Use of cd52 protein in the preparation of a medicament for the treatment of recurrent miscarriage

Abstract

The application discloses application of CD52 protein in preparation of a medicine for treating recurrent spontaneous abortion, and belongs to the technical field of biological medicine. It is found that the CD52 protein can target and inhibit abnormal activation of effector T cells at a maternal-fetal interface, reduce release of proinflammatory factors (such as IFN-gamma), thereby reversing immune tolerance imbalance and improving pregnancy outcome. The CD52 protein is applied to the treatment of recurrent spontaneous abortion for the first time, and has the advantages of high targeting and high safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of CD52 protein in preparation of a medicine for treating recurrent spontaneous abortion (RSA). BACKGROUND

[0002] Recurrent spontaneous abortion (RSA) is a pregnancy complication with complex causes, and about 50% of the cases are related to imbalance of immune tolerance at the maternal-fetal interface. At present, the commonly used immune regulation therapy (such as glucocorticoids, intravenous immunoglobulin, etc.) has two big bottlenecks:

[0003] (1) Non-specific immunosuppression: glucocorticoids and other drugs exert effects by inhibiting the immune system in a broad spectrum, which is easy to cause side effects such as infection and metabolic disorder;

[0004] (2) Lack of precise target: the existing treatment methods are not designed for the key molecular pathways of immune regulation at the maternal-fetal interface, and have limited efficacy and high recurrence rate.

[0005] Therefore, there is an urgent need to develop a new medicine for treating recurrent spontaneous abortion with good effect. SUMMARY

[0006] The application aims to provide application of CD52 protein in preparation of a medicine for treating recurrent spontaneous abortion. The application finds that CD52 can target and inhibit abnormal activation of effector T cells at the maternal-fetal interface, reduce release of proinflammatory factors (such as IFN-γ), thereby reversing immune tolerance imbalance and improving pregnancy outcome, by establishing a RSA mouse model and treating by exogenous supplement of CD52 recombinant protein. The application first applies CD52 recombinant protein to RSA treatment, and has the advantages of strong targeting and high safety.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0008] The application provides application of CD52 protein or biological material expressing CD52 protein in preparation of a medicine for treating recurrent spontaneous abortion. The amino acid sequence of the CD52 protein is shown as SEQ ID NO: 1; the CD52 protein further includes a variant having at least 90% homology with the amino acid sequence shown as SEQ ID NO: 1 and retaining an immune suppression function.

[0009] The biological material expressing CD52 protein includes any one of the following (1) to (4):

[0010] (1) a nucleic acid molecule encoding CD52 protein;

[0011] (2) an expression cassette containing the nucleic acid molecule of (1);

[0012] (3) a recombinant expression vector containing the nucleic acid molecule of (1), or a recombinant expression vector containing the expression cassette of (2);

[0013] (4) a host cell containing the nucleic acid molecule of (1), or a host cell containing the expression cassette of (2), or a host cell containing the recombinant expression vector of (3); the host cell includes a microbial cell, a plant cell, and an animal cell.

[0014] Further, the recurrent miscarriage is a recurrent miscarriage of CD4 + / CD8 + T cell abnormal activation subtype.

[0015] Further, the drug for treating recurrent miscarriage further comprises a pharmaceutically acceptable excipient.

[0016] Further, the excipient comprises at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener, or a colorant.

[0017] Further, the dosage form of the drug comprises at least one of a granule, a tablet, a pill, a capsule, an injection, or a dispersant.

[0018] Further, the administration dose of CD52 protein in the drug is 10-20 mg / kg.

[0019] Further, the CD52 protein in the drug is the main effective component of the drug.

[0020] Further, the CD52 protein in the drug is the only effective component of the drug.

[0021] The present application also provides a pharmaceutical composition comprising CD52 protein or the above-mentioned biological material expressing CD52 protein.

[0022] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0023] Further, the carrier comprises physiological saline, a phosphate buffer, or a stabilizer-containing injection.

[0024] The application also provides application of a CD52 detection reagent in preparation of a kit for diagnosing recurrent spontaneous abortion or monitoring therapeutic effect of recurrent spontaneous abortion, the kit can be used for CD52-Siglec-10 axis function typing of RSA patients by detecting CD52 (flow antibody marking CD3 / CD4 / CD8 and CD52 co-staining) on the cell surface of serum and peripheral blood mononuclear cells (PBMC) of the RSA patients, soluble CD52 (ELISA kit) and inflammatory factors IFN-gamma and GZMB (a matched detection module), and screening of CD52 targeted treatment suitable population. Meanwhile, the kit can be used for effect tracking by detecting at multiple time points before treatment (baseline), during treatment (1 / 3 / 6 weeks) and after treatment (pregnancy maintenance period) to generate a dynamic effect curve. In addition, CD52 level normalization is positively correlated with pregnancy outcome (such as reduced embryo resorption rate), and the treatment success rate can be predicted by the CD52 level.

[0025] In the peripheral blood (or decidua tissue) of the RSA patients, the following abnormalities can exist: (1) CD52 expression level abnormality: the CD52 expression on the surface of effector T cells (such as CD4 + , CD8 + T cells) is significantly increased or decreased, reflecting the imbalance of immune activation state; (2) soluble CD52 (sCD52) level change: sCD52 can be abnormally released in the decidua microenvironment, and is related to the intensity of inflammatory response. By detecting the CD52 expression level in the blood sample of the patient (such as flow cytometry for detecting the cell surface CD52 and ELISA for detecting the sCD52 concentration), it can be determined whether the immune abnormality is related to the dysfunction of the CD52-Siglec-10 signal axis, so as to screen out potential patients suitable for CD52 targeted treatment.

[0026] The application has at least the following technical effects or advantages:

[0027] (1) Strong targeting and significant effect

[0028] Reduced embryo resorption rate: in the CBA / JxDBA / 2J RSA mouse model, the embryo resorption rate of the CD52 recombinant protein treatment group is significantly reduced (P<0.01); P <0.01);

[0029] Inflammatory factor inhibition: the proportions of IFN-gamma + CD4 + T cells and GZMB + CD8 + T cells in the decidua tissue of the CD52 recombinant protein treatment group are significantly reduced compared with those in the RSA group.

[0030] (2) Avoiding non-specific immunosuppression

[0031] Precise regulation: specifically targeting CD52-Siglec-10 axis, only inhibiting abnormal activated effector T cells, without interfering with normal immune response.

[0032] (3) Flexible application, compatible with existing diagnosis and treatment system

[0033] Potential for combination therapy: can be combined with low molecular weight heparin, aspirin and other anticoagulation regimens to synergistically improve placental blood flow;

[0034] Diagnosis-treatment integration: CD52 detection kit can dynamically monitor the efficacy and guide individualized drug use. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Construction of CBA / J x DBA / 2J RSA mouse model and experimental procedures.

[0036] Figure 2 Effect of CD52 recombinant protein intervention on pregnancy outcome and placental structure in RSA pregnant mouse model. A: Representative images of uteruses of mice in each group. B: Quantitative analysis of embryo resorption rate (n=6 / group). C: ELISA detection of serum sCD52 level (n=6 / group). D: Histopathological evaluation of placental tissue (H&E staining, scale = 200 μm). E: Area statistics of uteroplacental units. Statistical significance is indicated as: .

[0037] Figure 3 Flow cytometry results and co-localization of CD52 protein and Siglec-10 at the maternal-fetal interface. A: Flow cytometry gating strategy for activated effector T cells. T cell subsets were divided by CD3 + CD4 / CD8 double labeling after gating; activation marker combination: effector memory T cells (TEM): CD62L low CD44 high ; central memory T cells (TCM): CD62L high CD44 low ; naive T cells; CD62L high CD44 low . B&C: Comparison of the proportion of activated T cells in each group. D: Flow cytometry analysis of CD4 + IFN-γ / TNF-α cytokine expression ratio and comparison in each group. E: Flow cytometry analysis of CD4 + GZMB / Perforin cytokine expression ratio and comparison in each group. F: Immunofluorescence co-localization analysis of CD4, Siglec-10 and CD52 in Con and RSA groups. G: Quantitative analysis and comparison of CD52 fluorescence intensity in Con and RSA groups. Statistical significance is indicated as: .

[0038] Figure 4 Effects of CD52 monoclonal antibody intervention on the pregnancy outcome and placental structure of normal pregnant mice. A: Representative images of the uterus of mice in each group. B: Quantitative analysis of embryo resorption rate (n=6 / group). C: Histopathological evaluation of placental tissue (H&E staining, scale bar=200 μm). D: Histogram of the area of the uteroplacental unit. E: Histogram of the diameter of the uteroplacental unit. Statistical significance is indicated as: . DETAILED DESCRIPTION

[0039] The advantages and various effects of the present application will be more clearly apparent from the following detailed description of the application in conjunction with the specific embodiments and examples. It should be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustration of the present application, and not to limit the present application.

[0040] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood in the manner as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application pertains. If there is a contradiction, the present specification takes precedence.

[0041] Unless otherwise specifically indicated, the various raw materials, reagents, instruments and equipment, etc. used in the present application can be obtained by market purchase or can be obtained by existing methods.

[0042] In order to solve the technical problems of the present application, the general idea of the present application is as follows:

[0043] The present application provides the use of CD52 protein in the preparation of a medicament for treating RSA.

[0044] Amino acid sequence of human CD52 protein:

[0045] MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS (SEQ ID NO: 1);

[0046] Amino acid sequence of mouse CD52 protein:

[0047] MKSFLLFLTIILLVVIQIQTGSLGQATTAASGTNKNSTSTKKTPLKSGASSIIDAGACSFLFFANTLMCLFYLS (SEQ ID NO: 2).

[0048] The CD52 protein can be a CD52 recombinant protein produced by a eukaryotic expression system (such as HEK293 cells) with a purity of ≥95%. The CD52 protein can also be obtained by commercial channels. The CD52 recombinant protein used in the following examples is purchased from MedChemExpress LLC, Catalog No: HY-P75398.

[0049] Formulation: CD52 recombinant protein (0.1-10 mg / mL) + physiological saline, PBS or 5% human serum albumin.

[0050] Dosing regimen: suitable for patients with immune recurrent spontaneous abortion or high-risk population, within 12 weeks of pregnancy after confirmation of pregnancy, by intravenous injection or local perfusion in the uterine cavity, the dose range is 0.1-10 mg / kg body weight, 1-3 times a week.

[0051] Efficacy monitoring: detection of CD52 levels in decidual tissue, proportion of effector T cells (Th1 / Th17 / CD8 + T cells), serum inflammatory factor (IFN-γ, IL-17A) concentration; evaluation criteria are ≥50% reduction in embryonic resorption rate and ≥30% recovery of placental vascular density.

[0052] Example 1: Therapeutic effect of CD52 recombinant protein on RSA mouse model

[0053] I. Experimental methods

[0054] 1. Female CBA / J mice aged 8-10 weeks and male DBA / 2J mice or BALB / c mice were mated in a ratio of 2:1 to establish CBA / J x DBA / 2J RSA model and CBA / J x BALB / c normal pregnancy model.

[0055] The day of detection of the negative plug (pregnancy day 0.5, GD0.5) was counted as pregnancy, and CD52 recombinant protein (10 μg / g body weight) or equal volume of PBS was injected intravenously on GD0.5, and samples were taken on GD13.5.

[0056] 2. Experimental grouping

[0057] Group A: normal pregnancy model + PBS injection, Con);

[0058] Group B: RSA model + PBS injection, RSA;

[0059] Group C: normal pregnancy model + CD52 recombinant protein intervention, Con+CD52;

[0060] Group D: RSA model + CD52 recombinant protein intervention, RSA+CD52.

[0061] 3. Detection index

[0062] 1) Observation of RSA pregnant mice pregnancy outcome: weighing each group of mice, after anesthesia, using cervical dislocation method to kill each group of mice, dissect the uterus, record the total embryo number and the number of absorbed embryos (necrosis or atrophy can be seen with the naked eye), calculate the embryo resorption rate, embryo resorption rate = absorbed embryo number / (absorbed embryo number + survival embryo number).

[0063] 2) Spleen, peripheral blood cytology detection: prepare mouse spleen mononuclear cell suspension by red blood cell lysis solution. Flow cytometry staining is performed on the above spleen single cell suspension and peripheral blood PBMC, and the surface molecules of effector T cells: CD62L, CD44, ICOS, KLRG-1, CX3CR1, Siglec-10; transcription factors: IRF4, ID2, BLIMP1, Ki67, STAT3, STAT4, STAT5; cytokines: IL-2, IFN-γ, TNF-α, PRF1, GZMB; CD52 are analyzed respectively. high T cell surface molecules: CD52, S100A4, CD24, CD127.

[0064] Effector T cells: CD4 + IFN-γ + (Th1), surface staining CD4-BV510, intracellular staining IFN-γ-PE; regulatory T cells: CD4 + Foxp3 + (Treg), surface staining CD4-BV510, CD25-FITC, intracellular staining Foxp3-Alexa647.

[0065] 3) Enzyme-linked immunosorbent assay (ELISA): blood is taken from the apex of the heart, serum is separated by centrifugation, and CD52 level is detected.

[0066] 4) RSA pregnant mouse uterine decidua tissue immunofluorescence staining: after paraffin embedding section of each group of specimens, immunofluorescence double staining CD52 and Siglec10 is performed to observe the co-localization expression and distribution of CD52 and Siglec10 in the decidua tissue.

[0067] II. Experimental results

[0068] The pregnancy outcome was observed and quantitatively analyzed, and the results are shown in Figure 2 , 3 .

[0069] The embryo loss rate of the RSA group was significantly higher than that of the healthy control group ( P <0.01), and the resorption rate after CD52 intervention was lower than that of the RSA group ( P<0.01 vs. RSA). Morphological evaluation further demonstrated that the utero-placental complex area of RSA group mice significantly atrophied ( P <0.01), partially recovered after CD52 treatment.

[0070] Flow cytometry analysis showed that the proportion of effector CD4 + and CD8 + T cells at the maternal-fetal interface of RSA model mice significantly increased, and the proportion of CD4 + IFN-γ and GZMB + T cells also significantly increased. After intervention with CD52 recombinant protein, the proportion of effector T cells in the spleen of RSA pregnant mice significantly decreased, which suggested that CD52 recombinant protein might improve pregnancy outcome by restoring immune tolerance. In addition, the results of immunofluorescence staining revealed the co-localization characteristics of CD52 protein and Siglec-10 at the maternal-fetal interface.

[0071] Example 2, Effect of CD52 monoclonal antibody intervention on normal pregnant mouse model

[0072] 1. Wild type C57BL / 6 female mice and BALB / c male mice were mated to obtain normal pregnant mice, and CD52 monoclonal antibody or isotype control antibody was injected into the tail vein at GD0.5, and samples were taken and indicators were detected at GD13.5.

[0073] The experimental groups are as follows:

[0074] Group A: Normal pregnancy + isotype control antibody (IgG), Con;

[0075] Group B: Normal pregnancy + CD52 monoclonal antibody (Anti-CD52 (CAMPATH-1) (Mouse) mAb, MBL), Con + anti-CD52.

[0076] 2. Normal pregnant mouse model was intervened by tail vein injection of CD52 monoclonal antibody, and embryonic resorption rate and decidual inflammation level were analyzed to preliminarily verify the effect of CD52 deletion on miscarriage. The detection indicators include: pregnancy outcome, placental pathological analysis, serum sCD52 level detection and decidual immune cell phenotype analysis.

[0077] 3. Results

[0078] By observing and quantitatively analyzing the pregnancy outcome, the results as shown in Figure 4 , the embryonic resorption rate of CD52 monoclonal antibody group was significantly higher than that of the control group, and morphological evaluation further demonstrated that the utero-placental complex area of CD52 monoclonal antibody group mice significantly atrophied ( P <0.01).

[0079] Finally, it should be noted that the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0080] While the preferred embodiments of the application have been described, additional variations and modifications can be made to the preferred embodiments by those of skill in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, the appended claims are intended to encompass within their scope all such variations and modifications as are within the scope of the application.

[0081] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. Use of a CD52 protein for the preparation of a medicament for the treatment of recurrent miscarriage, characterized in that, The amino acid sequence of the CD52 protein is shown in SEQ ID NO:

1.

2. Use of a biological material expressing a CD52 protein for the preparation of a medicament for the treatment of recurrent miscarriage, characterized in that, The biological material expressing the CD52 protein includes any one of the following (1) to (3): (1) an expression cassette containing a nucleic acid molecule encoding a CD52 protein; the amino acid sequence of the CD52 protein is shown in SEQ ID NO: 1; (2) a recombinant expression vector containing the expression cassette of (1); (3) a host cell containing the expression cassette of (1) or a host cell containing the recombinant expression vector of (2).

3. Use according to claim 1 or 2, characterized in that, The recurrent miscarriage is CD4 + / CD8 + T cell abnormal activation subtype of recurrent miscarriage.

4. Use according to claim 1 or 2, characterized in that, The medicament for treating recurrent miscarriage further includes a pharmaceutically acceptable adjuvant.

5. Use according to claim 1 or 2, characterized in that, The dosage form of the medicament for treating recurrent miscarriage includes at least one of granules, tablets, pills, capsules, injections, and dispersions.

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