Application of CD52 protein in preparation of medicine for treating recurrent abortion

Abstract

The invention discloses application of CD52 protein in preparation of a medicine for treating recurrent abortion, and belongs to the technical field of biological medicine. It is found that the CD52 protein can inhibit abnormal activation of maternal-fetal interfacial effect T cells in a targeted mode and reduce release of proinflammatory factors (such as IFN-gamma), so that immune tolerance imbalance is reversed, and the pregnancy outcome is improved. The CD52 protein is applied to treatment of the recurrent abortion for the first time, and the CD52 protein has the advantages of being high in targeting performance and high in safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of CD52 protein in preparing a drug for treating recurrent spontaneous abortion (RSA). Background Art

[0002] Recurrent spontaneous abortion (RSA) is a pregnancy complication with complex etiologies, with approximately 50% of cases associated with an imbalance in immune tolerance at the maternal-fetal interface. Currently, commonly used immunomodulatory therapies (such as glucocorticoids and intravenous immunoglobulin) in clinical practice face two major bottlenecks: (1) Nonspecific immunosuppression: Drugs such as glucocorticoids exert their effects by broadly suppressing the immune system, which can easily lead to side effects such as infection and metabolic disorders; (2) Lack of precise targets: Existing treatments are not designed to target the key molecular pathways regulating maternal-fetal immune regulation, resulting in limited efficacy and a high recurrence rate.

[0003] Therefore, there is an urgent need to develop a new and effective medicine for the treatment of recurrent spontaneous abortion. Summary of the Invention

[0004] The present invention aims to provide a method for the use of CD52 protein in the preparation of a drug for the treatment of recurrent spontaneous abortion (RSA). By establishing a RSA mouse model and exogenously supplementing with recombinant CD52 protein for treatment, the authors discovered that CD52 can target and inhibit the abnormal activation of effector T cells at the maternal-fetal interface, reducing the release of proinflammatory cytokines (such as IFN-γ), thereby reversing immune tolerance imbalance and improving pregnancy outcomes. This is the first application of recombinant CD52 protein in the treatment of RSA, offering the advantages of strong targeting and high safety.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides the use of a CD52 protein or a biomaterial expressing the CD52 protein in the preparation of a medicament for treating recurrent spontaneous abortion. The amino acid sequence of the CD52 protein is shown in SEQ ID NO: 1; the CD52 protein also includes variants having at least 90% homology to the amino acid sequence shown in SEQ ID NO: 1 and retaining immunosuppressive function.

[0006] The biological material expressing CD52 protein includes any one of the following (1) to (4): (1) a nucleic acid molecule encoding CD52 protein; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant expression vector containing the nucleic acid molecule described in (1), or a recombinant expression vector containing the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1), or a host cell containing the expression cassette described in (2), or a host cell containing the recombinant expression vector described in (3); the host cell includes a microbial cell, a plant cell, or an animal cell.

[0007] Furthermore, the recurrent spontaneous abortion is CD4 + / CD8 + Recurrent miscarriage with abnormal activation of T cell subtypes.

[0008] Furthermore, the medicine for treating recurrent spontaneous abortion also includes pharmaceutically acceptable excipients.

[0009] Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

[0010] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, injections or dispersions.

[0011] Furthermore, the dosage of CD52 protein in the drug is 10-20 mg / kg.

[0012] Furthermore, the CD52 protein in the medicine serves as the main active ingredient of the medicine.

[0013] Furthermore, the CD52 protein in the medicine is the only active ingredient of the medicine.

[0014] The present invention also provides a pharmaceutical composition, which comprises CD52 protein or the above-mentioned biological material expressing CD52 protein.

[0015] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0016] Furthermore, the carrier includes physiological saline, phosphate buffer or an injection containing a stabilizer.

[0017] The present invention also provides the use of a CD52 detection reagent in the preparation of a kit for diagnosing recurrent spontaneous abortion (RSA) or monitoring the efficacy of RSA treatment. This kit detects surface CD52 (CD52 co-stained with flow cytometry antibodies labeled CD3 / CD4 / CD8), soluble CD52 (ELISA kit), and inflammatory factors IFN-γ and GZMB (supporting detection module) in serum and peripheral blood mononuclear cells (PBMCs) of RSA patients. This allows for functional typing of the CD52-Siglec-10 axis in RSA patients and screening for patients suitable for CD52-targeted therapy. Furthermore, dynamic efficacy curves are generated by testing at multiple time points: before treatment (baseline), during treatment (weeks 1, 3, and 6), and after treatment (pregnancy maintenance period), for therapeutic efficacy tracking. Furthermore, normalized CD52 levels are positively correlated with pregnancy outcomes (e.g., reduced embryo resorption rate), allowing CD52 levels to predict treatment success.

[0018] The following abnormalities may be present in the peripheral blood (or decidual tissue) of RSA patients: (1) Abnormal expression level of CD52: effector T cells (such as CD4 + 、CD8 + (1) The expression of CD52 on the surface of T cells is significantly increased or decreased, reflecting an imbalance in the state of immune activation; (2) Changes in the level of soluble CD52 (sCD52): sCD52 may be abnormally released in the decidual microenvironment, which is related to the intensity of the inflammatory response. By detecting the expression level of CD52 in the patient's blood samples (such as flow cytometry to detect cell surface CD52 and ELISA to detect sCD52 concentration), it can help determine whether the immune abnormality is related to the dysfunction of the CD52-Siglec-10 signaling axis, thereby screening potential patients suitable for CD52 targeted therapy.

[0019] The present invention has at least the following technical effects or advantages: (1) Strong targeting and significant therapeutic effect Reduced embryo absorption rate: In the CBA / J×DBA / 2J RSA mouse model, the embryo absorption rate in the CD52 recombinant protein treatment group was significantly reduced ( P <0.01); Inhibition of inflammatory factors: IFN-γ in decidual tissue of CD52 recombinant protein treatment group + CD4 + T cells and GzmB + CD8 + The proportion of T cells was significantly decreased compared with the RSA group.

[0020] (2) Avoid nonspecific immunosuppression Precise regulation: Specifically targeting the CD52-Siglec-10 signaling axis, only inhibiting abnormally activated effector T cells without interfering with normal immune responses.

[0021] (3) Flexible application and compatible with the existing diagnosis and treatment system Potential for combined therapy: It can be used in combination with anticoagulant regimens such as low molecular weight heparin and aspirin to synergistically improve placental blood flow; Integrated diagnosis and treatment: The accompanying CD52 detection kit can dynamically monitor the therapeutic effect and guide individualized medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the construction and experimental process of the CBA / J×DBA / 2J RSA mouse model.

[0023] Figure 2 Effects of CD52 recombinant protein intervention on pregnancy outcomes and placental structure in the RSA mouse model. A: Representative images of the uterus in each group. B: Quantitative analysis of embryo resorption rate (n = 6 / group). C: ELISA analysis of serum sCD52 levels (n = 6 / group). D: Histopathological evaluation of the placenta (H&E staining, scale = 200 μm). E: Statistical plot of the uterine-placental unit area. Statistical significance is indicated by: .

[0024] Figure 3 Figure 1 shows the flow cytometry results and the 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: CD3 + After gating, T cell subsets were distinguished by CD4 / CD8 double labeling; 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 T cell activation ratio in each group. D: Flow cytometric analysis of CD4 + IFN-γ / TNF-α cytokine expression ratio and comparison among groups. E: Flow cytometric analysis of CD4 + GZMB / Perforin cytokine expression ratio and comparison among groups. F: Immunofluorescence colocalization analysis of CD4, Siglec-10, and CD52 in the Con and RSA groups. G: Quantitative analysis and comparison of CD52 fluorescence intensity in the Con and RSA groups. Statistical significance is indicated by: .

[0025] Figure 4The effects of CD52 monoclonal antibody treatment on pregnancy outcomes and placental structure in normal pregnant mice. A: Representative images of the uterus in each group. B: Quantitative analysis of embryo resorption rate (n = 6 / group). C: Histopathological evaluation of the placenta (H&E staining, scale = 200 μm). D: Statistical plot of the area of ​​the utero-placental unit. E: Statistical plot of the diameter of the utero-placental unit. Statistical significance is indicated by: . DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0027] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained through existing methods.

[0029] In order to solve the technical problems of the present invention, the overall concept of the present invention is as follows: The present invention provides the use of CD52 protein in preparing a medicine for treating RSA.

[0030] Amino acid sequence of human CD52 protein: MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFVANAIIHLFCFS (SEQ IDNO: 1); Amino acid sequence of mouse CD52 protein: MKSFLLFLTIILLVVIQIQTGSLGQATTAASGTNKNSTSTKKTPLKSGASSIIDAGACSFLFFANTLMCLFYLS (SEQ ID NO: 2).

[0031] The CD52 protein can be a recombinant CD52 protein produced via a eukaryotic expression system (e.g., HEK293 cells) with a purity of ≥95%. The CD52 protein can also be purchased commercially. The recombinant CD52 protein used in the following examples was purchased from MedChemExpress LLC, catalog number: HY-P75398.

[0032] Preparation formula: CD52 recombinant protein (0.1-10 mg / mL) + normal saline, PBS or 5% human serum albumin.

[0033] Dosage regimen: Suitable for patients with immune recurrent miscarriage or high-risk groups. It is administered by intravenous injection or local intrauterine perfusion from the time the pregnancy is confirmed to 12 weeks of pregnancy. The dosage range is 0.1-10 mg / kg body weight, 1-3 times a week.

[0034] Efficacy monitoring: Detect the CD52 level in decidual tissue, the ratio of effector T cells (Th1 / Th17 / CD8 + T cells), serum inflammatory factors (IFN-γ, IL-17A) concentrations; the evaluation criteria were a decrease in embryo absorption rate ≥50% and a recovery of placental vascular density ≥30%.

[0035] Example 1: Therapeutic effect of CD52 recombinant protein on RSA mouse model 1. Experimental Methods 1. 8-10 week old female CBA / J mice were mated with male DBA / 2J mice or BALB / c mice at a ratio of 2:1 to establish the CBA / J×DBA / 2J RSA model and the CBA / J×BALB / c normal pregnancy model.

[0036] The day when vaginal plug was detected (gestational day 0.5, GD0.5) was considered as pregnancy. CD52 recombinant protein (10 μg / g body weight) or an equal volume of PBS was injected into the tail vein on GD0.5, and samples were collected on GD13.5.

[0037] 2. Experimental Grouping Group A: normal pregnancy model + PBS injection, Con); Group B: RSA model + PBS injection, RSA; Group C: normal pregnancy model + CD52 recombinant protein intervention, Con+CD52; Group D: RSA model + CD52 recombinant protein intervention, RSA+CD52.

[0038] 3. Detection indicators 1) Observation of pregnancy outcomes in RSA pregnant mice: All mice were weighed and sacrificed by cervical dislocation after anesthesia. The uteri were dissected, and the total number of embryos and the number of resorbed embryos (necrotic or atrophic embryos visible to the naked eye) were recorded. The embryo resorption rate was calculated as: number of resorbed embryos / (number of resorbed embryos + number of surviving embryos).

[0039] 2) Spleen and peripheral blood cytology: Mouse spleen mononuclear cell suspension was prepared using red blood cell lysis buffer. The spleen mononuclear cell suspension and peripheral blood PBMC were stained by flow cytometry to analyze the effector T cell surface molecules: 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 high T cell surface molecules: CD52, S100A4, CD24, CD127.

[0040] Effector T cells: CD4 + IFN-γ + (Th1), surface staining CD4-BV510, intracellular staining IFN-γ-PE; regulatory T cells: CD4 + Foxp3 + (Treg), surface staining with CD4-BV510, CD25-FITC, intracellular staining with Foxp3-Alexa647.

[0041] 3) Enzyme-linked immunosorbent assay (ELISA): Blood was collected by puncture at the apex of the heart, and the serum was separated by centrifugation to detect the CD52 level.

[0042] 4) Immunofluorescence staining of uterine decidua tissue of pregnant RSA mice: After paraffin embedding and sectioning, the specimens of each group were double-stained with immunofluorescence to investigate the co-localization expression and distribution of CD52 and Siglec10 in the decidua tissue.

[0043] 2. Experimental Results The pregnancy outcomes were observed and quantitatively analyzed. Figure 2 、 3 shown.

[0044] The embryo loss rate in the RSA group was significantly higher than that in the healthy control group ( P <0.01) was significantly increased, while the absorption rate of CD52 decreased after intervention compared with the RSA group ( P <0.01 vs. RSA). Morphological evaluation further showed that the uterine-placental complex area of ​​mice in the RSA group was significantly shrunken ( P <0.01), and CD52 was partially restored after treatment.

[0045] Flow cytometry analysis showed that effector CD4 + and CD8 + The proportion of T cells increased significantly, and CD4 + IFN-γ and GZMB +T cells also increased significantly. Administration of recombinant CD52 protein significantly decreased the proportion of effector T cells in the spleen of pregnant RSA mice, suggesting that recombinant CD52 protein may improve pregnancy outcomes by restoring immune tolerance. Furthermore, immunofluorescence staining revealed colocalization of CD52 protein and Siglec-10 at the maternal-fetal interface.

[0046] Example 2: Effects of CD52 monoclonal antibody intervention on a normal pregnancy mouse model 1. Wild-type C57BL / 6 female mice were mated with BALB / c male mice to obtain normal pregnant mice. CD52 monoclonal antibody or isotype control antibody was injected into the tail vein on GD 0.5. Samples were collected and indicators were detected on GD 13.5.

[0047] The experimental groups are as follows: Group A: normal pregnancy + isotype control antibody (IgG), Con; Group B: normal pregnancy + CD52 monoclonal antibody (Anti-CD52 (CAMPATH-1) (Mouse) mAb, MBL), Con + anti-CD52.

[0048] 2. Using tail vein injection of CD52 monoclonal antibodies in a normal pregnant mouse model, we analyzed embryo resorption rates and decidual inflammation levels to preliminarily validate the effect of CD52 deficiency on miscarriage. Measurements included pregnancy outcome, placental pathology, serum sCD52 levels, and decidual immune cell phenotype analysis.

[0049] 3. Results Through observation and quantitative analysis of pregnancy outcomes, the results are as follows Figure 4 As shown in Figure 2, the embryo absorption rate in the CD52 monoclonal antibody group was significantly higher than that in the control group. Morphological evaluation further showed that the uterine-placental complex area of ​​the CD52 monoclonal antibody group mice was significantly shrunk ( P <0.01).

[0050] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0051] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0052] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of CD52 protein or a biomaterial expressing CD52 protein in the preparation of a drug for treating recurrent spontaneous abortion, characterized in that: The amino acid sequence of the CD52 protein is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The CD52 protein also includes variants that have at least 90% homology to the amino acid sequence shown in SEQ ID NO: 1 and retain the immunosuppressive function.

3. The use according to claim 1, characterized in that The biological material expressing CD52 protein includes any one of the following (1) to (4): (1) a nucleic acid molecule encoding CD52 protein; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant expression vector containing the nucleic acid molecule described in (1), or a recombinant expression vector containing the expression cassette described in (2); (4) A host cell containing the nucleic acid molecule described in (1), or a host cell containing the expression cassette described in (2), or a host cell containing the recombinant expression vector described in (3).

4. The use according to claim 1, characterized in that The recurrent spontaneous abortion is CD4 + / CD8 + Recurrent miscarriage with abnormal activation of T cell subtypes.

5. The use according to claim 1, characterized in that The medicine for treating recurrent spontaneous abortion also includes pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that The dosage form of the medicine for treating recurrent spontaneous abortion includes at least one of granules, tablets, pills, capsules, injections and dispersions.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises CD52 recombinant protein or biological material expressing CD52 protein.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical carrier includes physiological saline, phosphate buffer, or an injection containing a stabilizer.

10. Use of CD52 detection reagent in the preparation of a kit for diagnosing recurrent spontaneous abortion or monitoring the therapeutic effect of recurrent spontaneous abortion.

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