anti-for anti-CTLA-4 monoclonals Body.

6. application according to claim 5, it is characterised in that：The anti-CTLA-4 monoclonal antibodies are anti-for full people source CTLA-4 monoclonal antibodies.

7. application according to claim 6, it is characterised in that：The light chain of the anti-CTLA-4 monoclonal antibodies in full people source can Become CDR1, CDR2, CDR3 sequence in area respectively as the 46th to the 57th of SEQ ID No.1, the 73rd to the 79th, the Shown in 112 to the 120th, CDR1, CDR2, CDR3 of weight chain variable district are respectively such as the 45th to the 51st of SEQ ID No.2 Position, the 70th to the 76th, shown in the 118th to the 126th.

8. the application according to claim 6 or 7, it is characterised in that：The anti-CTLA-4 monoclonal antibodies in full people source it is light The coding gene sequence of CDR1, CDR2, CDR3 of chain variable region respectively as the 136th to the 171st of SEQ ID No.3, the 217 to the 237th, shown in the 334th to the 360th, the coding gene sequence of CDR1, CDR2, CDR3 of weight chain variable district Respectively such as SEQ ID No.4 the 133rd to the 153rd, the 208th to the 228th, shown in the 352nd to the 378th.

9. the application according to claim any one of 6-8, it is characterised in that：The anti-CTLA-4 monoclonal antibodies in full people source Light chain variable district amino acid sequence as shown in the 23rd to the 130th of SEQ ID No.1, the amino acid of weight chain variable district Sequence is as shown in the 20th to the 137th of SEQ ID No.2.

10. the application according to claim any one of 6-9, it is characterised in that：The anti-CTLA-4 monoclonals in full people source resist As shown in SEQ ID No.1, heavy chain amino acid sequence is as shown in SEQ ID No.2 for body light-chain amino acid sequence.