Immunomodulatory protein and application thereof in medicine preparation

Abstract

The invention relates to an immunomodulatory protein and application thereof in medicine preparation, and belongs to the technical field of biological medicine. The invention discloses application of a recombinant protein p33 in preparation of a medicine for treating autoimmune diseases. The diseases comprise systemic lupus erythematosus, skin type lupus erythematosus, acute graft versus host disease and chronic graft versus host disease. The human recombinant p33 protein is used for intravenous injection treatment of systemic lupus erythematosus mice and graft-versus-host disease mice for the first time, it is found that the recombinant p33 protein can significantly reduce lymphocyte cell proliferation and inhibit pathological damage of organs such as kidneys and livers caused by inflammatory cells, and therefore the effect of treating the systemic lupus erythematosus mice and the graft-versus-host disease mice is achieved. In addition, the recombinant p33 protein can relieve SLE mouse skin lesion and GVHD inflammation, and the survival rate is increased.

Description

Technical Field

[0001] The present invention relates to an immunomodulatory protein and application thereof in drug preparation, belonging to the technical field of biomedicine. Background Art

[0002] p33 is a multifunctional protein independently discovered by three different research groups. The protein has a Gene ID of 708, and aliases for the encoded protein include p32, gC1qR (globular C1Q receptor), HABP1 (hyaluronic acid binding protein 1), and C1QBP (complement C1Q binding protein). The protein exists as a proprotein containing 282 amino acid residues. After synthesis, the N-terminal 73 residues are hydrolyzed to form the mature p33 protein containing 209 amino acids. The mature protein is highly charged and acidic, with an isoelectric point of 4.15. The p33 protein is located intracellularly in the mitochondria, cytoplasm, nucleus, and plasma membrane, and can also be secreted extracellularly. The reported ligands of p33 protein include complement C1Q, hyaluronic acid, calreticulin, CD44, integrin, PKC, splicing factor ASF / SF2 and several bacterial and viral proteins, suggesting the diversity of the protein's functions.

[0003] Autoimmune diseases are a type of disease caused by abnormal activation of the immune system, which mistakenly attacks one's own tissues and organs. Under normal circumstances, the immune system can recognize and eliminate foreign pathogens, but in autoimmune diseases, the immune system loses tolerance to its own antigens, leading to an immune response against one's own tissues. This abnormal immune response is usually accompanied by chronic inflammation, manifested as excessive activation of immune cells (such as T cells and B cells), the production of autoantibodies, and the release of inflammatory factors (such as tumor necrosis factor-α, interleukin-6, etc.). Chronic inflammation not only directly damages tissues, but may also further aggravate disease progression through continued immune activation. Common autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. The treatment of these diseases usually involves strategies to suppress immune responses and relieve inflammation.

[0004] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by inflammation and immune-mediated damage to multiple organ systems, including the mucocutaneous, musculoskeletal, hematologic, renal, and nervous systems. Worldwide, 400,000 people are newly diagnosed with SLE each year. Approximately 3.4 million people are diagnosed with SLE worldwide. Typical clinical manifestations of SLE include fever (defined as a temperature >38.3°C in the classification criteria), alopecia, skin rash, oral ulcers, and joint pain and swelling. Laboratory abnormalities include anemia, thrombocytopenia, leukopenia, and hypocomplementemia. Serum and urine markers of lupus nephritis, such as proteinuria, hematuria, and elevated serum creatinine levels, are common. Specific autoantibodies (such as antiphospholipid antibodies, anti-double-stranded DNA antibodies, and anti-SMITH antibodies) and other autoantibodies (such as anti-ribonucleoprotein, anti-Ro / Sjögren's syndrome A, and anti-La / Sjögren's syndrome B antibodies) may be associated with specific clinical manifestations.

[0005] Cutaneous lupus erythematosus (CLE) is a manifestation of lupus erythematosus that primarily affects the skin. It may occur independently or in conjunction with systemic lupus erythematosus (SLE). The pathogenesis of CLE involves genetics, environmental factors (such as ultraviolet radiation exposure), and immune system abnormalities, particularly the production of autoantibodies and the deposition of immune complexes. Based on clinical manifestations and histopathological features, CLE can be divided into the following main types: acute cutaneous lupus erythematosus (ACLE), subacute cutaneous lupus erythematosus (SCLE), and chronic cutaneous lupus erythematosus (CCLE). Some patients may develop SLE. The diagnosis of CLE relies on clinical manifestations, skin biopsy, and immunological tests (such as antinuclear antibodies and anti-Ro / SSA antibodies). Treatment primarily involves topical and systemic medications, including topical corticosteroids, antimalarial drugs (such as hydroxychloroquine), immunosuppressants, and strict sun protection. Early diagnosis and appropriate treatment help control the disease and minimize skin damage and systemic complications.

[0006] The clinical treatment of lupus erythematosus faces many challenges and shortcomings. First, the disease lacks a radical cure, and the current treatment of SLE mainly focuses on controlling symptoms, relieving inflammation and suppressing immune response, which cannot completely cure the disease. Patients need long-term or even lifelong medication, which increases the treatment burden and compliance problems. SLE often presents alternating characteristics of recurrence and remission, even in the remission period, patients still need continuous treatment to prevent recurrence, and recurrence may cause more serious organ damage. Second, the side effects of drugs are large, and commonly used drugs such as glucocorticoids and immunosuppressants are effective, but may cause serious side effects, such as osteoporosis, increased risk of infection, metabolic disorders, and affect the quality of life of patients. Third, early diagnosis is difficult, the early symptoms of SLE are atypical, easy to misdiagnose or miss, leading to delayed treatment and increasing the risk of irreversible organ damage. Therefore, the treatment of SLE needs to be further optimized, including developing more accurate individualized treatment plans, reducing drug side effects, exploring radical means, and strengthening patient education and psychological support. Therefore, finding effective target drugs to treat SLE and other autoimmune diseases, and forming a combination with current clinical drugs is a problem that needs to be solved.

[0007] Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation, which is caused by excessive activation of donor T cells. Its inflammatory process involves a complex cascade of reactions: after donor T cells recognize host antigens, they proliferate and differentiate, release pro-inflammatory factors such as IFN-γ and TNF-α, activate monocytes and dendritic cells, further expand the cytokine storm (such as IL-1, IL-6), leading to endothelial damage, tissue infiltration, and immunopathological damage to target organs (skin, liver, intestine). Chronic GVHD presents an autoimmune-like feature with fibrosis progression. The current treatment dilemma of GVHD is the immunosuppression paradox: glucocorticoids, the first-line treatment, may increase the risk of infection and be ineffective in some patients; new drugs such as JAK inhibitors can regulate inflammatory pathways, but it is difficult to balance anti-GVHD and retain graft-versus-leukemia effect (GVL), especially with large disease heterogeneity and individual inflammatory microenvironment differences, leading to the lack of precision treatment guided by biomarkers. Therefore, more specific intervention strategies other than hormones need to be explored in this technical field. SUMMARY

[0008] The purpose of the present application is to solve the technical problem of how to obtain an effective drug and drug combination for treating autoimmune diseases.

[0009] To achieve the purpose of solving the above problems, the present invention provides a recombinant protein p33, also known as p32, gC1qR, HABP1 and C1QBP, the amino acid sequence of which is shown in SEQ ID No. 1; the classification identifier in the NCBI international nucleotide sequence database, i.e., Gene ID 708.

[0010] SEQ ID No. 1:

[0011] 1-LHTDGDKAFVDFLSDEIKEERKIQKHKTLPKMSGGWELELNGTEAKLVRKVAGEKITVTFNIN NSIPPTFDGEEEEPSQGQKVEEQEPELTSTPNFVVEVIKNDDGKKALVLDCHYPEDEVGQEDEAESDIFS IREVSFQSTGESEWKDTNYTLNTDSLDWALYDHLMDFLADRGVDNTFADELVELSTALEHQEYITFLED LKSFVKSQ-209

[0012] The present invention provides an expression vector comprising a nucleotide sequence corresponding to the amino acid sequence of the recombinant protein p33.

[0013] The present invention provides use of a recombinant protein p33 in preparing a medicine for treating autoimmune diseases, wherein the diseases include systemic lupus erythematosus, cutaneous lupus erythematosus, acute graft-versus-host disease and chronic graft-versus-host disease.

[0014] The present invention provides a use of a recombinant protein p33 in preparing a medicine for treating autoimmune diseases by inhibiting the proliferation of lymphocytes and the action of inhibiting inflammatory cells.

[0015] The present invention provides use of a recombinant protein p33 in preparing a drug for treating autoimmune diseases by inducing granulocyte-like myeloid suppressor cells (G-MDSCs).

[0016] The present invention provides use of a recombinant protein p33 in preparing a medicine for treating autoimmune diseases by inhibiting T cells.

[0017] The present invention provides a method for preparing a recombinant protein p33, which comprises constructing a nucleotide sequence encoding the protein p33; introducing the nucleotide sequence into an expression vector; transforming the expression vector into a suitable host cell, and then inducing the host cell to express the recombinant protein p33; the host cell comprises Escherichia coli, CHO cells or 293 cells.

[0018] The present invention provides a modified recombinant protein p33, wherein the p33 protein and mutants described above after expression and purification are modified, and the modification conjugate used for modification includes any one of horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocyanate, Cy3 or Cy5, or a combination of at least two of them.

[0019] The present invention provides a coupled recombinant protein p33, which is coupled to the p33 protein and mutants described above after expression and purification, wherein the coupling portion includes a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, and the protein portion and the coupling portion are coupled via a chemical bond or a linker.

[0020] The present invention provides a pharmaceutical composition comprising the protein p33 as described above, and any one or a combination of several other first-line therapeutic drugs clinically used for systemic lupus erythematosus, cutaneous lupus erythematosus, acute graft-versus-host disease or chronic graft-versus-host disease.

[0021] The present invention provides a sequence-modified recombinant p33 protein, wherein a signal peptide is added to the p33 protein to enable its expression in a specific space and time, and / or amino acids and domains having no effect on function are deleted on any side of SEQ ID No. 1 or within the sequence.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This study, published in the journal Nature Communications, used recombinant human p33 protein for the first time in mice with systemic lupus erythematosus (SLE) and graft-versus-host disease (GVHD). The researchers found that recombinant p33 significantly reduced lymphocyte proliferation and inhibited inflammatory cell-induced pathological damage to organs such as the kidneys and liver, thereby effectively treating both diseases. Furthermore, recombinant p33 protein alleviated skin lesions and GVHD inflammation in SLE mice, improving their survival rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results show that recombinant p33 protein alleviates the symptoms of MRL / lpr lupus mice;

[0025] Figure 2 This figure shows the results of recombinant p33 protein alleviating renal pathological damage in MRL / lpr lupus mice;

[0026] Figure 3 This is the result of recombinant p33 protein alleviating renal IgG deposition in MRL / lpr lupus mice;

[0027] Figure 4 This is the result of recombinant p33 protein reducing the urine protein-creatinine ratio in MRL / lpr lupus mice;

[0028] Figure 5 This is the result graph showing that recombinant p33 protein reduces the proportion of abnormal autoreactive T cells in MRL / lpr lupus mice;

[0029] Figure 6 This is the result of single-cell sequencing detection of the proportion of immune cells in MRL / lpr lupus mice after treatment with recombinant p33 protein;

[0030] Figure 7 This is the result of recombinant p33 protein inhibiting the damage of cervical lymph nodes and glomeruli in pristane-induced lupus mice;

[0031] Figure 8 This is the result of recombinant p33 protein alleviating the pathogenesis of GVHD in mice; DETAILED DESCRIPTION

[0032] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0033] The present invention discloses a recombinant p33 protein that can be used to prepare drugs for treating systemic lupus erythematosus, cutaneous lupus erythematosus, acute graft-versus-host disease, and chronic graft-versus-host disease. The protein can be used in combination with glucocorticoids, a first-line clinical drug, to reduce hormone dosage while enhancing therapeutic efficacy.

[0034] Specifically, in a first aspect, the present invention provides a recombinant p33, the amino acid sequence of which is shown in SEQ ID No. 1.

[0035] In a second aspect, the present invention provides polynucleotide sequences encoding the recombinant p33 protein, i.e., C1QBP, according to the first aspect of the present invention. Herein, the polynucleotide may be in the form of DNA or RNA, preferably DNA. DNA forms include natural cDNA and synthetic cDNA, and the DNA may be a coding strand or a template strand. Using conventional techniques, such as PCR, recombinant methods, or synthetic methods, those skilled in the art can readily obtain nucleic acid polynucleotides or fragments thereof encoding the mutants of the present invention. Once obtained, these sequences can be cloned into vectors, transformed or transfected into appropriate cells, and then propagated using conventional host cells to isolate and obtain a large number of nucleic acid molecules.

[0036] In a third aspect, the present invention provides an expression vector, including expression vectors and cloning vectors, which refer to bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors commonly used in the art. Vectors suitable for use in the present invention include, but are not limited to, vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (such as Pichia pastoris vectors and Hansenula vectors), baculovirus vectors for expression in insect cells, vectors for expression in mammalian cells (vaccinia virus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, etc.), plant virus vectors for expression in plants, and various vectors for expression in mammalian mammary glands. In short, any plasmid or vector can be used as long as it can stably replicate in the host cell. Preferred expression vectors contain selectable marker genes, such as bacterial ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, streptomycin resistance genes, and chloramphenicol resistance genes; yeast neomycin resistance genes and Zeocin resistance genes, or yeast defective selectable markers such as His, Leu, and Trp; and eukaryotic neomycin resistance genes, Zeocin resistance genes, dihydrofolate reductase genes, and fluorescent protein marker genes. In a specific embodiment of the present invention, the commercially available pSUMO3 plasmid is used.

[0037] In a fourth aspect, the present invention provides a method for preparing a recombinant p33 protein, comprising the following steps: culturing a transformant, expressing and purifying the culture to obtain the recombinant p33 protein. The recombinant expression of the p33 protein primarily involves: constructing a nucleic acid sequence encoding the p33 protein; ligating the sequence into a pSUMO3 expression vector; and transforming the expression vector into an appropriate host cell for induced expression. The specific steps include:

[0038] (1) The nucleic acid sequence encoding p33 was amplified by PCR and cloned into the pSUMO3 expression vector.

[0039] It should be understood that those skilled in the art can synthesize the corresponding nucleotide sequence based on codon incompatibility and expression preferences in different species, clone it into an appropriate expression vector, and transform the corresponding host. For example, the nucleic acid sequence shown in SEQ ID NO. 1 can be synthesized, cloned into the pET-20b expression vector, and transformed into BL21(DE3) competent cells.

[0040] (2) The transformed host cells were cultured to induce the expression of SUMO3-p33, SUMO3-p33 was isolated and purified, the SUMO3 tag portion was removed, and p33 was obtained by FPLC purification.

[0041] Fifthly, in practical applications, known protein modification schemes can be used to modify the structure and sequence of the recombinant p33 protein as needed to obtain the desired properties. The modification of the recombinant p33 protein includes:

[0042] (1) Marker protein

[0043] Proteins can be labeled for easy detection. The labeling method can be fluorescent labeling, chemiluminescent labeling, radioactive labeling, enzyme-linked labeling, biotin / avidin labeling, magnetic bead labeling, or nanoparticle labeling.

[0044] Preferably, the purified p33 protein is modified, and the modification conjugate used for the modification includes any one of horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocyanate, Cy3 or Cy5, Cy5.5, or a combination of at least two thereof.

[0045] Preferably, the purified p33 protein is modified, and the modified conjugate used in the modification includes the following cytotoxic compounds:

[0046] a) Traditional chemotherapy drugs such as methotrexate, vinblastine, and doxorubicin as cytotoxic payloads.

[0047] b) Tubulin inhibitors include the following categories: ① Tubulin polymerization enhancers, such as the auristatin compounds MMAE (monomethyl auristatin E) and monomethyl auristatin F (MMAF), which act on the β subunit of α- and β-tubulin dimers. ② Tubulin polymerization inhibitors, such as the maytansine compounds DM1 and DM4. ③ Eribuli compounds, i.e., natural products of the polyether macrolide class such as Halichondrin B; ④ Tubulysins, natural antimitotic peptides isolated from slime mold cultures; ⑤ Cryptophycin-1 and cryptophycin-25; ⑥ Kinesin (KSP / EG5 / KIF11) inhibitors.

[0048] c) DNA-damaging agents acting during the cell cycle: topoisomerase I inhibitors, such as DXd (DX-8951 derivative); Enediyne; PDB (Pyrrolo[2,1-c][1,4]benzodiazepines), a pyrrolobenzodiazepine compound found in Streptomyces; Duocarmycin A, a strong DNA alkylating agent isolated from Streptomyces;

[0049] d) RNA-targeting payloads: RNA splicing inhibitors (Thailanstatin and its analogs) and RNA polymerase II inhibitors (Amatoxins).

[0050] e) Radionuclides, including radioisotopes such as I-131, Y-90, Lu-177, Re-188, Bi-213, and At-211. For example, tiuxetan (Y-90), a pure beta-emitting radionuclide, has been used clinically.

[0051] (2) Structural transformation

[0052] The structure of the recombinant p33 protein can be modified, including adding a signal peptide and reducing the domains that have no effect on the function to reduce the size of the protein.

[0053] In a sixth aspect, the present invention provides a recombinant p33 protein and a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic filler, stabilizer, diluent, adjuvant, or other formulation excipient. According to known techniques in the art, the pharmaceutical composition can be prepared into various dosage forms depending on the therapeutic purpose and route of administration. Preferably, the composition is in a unit dosage form, such as a lyophilized agent, tablet, capsule, powder, emulsion, injection, or spray. More preferably, the pharmaceutical composition is in an injectable form, such as a lyophilized powder injection. It is also preferred that the pharmaceutical composition be a liquid formulation, such as one that may contain a buffer (e.g., phosphate buffer, Tris-HCl buffer).

[0054] In a seventh aspect, the recombinant protein, the conjugate, and the pharmaceutical composition are used in the preparation of a drug for treating SLE and GVHD. The pharmaceutical composition comprises:

[0055] First-line treatment for systemic lupus erythematosus (SLE) includes glucocorticoids (methylprednisolone or prednisone), hydroxychloroquine, cyclophosphamide, azathioprine, and the biologic agent belimumab. First-line treatment for graft-versus-host disease (GVHD) includes glucocorticoids (methylprednisolone or prednisone), calcineurin inhibitors (cyclosporine A or tacrolimus), and antithymocyte globulin (ATG).

[0056] Example 1

[0057] Observation of the in vivo efficacy of injection of recombinant p33 protein in treating MRL / lpr lupus mice;

[0058] 1.1 Experimental Animals: 6- to 8-week-old female MRL / lpr mice, weighing 30-40 g, were provided by Spefox (Beijing) Biotechnology Co., Ltd. They were housed in the Central Laboratory Animal Facility (SPF) at the Shanghai Jiao Tong University School of Medicine. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Shanghai Jiao Tong University School of Medicine.

[0059] 1.2 Experimental Procedures and Methods: MRL / lpr mice were randomly divided into two groups: a negative control group (n=10) and a protein treatment group (n=10). The protein treatment group received intravenous injections of recombinant p33 protein (500 μg / mouse) twice weekly through the tail vein; the negative control group received PBS through the tail vein at the same time points. At the end of the experiment, mice were sacrificed, and the following seven assessments were performed to assess the effect of p33 protein on lupus inflammation: ① Organs were observed and photographed. These included the spleen, liver, kidneys, and skin, and images were taken to compare organ size and inflammatory infiltration. ② Lymph nodes were observed and photographed throughout the mice, and the size and number of lymph nodes were counted. ③ Kidney tissue was fixed, embedded in paraffin, and stained with hematoxylin and eosin (HE) to assess glomerular damage. ④ Kidney tissue was fixed, embedded, and stained with immunofluorescence (antibody: mouse IgG) to assess IgG deposition and damage in the glomeruli caused by lupus inflammation. ⑤ Urine was collected from the mice for protein and creatinine testing. ⑥ During the treatment process, peripheral blood was collected from mice weekly for antibody labeling and flow cytometry. The proportion of abnormal autoreactive T cells was observed and a time-dependent effect curve was plotted. At the end of the experiment, peripheral blood and spleens were collected from mice for antibody labeling and flow cytometry, and the proportion of abnormal autoreactive T cells was recorded. Through the above two flow cytometry assays, the effect of p33 protein injection on inflammatory cells in vivo can be clearly determined. ⑦ Single-cell suspensions were prepared from the spleens of mice and then single-cell RNA-seq was performed to determine the effect of the protein on inflammatory cells in vivo at the single-cell level, further providing evidence for the therapeutic effect of p33 protein.

[0060] 1.3 Experimental results:

[0061] 1.3.1 Recombinant p33 protein alleviates systemic organ damage in MRL / lpr lupus mice;

[0062] like Figure 1 As shown, the liver, spleen and kidney of MRL / lpr lupus mice treated with p33 protein were reduced in size ( Figure 1 The skin lesions on the body surface were significantly improved; the number and size of lymph nodes were reduced ( Figure 1 Figures B and C in the middle showed that the infiltration of inflammatory cells was reduced, indicating that inflammatory cells were effectively suppressed.

[0063] 1.3.2 Recombinant p33 protein alleviates renal pathological damage in MRL / lpr lupus mice;

[0064] We provide three pieces of evidence. First, Figure 2 As shown in the figure, the glomerular volume, cell number and basement membrane of the control mice increased. The glomerular tissue morphology of the mice treated with p33 protein returned to normal.Figure 3 As shown, laser confocal microscopy observed that the widely distributed red fluorescent signal in the kidney of the control group represented the deposition of IgG. The deposition of IgG in the glomeruli of mice treated with p33 protein injection was significantly reduced. Third, urine protein was tested weekly using the Urit protein urine test strip, urine protein was tested using the protein quantitative detection kit, and urine creatinine was tested using the creatinine detection kit, and the urine protein / urine creatinine value was calculated. Figure 4 As shown, the protein / creatinine ratio of mice treated with p33 protein injection was significantly reduced, indicating that renal pathological damage was improved.

[0065] 1.3.3 Recombinant p33 protein reduces the proportion of abnormal autoreactive T cells in MRL / lpr lupus mice; Figure 5 The results showed that after injection of p33 protein, abnormal autoreactive T cells (CD3 + B220 + ) ratio decreased significantly ( Figure 5 A), and with time-dependent effects of treatment ( Figure 5 Middle panel B), showing that injection of p33 protein reduced the number of autoreactive T cells, thereby alleviating inflammation.

[0066] 1.3.4 The immunomodulatory effect of recombinant p33 protein is to inhibit T cell inflammation by inducing the production of suppressor granulocytes. Figure 6 As shown in the results, the single-cell RNA-seq results of the spleen single-cell suspension of MRL / lpr lupus mice showed that the proportion of autoreactive T cell populations decreased significantly after treatment, while the proportion of a group of cells with immunosuppressive activity, namely granulocyte-like myeloid-derived suppressor cells (G-MDSCs), increased significantly, indicating that recombinant p33 protein inhibited the activity of autoreactive T cells by inducing G-MDSCs, thereby alleviating lupus damage.

[0067] Example 2

[0068] In vivo observation of the therapeutic effect of injection of recombinant p33 protein in the treatment of pristane-induced lupus mice:

[0069] 2.1 Experimental Animals: Pristane-induced lupus mice (also known as pristane-induced lupus mice): C57BL / 6J mice were injected intraperitoneally with 500 μL of pristane per mouse for lupus modeling. The mice were approximately 6-8 weeks old and weighed 20-30 g. They were housed in the Central Laboratory Animal Facility (SPF) at Shanghai Jiao Tong University School of Medicine. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of Shanghai Jiao Tong University School of Medicine.

[0070] 2.2 Experimental Procedures and Methods: Mice with pristane-induced lupus were randomly divided into two groups: a negative control group (n=8) and a protein-treated group (n=8). The protein-treated group received intravenous injections of recombinant p33 protein (500 μg / mouse) twice weekly via the tail vein; the negative control group received PBS via the tail vein at the same time points. At the end of the experiment, mice were sacrificed, and the following assays were performed to assess the effect of p33 protein on lupus inflammation: ① Lymph nodes were harvested and photographed, and the size and number of lymph nodes were counted. ② Kidney tissue was fixed, embedded in paraffin, and stained with hematoxylin and eosin (HE) to assess glomerular damage.

[0071] 2.3 Experimental results:

[0072] Recombinant p33 protein alleviates renal damage and systemic inflammatory cell infiltration in MRL / lpr lupus mice. Figure 7 As shown, the number and size of lymph nodes in lupus mice treated with p33 protein were reduced ( Figure 7 Figure A in the middle panel indicates that inflammatory cell infiltration was reduced, indicating that inflammatory cells were effectively suppressed. At the same time, the glomerular volume of the control group mice increased, the basement membrane thickened and was incomplete, while the glomerular tissue morphology of the mice in the recombinant p33 protein treatment group returned to normal ( Figure 7 (B in the middle). The results showed that p33 protein injection significantly inhibited the proliferation of inflammatory cells, reduced inflammatory cell infiltration in organs, and alleviated renal pathological damage in mice with ulcers.

[0073] Example 3

[0074] Observation of the in vivo efficacy of injection of recombinant p33 protein in treating GVHD mice;

[0075] 3.1 GVHD model construction:

[0076] 3.1.1 Experimental Animals: 6-8 week old female NSG immunodeficient mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. License number: SCXK(Su)-0008. Animals were housed in the Central Laboratory Animal Facility (SPF) at Shanghai Jiao Tong University School of Medicine. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of Shanghai Jiao Tong University School of Medicine.

[0077] 3.1.2 GVHD modeling steps:

[0078] (1) Collect 5-10 ml of healthy donor peripheral blood from the patient and dilute it by adding an equal volume of 1× PBS buffer. Use a pipette to slowly transfer the diluted peripheral blood along the tube wall to the upper layer of Ficoll separation solution (lymphocyte separation solution).

[0079] (2) Centrifuge at 2,000 rpm for 30 min in a low-temperature horizontal centrifuge. Aspirate the white, cloudy layer of cells at the interface between the upper and middle liquid layers, which are mononuclear cells. Wash twice with saline at low speed, resuspend in 0.5 mL of saline or 1× PBS buffer, and count the cells.

[0080] (3) Human mononuclear cells were injected into the tail vein, and 1×10 6 Before injection, cells were resuspended in PBS and the injection volume was 200 μL.

[0081] (4) Modeling success evaluation indicators:

[0082] It includes general indicators and cell indicators. General indicators refer to the observation of mice after inoculation for symptoms such as decreased activity, weight loss, hunched back, diarrhea, hair loss, hemiplegia, etc. The mice were weighed and recorded weekly. Cell indicators refer to the detection of human cells (hCD45) by flow cytometry in peripheral blood collected from mice 2 weeks after inoculation. + )Proportion.

[0083] 3.2 Experimental procedures and methods: GVHD model mice were randomly divided into two groups: a negative control group of 8 mice and a protein treatment group of 8 mice. The protein treatment group was intravenously injected with recombinant p33 protein, 500 μg / mouse, twice a week through the tail vein; the negative control group was injected with PBS through the tail vein at the same time point. At the end of the experiment, the mice were sacrificed and then tested to determine the effect of p33 protein on lupus inflammation: ① During the treatment, peripheral blood of the mice was collected for antibody labeling and flow cytometry detection. By observing the expression of human T cells (hCD3 + ) ratio to determine the effect of p33 protein injection on inflammatory cells in vivo. ② Mouse livers were harvested for immunohistochemistry (IHC) staining (antibody: human hCD45) to determine the proportion of human cell infiltration. A higher proportion of human cell infiltration indicates more severe GVHD. ③ The survival of mice after the GVHD model was established was recorded to evaluate the long-term efficacy of recombinant p33 protein against GVHD.

[0084] 2.3 Experimental results:

[0085] Recombinant p33 protein reduces systemic inflammatory cell infiltration and survival of GVHD mice. Figure 8 As shown, human T cells (hCD3 + ) ratio decreased significantly ( Figure 8 Figure A), the survival time of mice was significantly prolonged ( Figure 8 Figure B), and immunohistochemistry IHC staining (antibody: human hCD45) showed that human inflammatory cells (hCD45 +The ratio of CD4+T cells to CD8+T cells decreased significantly. The results showed that injection of p33 protein significantly reduced the number of inflammatory T cells in GVHD model and had a significant effect on GVHD.

[0086] In the drawings, Figure 1 The results of recombinant p33 protein reducing the pathogenic symptoms of MRL / lpr lupus mice. In the A figure, the gross photographs of the organs of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) are shown. In the B figure, the gross photographs of the external appearance of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) are shown. The red circles indicate the lymph nodes and lupus lesions on the surface, respectively. In the C figure, the photographs of the lymph nodes of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) are shown. The results show that the organs of the mice treated with p33 protein are smaller, the number and size of the lymph nodes are reduced, and the skin lesions on the surface are significantly improved, indicating that the inflammatory cells are effectively inhibited.

[0087] Figure 2 The results of recombinant p33 protein reducing the pathological damage of the kidneys of MRL / lpr lupus mice. The results of HE staining of the kidneys of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) are compared. The glomerular volume of the control group mice is increased, the number of cells is increased, and the basement membrane is thickened. The glomerular tissue morphology of the mice treated with p33 protein returns to normal.

[0088] Figure 3 The results of recombinant p33 protein reducing the IgG deposition in the kidneys of MRL / lpr lupus mice. The results of immunofluorescence detection of IgG deposition in the kidneys of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) are compared. The kidney sections of the mice are immunostained with specific antibody IgG, the secondary antibody is coupled with red fluorescence, and laser confocal microscopy is used for observation. It can be observed that the control group has a wide distribution of red fluorescence signals representing IgG deposition in the kidneys, and the deposition of IgG in the glomeruli of the mice treated with p33 protein is significantly reduced, indicating that the kidney damage of the lupus mice is improved.

[0089] Figure 4 The results of recombinant p33 protein reducing the protein-to-creatinine ratio in the urine of MRL / lpr lupus mice. The urine of MRL / lpr lupus mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein) is detected, and the results show that the protein-to-creatinine ratio of the mice treated with p33 protein is significantly reduced, indicating that the pathological damage of the kidneys is improved.

[0090] Figure 5The results of the recombinant p33 protein reducing the proportion of abnormal autoreactive T cells in MRL / lpr lupus mice; peripheral blood and spleen were collected from the control group (PBS injection) and protein treatment group (p33 protein injection) of MRL / lpr lupus mice, and single cell suspensions were prepared and labeled with antibodies for flow cytometry analysis. The results showed that abnormal autoreactive T cells (CD3 + B220 + ) ratio was significantly reduced in the protein treatment group ( Figure 5 A), and with time-dependent effects of treatment ( Figure 5 Middle panel B), showing that injection of p33 protein reduced the number of autoreactive T cells, thereby alleviating inflammation.

[0091] Figure 6 Figure 3. Single-cell sequencing analysis of immune cell proportions in MRL / lpr lupus mice after treatment with recombinant p33 protein. Spleens were collected from MRL / lpr lupus mice in the control group (injected with PBS) and the protein-treated group (injected with p33 protein), and single-cell suspensions were prepared for single-cell RNA-seq. The results showed a significant decrease in the proportion of autoreactive T cell populations after treatment, while a significant increase in the proportion of granulocyte-like myeloid-derived suppressor cells (G-MDSCs), a group of cells with immunosuppressive activity, was observed. This suggests that recombinant p33 protein suppresses the activity of autoreactive T cells by inducing G-MDSCs, thereby alleviating lupus damage.

[0092] Figure 7 Figures show the results of recombinant p33 protein inhibiting pristane-induced cervical lymph node and glomerular damage in lupus mice. Panel A shows the number of lymph nodes in the control group (PBS injection) and the protein-treated group (p33 protein injection) of lupus mice. Panel B shows HE staining of the kidneys of lupus mice in the control group (PBS injection) and the protein-treated group (p33 protein injection) of lupus mice. The results show that p33 protein treatment reduced the number and size of lymph nodes in pristane-induced lupus mice, and significantly improved kidney pathological damage.

[0093] Figure 8Results of reducing the incidence of GVHD in mice by recombinant p33 protein. NSG mice were injected with human peripheral blood mononuclear cells to induce a GVHD model, and then treated with PBS (control group) or recombinant p33 protein (protein treatment group). In which, Figure A is the proportion of human T cells in the spleen of GVHD mice in the control group and the protein treatment group. Figure B is a survival curve of GVHD mice in the control group (injected with PBS) and the protein treatment group (injected with p33 protein). Figure C is an immunohistochemical (IHC) staining with hCD45 antibody to show the infiltration of human immune cells in the liver. The results show that the effector cells of GVHD, i.e. human T cells, are significantly inhibited after p33 protein treatment, thereby reducing inflammatory damage and prolonging the survival of mice.

[0094] The above description is only the preferred embodiment of the present application, and is not any form and substantial limitation of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application. For those skilled in the art, without departing from the spirit and scope of the present application, some changes, modifications and equivalent changes made by using the above disclosed technical content are equivalent embodiments of the present application; at the same time, any equivalent changes, modifications and evolution of the above-mentioned embodiments according to the essential technology of the present application are still within the scope of the technical solutions of the present application.

Claims

1. A recombinant protein p33, also known as p32, gC1qR, HABP1 and C1QBP, characterized in that The p33 amino acid sequence is shown in SEQ ID No. 1; the classification identifier in the NCBI international nucleotide sequence database, namely, Gene ID is 708.

2. An expression vector, characterized in that: The expression vector comprises a nucleotide sequence corresponding to the amino acid sequence of the recombinant protein p33 according to claim 1.

3. Use of recombinant protein p33 in the preparation of a medicament for treating autoimmune diseases, characterized in that: Such diseases include systemic lupus erythematosus, cutaneous lupus erythematosus, acute graft-versus-host disease, and chronic graft-versus-host disease.

4. Use of recombinant protein p33 in the preparation of drugs for treating autoimmune diseases by inhibiting lymphocyte proliferation and inflammatory cells.

5. Use of recombinant protein p33 in the preparation of a drug for treating autoimmune diseases by inducing granulocytic myeloid-derived suppressor cells.

6. Use of recombinant protein p33 in the preparation of drugs for treating autoimmune diseases by inhibiting T cells.

7. A method for preparing recombinant protein p33, characterized in that: The preparation method comprises constructing a nucleotide sequence encoding protein p33; introducing the nucleotide sequence into an expression vector; transforming the expression vector into a suitable host cell, and then inducing the host cell to express the recombinant protein p33; the host cell comprises Escherichia coli, CHO cells or 293 cells.

8. A modified recombinant protein p33, characterized in that The p33 protein and mutant according to claim 1 are modified after expression and purification, wherein the modification conjugate used in the modification comprises any one of horseradish peroxidase, alkaline phosphatase, biotin, fluorescein isothiocyanate, Cy3 or Cy5, or a combination of at least two thereof.

9. A coupled recombinant protein p33, characterized in that The p33 protein and mutant according to claim 1 after expression and purification are coupled, wherein the coupling portion includes a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, and the protein portion and the coupling portion are coupled via a chemical bond or a linker.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the protein p33 as claimed in claim 1 and any one or a combination of other first-line therapeutic drugs clinically used for systemic lupus erythematosus, cutaneous lupus erythematosus, acute graft-versus-host disease or chronic graft-versus-host disease.

11. A sequence-engineered recombinant p33 protein, characterized in that: A signal peptide is added to the p33 protein to enable its expression in a specific space and time, and / or amino acids and structural domains that have no effect on the function are reduced on any side of SEQ ID No. 1 or within the sequence.

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