An immunosuppressive molecule and its application in inhibiting T cell activation

Abstract

The present invention discloses an immunosuppressive molecule and its application in inhibiting T cell activation. The immunosuppressive molecule is sVASN, and its corresponding amino acid sequence is shown in SEQ ID NO: 1. The present invention finds for the first time that sVASN can effectively inhibit T cell activation, provides a new treatment scheme for the research field of hyperimmune response treatment and tumor treatment, and provides new ideas and strategies for developing drugs that inhibit hyperimmune response and fight tumors. The present invention has important scientific significance and clinical application value, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an immunosuppressive molecule and its application in inhibiting T cell activation. More specifically, the immunosuppressive molecule is sVASN. Background Art

[0002] Tumor is one of the most deadly diseases at present. In the past decade, immunotherapy has set off a new revolution in the field of tumor treatment and brought new hope to cancer patients. Immune checkpoint inhibitors (ICIs) therapy is one of the most popular tumor immunotherapies and has been approved as a first-line drug for the treatment of various tumors. However, single ICIs therapy has defects such as limited efficacy and easy drug resistance. Therefore, the development of combined treatment strategies to improve the efficacy of ICIs has become a research hotspot in the anti-tumor field. Human VASN protein is a typical type I transmembrane glycoprotein with a full length of 673 amino acids. Bioinformatics predicts a relative molecular weight of approximately 72kD. Its extracellular domain can be cleaved and detached by adisintegrin and metalloprotease 17 (ADAM17) to become soluble VASN (sVASN). VASN was first discovered and reported as a TGF-β binding protein in 2004. It is mainly expressed in the aorta, the vascular-rich placenta, and the kidneys. It can regulate the aortic injury response during vascular injury repair by participating in the TGF-β pathway. VASN is highly expressed in breast cancer cells, glioma cells, and liver cancer cells.

[0003] The present invention treats activated human T cells (Jurkat) by adding exogenous sVASN in vitro, and finds that T cell activation is inhibited, which is manifested by the reduction of IL-2 expression and the reduction of cell surface CD26 expression (CD26 is a marker of T cell activation), and the changes are sVASN dose-dependent. After blocking the cell surface CD71 with CD71 blocking peptide, the inhibitory effect of sVASN on T cell activation is weakened. The VASN gene and TGFβ1 gene in the VASN high-expressing cell U118 (human glioma cell) were knocked down in a gradient manner, and then co-cultured with T cells. The IL-2 secretion was detected and it was found that the secretion of IL-2 was affected by the gradient-changing VASN of tumor cells and showed a gradient change, but was unrelated to the change of TGFβ1, indicating that the immunosuppressive effect of Jurkat cells is caused by the change of VASN in tumor cells rather than TGFβ1. Using VASN-high-expressing liver cancer cells HepG2, the culture supernatant was tested and it was found that the sVASN content was high. HepG2 cells were treated with ADAM17 (which can cleave membrane-type VASN to form sVASN and release it into the cytoplasm) inhibitors (BB94 or TAPI), and the cell culture supernatant was taken or directly co-cultured with T cells to detect IL-2 secretion. The results showed that ADAM17 was inhibited, VASN cleavage was reduced, that is, sVASN was reduced, the immunosuppressive effect was weakened, and IL-2 increased. The expression of IL-2 was detected by co-culturing HepG2 cell culture supernatants with different concentrations with Jurkat cells. The results showed that the degree of inhibition of T cell activation by HepG2 culture supernatants with different concentrations was different. The more concentrated the concentration (the higher the sVASN concentration), the more obvious the activation inhibition effect, and the more IL-2 was reduced. A variety of tumor cells (liver cancer cell HepG2, glioma cell U251, colorectal cancer cell HCT116, human endometrial cancer cell Ishikaw) were co-cultured with T cells after knocking down or overexpressing VASN, and the changes in T cell secretion of IL-2 were detected. The results showed that in different tumor cells, knocking down VASN caused increased activation of Jurkat cells, while overexpression of VASN caused inhibition of Jurkat cell activation.

[0004] After overexpression of VASN in tumor cells or epithelial cells, the changes in VASN in feeder cells (tumor cells or epithelial cells) and T cells were detected with Jurkat cells. In unactivated T cells, overexpression of VASN in feeder cells did not significantly change the VASN level in T cells; while in activated T cells, overexpression of VASN in feeder cells significantly increased the expression of VASN in T cells, indicating that the inhibitory effect of VASN on T cells is regulated after entering T cells. The changes in mTOR were detected by adding exogenous sVASN to activated T cells in vitro. As the concentration of sVASN increased, mTOR (total amount) remained unchanged, and phosphorylated mTOR (p-mTOR) gradually decreased, indicating that the inhibitory effect of sVASN on activated T cells was achieved by affecting phosphorylated mTOR. In summary, the inventors of the present invention application proposed that VASN (sVASN) can be used as a new immunosuppressive molecule, and proposed its potential application in combined immunotherapy strategies with CD71. Summary of the invention

[0005] The purpose of the present invention is to provide an immunosuppressive molecule sVASN and its application in inhibiting T cell activation. The present invention discovers for the first time that sVASN can effectively inhibit T cell activation, providing new ideas and strategies for the development of drugs that inhibit hyperimmune response and anti-tumor, and has broad prospects for clinical application.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides use of sVASN in inhibiting T cell activation.

[0008] Furthermore, the amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0009] Preferably, the sVASN inhibits T cell activation by inhibiting the mTOR signaling pathway;

[0010] Preferably, the functional receptors of the sVASN that inhibit T cell activation include CD71;

[0011] More preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0012] In the present invention, the sVASN refers to soluble VASN (sVASN) secreted by tumor cells, and its corresponding amino acid sequence is shown in SEQ ID NO: 1. The present invention first experimentally verifies that the sVASN can be used as an immunosuppressive molecule that effectively inhibits T cell activation. In a specific embodiment of the present invention, the sVASN includes exogenously expressed sVASN and / or sVASN endogenously expressed and secreted by tumor cells.

[0013] In the present invention, the VASN refers to the soluble form of endogenous VASN expressed by tumor cells or the soluble form of VASN expressed and purified by CHO and HEK293T cells in vitro. The Gene ID of VASN in NCBI (https: / / www.ncbi.nlm.nih.gov / ) is 114990, and its corresponding amino acid sequence is shown in SEQ ID NO: 2. Human VASN protein is a typical type I transmembrane glycoprotein with a total length of 673 amino acids. Its extracellular domain can be cleaved and detached by disintegrin and metalloprotease 17 (ADAM17) to become soluble VASN (soluble VASN, sVASN). sVASN contains important functional domains such as tandem leucine-rich (LRR) domains, epidermal growth factor (EGF)-like domains and fibronectin III (FN3) domains, which are the structural basis of its biological function.

[0014] In some embodiments, the inhibition includes in vitro inhibition and in vivo inhibition. In a preferred embodiment, the inhibition is in vitro inhibition.

[0015] In addition, the present invention also provides use of the sVASN described in the first aspect of the present invention in the preparation of a drug for inhibiting T cell activation.

[0016] The second aspect of the present invention provides the use of any one or any combination of CD71, CD71 promoter, sVASN, and sVASN promoter in inhibiting T cell activation.

[0017] Furthermore, the amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0018] Preferably, the CD71 promoter includes an active peptide that promotes CD71 expression, a protein that promotes CD71 expression, an oligonucleotide that promotes CD71 expression, a small molecule compound that promotes CD71 expression, and / or a vector that overexpresses CD71;

[0019] Preferably, the sVASN promoter includes an active peptide that promotes sVASN expression, a protein that promotes sVASN expression, an oligonucleotide that promotes sVASN expression, a small molecule compound that promotes sVASN expression, and / or a vector that overexpresses sVASN;

[0020] More preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0021] In the present invention, the CD71 is a glycoprotein expressed on the cell surface, which is composed of two homodimeric subunits connected by disulfide bonds. CD71 contains 760 amino acids, including a short N-terminal intracellular domain (amino acids 1 to 67), a hydrophobic transmembrane domain (amino acids 68 to 88) and a large globular extracellular domain (amino acids 89 to 760) containing a transferrin binding site at the C-terminus. CD71 is the main receptor responsible for regulating the cellular uptake of iron in transferrin, and is usually highly expressed in proliferating cells and cancer cells in the pancreas, colon, lung, breast, bladder and lymphoid organs.

[0022] In some embodiments, the CD71 promoter or sVASN promoter refers to any substance that can upregulate the expression of CD71 or sVASN, increase the activity of CD71 or sVASN, improve the stability of CD71 or sVASN, increase the effective action time of CD71 or sVASN, or promote the transcription and translation of CD71 or sVASN, and these substances can be used in the present invention. The CD71 or sVASN promoter includes but is not limited to: nucleic acid promoter, protein promoter, for example, a vector or construct overexpressing CD71 or sVASN, CD71 or sVASN protein or its active peptide, etc.

[0023] In the present invention, those skilled in the art can select the specific type of the inhibitor or promoter according to needs and common knowledge in the art. The selection of other types of inhibitors or promoters for specific targets that can exert similar effects to the inhibitors or promoters used in the embodiments of the present invention can be obtained without the need for creative work by those skilled in the art.

[0024] In some embodiments, the inhibition includes in vitro inhibition and in vivo inhibition. In a preferred embodiment, the inhibition is in vitro inhibition.

[0025] In addition, the present invention also provides the use of any one or any combination of CD71, CD71 promoter, sVASN, and sVASN promoter described in the second aspect of the present invention in the preparation of a drug for inhibiting T cell activation.

[0026] The third aspect of the present invention provides the use of any one or any combination of sVASN inhibitors, CD71 inhibitors, and agents that block the binding of sVASN to its functional receptor CD71 in promoting T cell activation.

[0027] Furthermore, the amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0028] Preferably, the sVASN inhibitor includes a blocking peptide targeting sVASN, an antibody specifically targeting sVASN, an interfering molecule specifically interfering with sVASN expression, a CRISPR gene editing reagent for sVASN, a homologous recombination reagent for sVASN, and / or a site-directed mutagenesis reagent for sVASN;

[0029] Preferably, the CD71 inhibitor includes a blocking peptide specifically targeting CD71, an inhibitor specifically promoting CD71 degradation, an antibody specifically targeting CD71, an interfering molecule specifically interfering with CD71 expression, a CRISPR gene editing reagent for CD71, a homologous recombination reagent for CD71 and / or a site-directed mutagenesis reagent for CD71;

[0030] More preferably, the interfering molecule comprises shRNA, siRNA, miRNA, antisense nucleic acid and / or nucleic acid aptamer;

[0031] More preferably, the inhibitor that specifically promotes CD71 degradation is Ferristatin II;

[0032] More preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0033] In some embodiments, the sVASN inhibitor or CD71 inhibitor refers to any substance that can downregulate the expression of sVASN or CD71, reduce the activity of sVASN or CD71, reduce the stability of sVASN or CD71, reduce the effective action time of sVASN or CD71, or inhibit the transcription and translation of sVASN or CD71, and these substances can be used in the present invention. The sVASN or CD71 inhibitor includes, but is not limited to: agents that inhibit the transcriptional activity of sVASN or CD71, agents that inhibit the transcriptional level of sVASN or CD71 mRNA, agents that promote the degradation of sVASN or CD71 mRNA, siRNA targeting sVASN or CD71, shRNA targeting sVASN or CD71, agents that inhibit the translation of sVASN or CD71 mRNA, agents that specifically recognize the guide nucleic acid of sVASN or CD71 and cleave it to reduce its expression level, dsRNA targeting sVASN or CD71, miRNA targeting sVASN or CD71, antisense nucleic acid targeting sVASN or CD71, specific antibodies targeting sVASN or CD71, small molecule compounds that inhibit the activity of sVASN or CD71 protein, related agents for knocking out sVASN or CD71 by administering targeting vectors, and related agents for reducing or losing the activity of sVASN or CD71 protein by introducing mutations into the protein, etc.

[0034] In some embodiments, the promotion includes in vitro promotion and in vivo promotion. In a preferred embodiment, the promotion is in vitro promotion.

[0035] In addition, the present invention also provides the use of any one or any combination of the sVASN inhibitors, CD71 inhibitors, and agents that block the binding of sVASN to its functional receptor CD71 described in the third aspect of the present invention in the preparation of a drug for promoting T cell activation.

[0036] The fourth aspect of the present invention provides the use of a VASN promoter in inhibiting T cell activation;

[0037] Preferably, the VASN promoter includes a vector that overexpresses VASN, an active peptide that promotes VASN expression, a protein that promotes VASN expression, an oligonucleotide that promotes VASN expression, and / or a small molecule compound that promotes VASN expression.

[0038] In some embodiments, the VASN promoter refers to any substance that can upregulate the expression of VASN, increase the activity of VASN, improve the stability of VASN, increase the effective action time of VASN, or promote the transcription and translation of VASN, and these substances can be used in the present invention. The VASN promoter includes but is not limited to: nucleic acid promoters, protein promoters, for example, vectors or constructs that overexpress VASN, VASN proteins or active peptides thereof, etc.

[0039] In some embodiments, the inhibition includes in vitro inhibition and in vivo inhibition. In a preferred embodiment, the inhibition is in vitro inhibition.

[0040] In addition, the present invention also provides the use of the VASN promoter described in the fourth aspect of the present invention in the preparation of a drug for inhibiting T cell activation.

[0041] The fifth aspect of the present invention provides the use of VASN inhibitors in promoting T cell activation;

[0042] Preferably, the VASN inhibitor includes a blocking peptide targeting VASN, an antibody specifically targeting VASN, an interfering molecule specifically interfering with VASN expression, a CRISPR gene editing reagent targeting VASN, a homologous recombination reagent targeting VASN, and / or a site-directed mutagenesis reagent targeting VASN;

[0043] More preferably, the interfering molecule comprises shRNA, siRNA, miRNA, antisense nucleic acid and / or nucleic acid aptamer.

[0044] In some embodiments, the VASN inhibitor refers to any substance that can downregulate the expression of VASN, reduce the activity of VASN, reduce the stability of VASN, reduce the effective action time of VASN, or inhibit the transcription and translation of VASN, and these substances can be used in the present invention. The VASN inhibitor includes but is not limited to: agents that inhibit the transcriptional activity of VASN, agents that inhibit the transcription level of VASN mRNA, agents that promote the degradation of VASN mRNA, siRNA for VASN, shRNA for VASN, agents that inhibit the translation of VASN mRNA, agents that specifically recognize the guide nucleic acid of VASN and cut it to reduce its expression level, dsRNA for VASN, miRNA for VASN, antisense nucleic acid for VASN, specific antibodies targeting VASN, small molecule compounds that inhibit the activity of VASN protein, related agents that knock out VASN by administering targeting vectors, and related agents that reduce or lose the activity of VASN protein by introducing mutations in VASN protein, etc.

[0045] In some embodiments, the promotion includes in vitro promotion and in vivo promotion. In a preferred embodiment, the promotion is in vitro promotion.

[0046] In addition, the present invention also provides use of the VASN inhibitor described in the fifth aspect of the present invention in the preparation of a drug for promoting T cell activation.

[0047] The sixth aspect of the present invention provides an immunosuppressive molecule sVASN.

[0048] Furthermore, the amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0049] Preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0050] The seventh aspect of the present invention provides any one of the following pharmaceutical compositions:

[0051] (1) A pharmaceutical composition comprising any one or more of sVASN, sVASN promoter, CD71, CD71 promoter, and VASN promoter;

[0052] In some embodiments, the pharmaceutical composition may further comprise a second therapeutic agent, which is other immunosuppressants that can be used to inhibit T cell activation, including but not limited to cyclosporine A, tacrolimus, sirolimus, everolimus, cyclophosphamide, rapamycin, methotrexate, azathioprine, mycophenolate mofetil, dexamethasone, and senlitep.

[0053] (2) A pharmaceutical composition comprising any one or more of a sVASN inhibitor, a CD71 inhibitor, an agent that blocks the binding of sVASN to its functional receptor, and a VASN inhibitor;

[0054] The amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0055] Preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0056] In some embodiments, the pharmaceutical composition may further comprise a second therapeutic agent, which is other drugs that can be used to treat and / or prevent tumors. The other drugs that can be used to treat and / or prevent tumors include, but are not limited to, chemotherapy drugs (e.g., paclitaxel, docetaxel, cabazitaxel, S-1 tablets, capecitabine tablets, vinorelbine tablets, doxorubicin, epirubicin, daunorubicin, pirarubicin, etoposide, irinotecan, mitoxantrone, cisplatin, carboplatin, lobaplatin, nedaplatin, oxaliplatin, gemcitabine, 5-fluorouracil, cyclophosphamide, ifosfamide), targeted drugs (e.g., gefitinib tablets, erlotinib tablets), anti-vascular drugs (e.g., apatinib tablets, anlotinib tablets), and endocrine therapy drugs (e.g., letrozole tablets, anastrozole tablets, exemestane tablets).

[0057] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0058] An eighth aspect of the present invention provides a pharmaceutical preparation.

[0059] Further, the pharmaceutical preparation comprises any one of the pharmaceutical compositions described in the seventh aspect of the present invention;

[0060] Preferably, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or excipient;

[0061] More preferably, the pharmaceutically acceptable carrier and / or excipient comprises a diluent, a binder, a surfactant, a humectant, an adsorbent carrier, a lubricant, a filler and / or a disintegrant.

[0062] Preferably, the dosage form of the pharmaceutical preparation includes injection, tablet, capsule, powder, granule, ointment, suppository, inhalant, gel, microsphere and / or aerosol.

[0063] In some embodiments, the diluent includes but is not limited to: lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to: sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. The humectant includes but is not limited to: glycerol, starch, etc. The adsorption carrier includes but is not limited to: starch, lactose, bentonite, soap clay, etc. The lubricant includes but is not limited to: zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose acid ester, magnesium lauryl sulfate, etc. The filler includes but is not limited to: mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrants include, but are not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides, and the like.

[0064] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0065] In some embodiments, the administration methods of the pharmaceutical composition or pharmaceutical preparation include but are not limited to: subcutaneous injection, intramuscular injection, intravenous injection, oral administration, rectal administration, vaginal administration, nasal administration, transdermal administration, subconjunctival administration, intraocular administration, orbital administration, retro-ocular administration, retinal administration, choroidal administration, intrathecal injection, and intraperitoneal injection.

[0066] In some embodiments, the dosage form of the pharmaceutical composition or pharmaceutical preparation is not particularly limited. In some embodiments, the pharmaceutical composition or pharmaceutical preparation can be prepared into an injection or oral preparation, including injection, tablet, capsule, pill, suppository, aerosol, oral liquid preparation, granule, powder, sustained release, nano preparation, syrup, wine, tincture, dew. Those skilled in the art can select a suitable dosage form known in the art according to actual needs (e.g., improving the bioavailability of the drug, improving the patient's medication compliance, etc.).

[0067] In some embodiments, the dosage of the pharmaceutical composition or pharmaceutical preparation of the present invention is not limited, as long as the desired therapeutic effect or preventive effect can be obtained, and can be appropriately determined according to the subject's symptoms, gender, age, etc. The dosage of the pharmaceutical composition or pharmaceutical preparation of the present invention can be determined in detail using, for example, the therapeutic effect or preventive effect on the disease as an indicator.

[0068] A ninth aspect of the present invention provides any of the following methods:

[0069] (1) A method for inhibiting T cell activation in vitro, the method comprising administering any one or more of sVASN, sVASN promoter, CD71, CD71 promoter, and VASN promoter to a system containing T cells in need thereof;

[0070] (2) A method for promoting T cell activation in vitro, the method comprising administering any one or more of a sVASN inhibitor, a CD71 inhibitor, an agent that blocks the binding of sVASN to its functional receptor, and a VASN inhibitor to a system containing T cells in need thereof;

[0071] The amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0072] Preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells.

[0073] In some embodiments, the system comprising T cells is selected from: a cell system comprising T cells, a solution system comprising T cells, a tissue system comprising T cells, an organ system comprising T cells, or an animal system comprising T cells.

[0074] The tenth aspect of the present invention provides any of the following applications:

[0075] (1) Use of any one or more of sVASN, sVASN promoter, CD71, CD71 promoter, and VASN promoter in the preparation of a drug for treating and / or preventing diseases related to T cell hyperactivation;

[0076] (2) Use of any one or more of a sVASN inhibitor, a CD71 inhibitor, an agent that blocks the binding of sVASN to its functional receptor, or a VASN inhibitor in the preparation of a drug for treating and / or preventing tumors;

[0077] The amino acid sequence of the sVASN is shown in SEQ ID NO: 1;

[0078] Preferably, the sVASN promoter includes an active peptide that promotes sVASN expression, a protein that promotes sVASN expression, an oligonucleotide that promotes sVASN expression, a small molecule compound that promotes sVASN expression, and / or a vector that overexpresses sVASN;

[0079] Preferably, the CD71 promoter includes an active peptide that promotes CD71 expression, a protein that promotes CD71 expression, an oligonucleotide that promotes CD71 expression, a small molecule compound that promotes CD71 expression, and / or a vector that overexpresses CD71;

[0080] Preferably, the sVASN inhibitor includes a blocking peptide targeting sVASN, an antibody specifically targeting sVASN, an interfering molecule specifically interfering with sVASN expression, a CRISPR gene editing reagent for sVASN, a homologous recombination reagent for sVASN, and / or a site-directed mutagenesis reagent for sVASN;

[0081] Preferably, the CD71 inhibitor includes a blocking peptide specifically targeting CD71, an inhibitor specifically promoting CD71 degradation, an antibody specifically targeting CD71, an interfering molecule specifically interfering with CD71 expression, a CRISPR gene editing reagent for CD71, a homologous recombination reagent for CD71 and / or a site-directed mutagenesis reagent for CD71;

[0082] More preferably, the interfering molecule comprises shRNA, siRNA, miRNA and / or antisense nucleic acid;

[0083] More preferably, the inhibitor that specifically promotes CD71 degradation is Ferristatin II;

[0084] Preferably, the VASN promoter includes a vector that overexpresses VASN, an active peptide that promotes VASN expression, a protein that promotes VASN expression, an oligonucleotide that promotes VASN expression, and / or a small molecule compound that promotes VASN expression;

[0085] Preferably, the VASN inhibitor includes a blocking peptide targeting VASN, an antibody specifically targeting VASN, an interfering molecule specifically interfering with VASN expression, a CRISPR gene editing reagent targeting VASN, a homologous recombination reagent targeting VASN, and / or a site-directed mutagenesis reagent targeting VASN;

[0086] More preferably, the interfering molecule comprises shRNA, siRNA, miRNA, antisense nucleic acid and / or nucleic acid aptamer;

[0087] More preferably, the sVASN includes exogenously expressed sVASN and sVASN endogenously expressed and secreted by tumor cells;

[0088] Preferably, the T cell overactivation-related diseases include rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, ulcerative colitis, osteoarthritis, atopic dermatitis, psoriasis, alopecia areata, asthma, eczema, allergic pneumonia, allergic rhinitis, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, hyperthyroidism, ankylosing spondylitis, fibrositis, polyarteritis nodosa, inflammatory bowel disease, graft-versus-host disease and / or organ transplant rejection;

[0089] Preferably, the tumor comprises lung cancer, liver cancer, gastric cancer, breast cancer, esophageal cancer, thyroid cancer, uterine body cancer, cervical cancer, pancreatic cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, lymphoma, kidney cancer, gallbladder cancer, ovarian cancer, oral cancer, laryngeal cancer, melanoma, testicular cancer, multiple myeloma, astrocytoma, glioma, medulloblastoma, pituitary tumor, endometrial cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma and / or anaplastic large cell lymphoma.

[0090] In addition, the present invention also provides a method for treating and / or preventing diseases related to T cell overactivation, the method comprising the following steps: administering a therapeutically and / or preventive effective amount of any one or any combination of the sVASN, sVASN promoter, CD71, CD71 promoter, VASN promoter, pharmaceutical composition, and pharmaceutical preparation described in the present invention to a subject in need.

[0091] In addition, the present invention also provides a method for treating and / or preventing tumors, which comprises the following steps: administering a therapeutically and / or preventively effective amount of any one or any combination of the sVASN inhibitors, CD71 inhibitors, agents that block the binding of sVASN to its functional receptors, and VASN inhibitors described in the present invention to a subject in need thereof.

[0092] Compared with the prior art, the present invention has the following beneficial effects:

[0093] The present invention discovered for the first time that sVASN can effectively inhibit the activation of T cells, providing a new treatment plan for the research field of hyperimmune response treatment and tumor treatment, and providing new ideas and strategies for the development of drugs that inhibit hyperimmune response and fight tumors. It has important scientific significance and clinical application value, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 :HepG2 culture supernatant inhibits T cell activation (supernatant at different concentration multiples);

[0095] Figure 2 : The HepG2 culture supernatant that inhibits metalloproteinases (BB94 and TAPI) weakens the inhibition of T cell activation, where the left picture is the supernatant culture, and the right picture is the co-culture of HepG2 and Jurkat cells;

[0096] Figure 3 :sVASN inhibits T cell activation and reduces IL-2 levels;

[0097] Figure 4 :CD26 on the surface of Jurkat cells decreased with the increase of sVASN concentration;

[0098] Figure 5 : The immunosuppressive effect is caused by sVASN and is independent of TGFβ;

[0099] Figure 6 : After inhibiting CD71, the activation inhibition of Jurkat by HepG2 supernatant at different concentrations was not affected by the concentration;

[0100] Figure 7 :After inhibiting CD71, different concentrations of sVASN inhibited Jurkat activation;

[0101] Figure 8 : After CD71 blocking peptide blocked CD71, the inhibitory ability of sVASN on Jurkat activation was weakened;

[0102] Fig. 9 :Co-culture of different tumor cells with activated Jurkat cells, knockdown or overexpression of VASN caused changes in immunosuppression;

[0103] Fig.10 : sVASN inhibits T cell activation by inhibiting mTOR phosphorylation. DETAILED DESCRIPTION

[0104] The present invention is further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. Those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents. Here, some of the terms involved in the present invention are explained as follows:

[0105] The term "shRNA" used in this article refers to short hairpin RNA, which includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure controlled by the polⅢ promoter. Subsequently, 5-6 Ts are connected as transcription terminators for RNA polymerase Ⅲ. shRNA can be stably integrated into the genome of the cell, allowing long-term gene knockout.

[0106] The term "siRNA" as used herein refers to small interfering ribonucleic acid, i.e., a relatively short-length double-stranded nucleic acid or optionally a longer precursor thereof. In some embodiments, the length of the available siRNA in the present invention is preferably a length of about 20-50bp. However, there is no particular limitation on the length of the available siRNA in the present invention. For example, the siRNA may initially be present in a precursor form in a cell, and the final or processed form of the siRNA that exhibits and exerts gene silencing activity when or after being delivered to a target cell is substantially different. For example, the precursor form of the siRNA may include a precursor sequence element, which may be processed, degraded, altered, or cut when or after being delivered to produce an siRNA that mediates gene silencing activity in the cell.

[0107] The term "miRNA" used in this article refers to microRNA, which is a non-coding RNA of about 22 nt in length and widely present in various organisms from viruses to humans. The main function of mature miRNA is to negatively regulate the post-transcriptional level of genes, and participate in many life processes such as cell proliferation, apoptosis, immunity, neuroendocrine and stem cell differentiation by causing the degradation of its target mRNA or interruption of the translation process.

[0108] The term "antisense nucleic acid" as used herein refers to a nucleic acid containing a sequence complementary to the coding target gene. Antisense nucleic acids can be composed of DNA, RNA or both. Antisense nucleic acids can contain non-complementary bases, as long as they can specifically hybridize under stringent conditions. When antisense nucleic acids are introduced into cells, they bind to target polynucleotides and inhibit transcription, RNA processing, or stability. In addition to antisense polynucleotides, antisense nucleic acids also include polynucleotide mimics, which contain modified main chains and 3' and 5' end portions. Such antisense nucleic acids can be appropriately designed according to the sequence information of the target gene and prepared using methods well known to those skilled in the art.

[0109] The term "dsRNA" as used herein refers to double-stranded ribonucleic acid, an RNA molecule formed by the renaturation of two complementary strands, which can be cleaved by the Dicer enzyme to form siRNA. dsRNA inhibits gene expression through RNA interference (RNAi), and dsRNA does not need to have 100% homology with the target gene sequence, as long as it can inhibit the expression of the target gene.

[0110] The term "pharmaceutically acceptable carrier and / or excipient" as used herein is described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the drug or to help improve the activity of the effective active ingredients in the drug as needed. The effective ingredients that can be used in such pharmaceutical compositions or pharmaceutical preparations can be in the form of their original compounds themselves, or optionally in the form of pharmaceutically acceptable salts thereof. The pharmaceutical compositions or pharmaceutical preparations thus formulated can be administered in any appropriate manner known to those skilled in the art as needed.

[0111] In some embodiments, the "pharmaceutically acceptable carrier and / or excipient" may additionally contain a liquid such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers or pH buffer substances may be present in the composition. These carriers enable the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by patients.

[0112] In some embodiments, the suitable application form of the pharmaceutical composition or pharmaceutical preparation provided by the present invention includes a form suitable for parenteral administration, such as by injection or infusion, such as by rapid injection or continuous infusion, intravenous, inhalable or subcutaneous form. In the case where the product is used for injection or infusion, it can adopt the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it can contain a formulation agent, such as a suspending agent, a preservative, a stabilizer and / or a dispersant. Alternatively, the pharmaceutical composition or pharmaceutical preparation according to the present invention can be in a dry form, and can be further reconstituted with a suitable sterile liquid before use. It is also possible to prepare a solid form suitable for dissolving or being suspended in a liquid vehicle before injection.

[0113] In some embodiments, the pharmaceutical composition or pharmaceutical preparation described in the present invention can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of an effective ingredient (e.g., sVASN, sVASN promoter, CD71, CD71 promoter, VASN promoter, sVASN inhibitor, CD71 inhibitor, an agent that blocks the binding of sVASN to its functional receptor, a VASN inhibitor) that produces a therapeutic effect. Typically, out of 100%, the amount will range from about 1% to about 90% of the active ingredient, preferably about 5% to about 80%.

[0114] The term "T cell hyperactivation-related disease" as used herein refers to any disease associated with T cell hyperactivation, abnormal T cell proliferation, and differentiation, including but not limited to autoimmune diseases, transplant organ rejection, and atopic diseases.

[0115] In some embodiments, the autoimmune diseases include, but are not limited to, acute transverse myelitis, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, atherosclerosis, autoimmune Addison's disease, autoimmune hemolytic anemia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, cerebellar spinocerebellar disease, spinocerebellar degeneration (spinal ataxia, Friedreich's ataxia, cerebellar cortical degeneration), chronic alcoholism, alcohol-induced hepatitis, autoimmune hepatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory bowel disease , chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Creutzfeldt-Jakob disease, Crohn's disease, De-Tooth syndrome, dementia pugilistica, diabetes mellitus, diffuse Lewy body disease, discoid lupus, basal ganglia disease, disseminated intravascular coagulation, middle-aged Down syndrome, drug-induced movement disorders, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, graft-versus-host disease, Graves' disease, acute febrile polyneuritis, Hart-Strauss disease, Hashimoto's thyroiditis, Huntington's senile chorea, idiopathic pulmonary fibrosis, idiopathic platelet ITP, IgA nephropathy, spinal muscular atrophy in infancy or adolescence, insulin-dependent diabetes mellitus, juvenile arthritis, Kawasaki disease, corticospinal system disease, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, multisystem degeneration (Mencel, Dave-Too syndrome, Shy-Drager syndrome, Azores disease of the nervous system), myasthenia gravis, neurogenic muscular atrophy, Parkinson's disease, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis dermatomyositis, primary alopecia Aglobulinemia, primary biliary cirrhosis, progressive supranuclear palsy, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Alzheimer's disease with Lewy bodies, Sjögren's syndrome, stiff-person syndrome, subacute sclerosing panencephalitis, systemic diseases (Refsum's disease, betalipin deficiency, ataxia, telangiectasia, multisystem mitochondrial disease), systemic lupus erythematosus (SLE), catalase deficiency, temporal arteritis / giant cell arteritis, thyroiditis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and Wegener-Columbia syndrome.

[0116] In some embodiments, the atopic diseases include, but are not limited to: chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology; and vascular inflammatory pathologies, such as but not limited to disseminated intravascular coagulation, atherosclerosis, and Kawasaki disease.

[0117] As used herein, the term "administering" refers to contact of an exogenous drug, pharmaceutical composition, or pharmaceutical formulation with an animal, human, subject, cell, tissue, organ, or biological fluid when applied to the subject. "Administering" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid. "Administering" also means treating a cell in vitro and ex vivo by an agent, composition, or by another cell, and when the "treatment" is applied to a human, veterinary, or research subject, it refers to therapeutic treatment, prophylactic or preventive measures, research, and diagnostic applications.

[0118] As used herein, the terms "treat and / or prevent" refer to delaying the development of a disease, preventing the development of a disease, and / or reducing the severity of symptoms that will develop or are expected to develop. Thus, these terms include ameliorating existing disease symptoms, preventing additional symptoms, ameliorating or preventing potential metabolic causes of symptoms, inhibiting the disorder or disease, e.g., preventing the development of the disorder or disease, alleviating the disorder or disease, regressing the disorder or disease, alleviating the symptoms caused by the disease or disorder, or stopping the symptoms of the disease or disorder.

[0119] The term "effective amount" as used herein refers to the amount of the compound that can effectively produce the desired prevention, alleviation or treatment effect on the corresponding diseases described in the present invention. Depending on the compound, the symptoms and their severity, the age of the treated mammal and other factors, the amount of the pharmaceutical composition or pharmaceutical preparation of the present invention to achieve the effective amount will also vary accordingly, but the specific amount can be routinely determined by a person of ordinary skill in the art based on the knowledge in the art mastered by him / her and the content disclosed in the present invention, and the dosage that can produce the above-mentioned effects is within the scope of protection of the present invention.

[0120] The term "subject" as used herein includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment of the present invention, the subject is preferably a human. The term subject includes patients diagnosed with the corresponding diseases described in the present invention.

[0121] The drugs, reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0122] Example Immunosuppressive molecule sVASN and its application in inhibiting T cell activation

[0123] 1. Experimental Materials

[0124] Phorbol 12-myristate 13-acetate (PMA, MCE, HY-18739), ionomycin (Iono, MCE, HY-13434), IL-2 high sensitivity ELISA detection kit (RK00002, Abclonal).

[0125] The amino acid sequence of sVASN is as follows: MCSRVPLLLPLLLLLALGPGVQGCPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDAGSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETFRGLRRLERLYLGKNRIRHIQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQLDEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSLQALPGDLSGLFPRLRLLAAARNPFNCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYADFGCPATTTTATVPTTRPVVREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTVGPVPQPQDCPPSTCLNGGTCHLGTRHHLACLCPEGFTGLYCESQMGQGTRPSPTPVTPRPPRSLTLGIEPVSPTSLRVGLQRYLQGSSVQLRSLRLTYRNLSGPDKRLVTLRLPASLAEYTVTQLRPNATYSVCVMPLGPGRVPEGEEACGEAHTPPAVHSNHAPVTQAREGNLP (SEQ ID NO:1).

[0126] The amino acid sequence of VASN is shown below: MCSRVPLLLPLLLLLALGPGVQGCPSGCQ (SEQ ID NO: 2).

[0127] 2. Experimental Methods

[0128] 1. Jurkat cell activation and IL-2 detection

[0129] (1) Jurkat cells were cultured normally. Cells were counted in the logarithmic growth phase and 2×10 cells were seeded in a T25 cell culture flask. 5 cell;

[0130] (2) One group was controlled, and one group was stimulated with PMA (200 ng / mL) and Iono (300 ng / mL) for 24 hours; specific dosage: 1 μL of 10 mg / mL PMA stock solution and 0.5 μL of 5 mM Iono were added to 5 mL of 1640 medium;

[0131] (3) After 24 h, the cells were collected and centrifuged, the supernatant was removed, new low-serum cell culture medium was added, and then different concentrations of sVASN were added and the treatment continued for 24 h;

[0132] (4) Collect the cell supernatant, centrifuge at 1000 rpm for 10 min, and take the supernatant for ELISA detection;

[0133] (5) The ELISA operation method is as follows:

[0134] 1) Take out the kit from the refrigerator 20 minutes in advance and equilibrate to room temperature.

[0135] 2) Preparation of standard gradient working solution: Add 1 mL of sample diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve and then gently mix (concentration is 500 pg / mL), then dilute to the following concentrations: 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.12 pg / mL, 1.56 pg / mL, 0.78 pg / mL, 0 pg / mL.

[0136] 3) Wash the plate with wash buffer 3 times.

[0137] 4) Add 100 μL of the sample diluted with the standard / sample diluent, cover with a film to prevent the liquid from evaporating, and incubate at 37°C for 2 h.

[0138] 5) Prepare biotin-conjugated antibody and dilute 100-fold with antibody diluent.

[0139] 6) Wash the plate with wash buffer 3 times.

[0140] 7) Add 100 μL of biotin-conjugated antibody, cover with film to prevent liquid evaporation, and incubate at 37°C for 1 h.

[0141] 8) Prepare streptavidin-HRP working solution and dilute it with sample diluent.

[0142] 9) Wash the plate with wash buffer 3 times.

[0143] 10) Add 100 μL of streptavidin-HRP working solution, cover with a film to prevent the liquid from evaporating, and incubate at 37°C for 0.5 h.

[0144] 11) Wash the plate with wash buffer 3 times.

[0145] 12) Add 100 μL TMB substrate and incubate at 37°C for 15-20 min.

[0146] 13) Add 50 μL of stop solution, measure the absorbance at 450 nm within 5 min, and calculate the concentration of the sample based on the standard curve drawn according to the standard.

[0147] 2. Co-culture of tumor cells and Jurkat cells

[0148] (1) Direct co-culture

[0149] 1) Select Jurkat cells with good growth status and density of 80-90%, count and add PRMI-1640 complete medium, the Jurkat concentration is 5×10 5 Pieces / mL;

[0150] 2) Add PMA (200 ng / mL) and Iono (300 ng / mL) and mix thoroughly by pipetting, place in a 5% CO2, 37°C cell culture incubator for 24 h, centrifuge after 24 h, discard the supernatant, and add new 1640 complete medium;

[0151] 3) Take well-growing tumor cells, wash them three times with PBS, add basal culture medium and culture for 24 hours, take the supernatant and centrifuge it at 3000 rpm at 4°C for 20 minutes, slowly aspirate the supernatant after centrifugation as co-culture conditioned medium and add it to Jurkat cells, co-culture for 24 hours, and carry out subsequent experiments.

[0152] (2) Indirect co-culture

[0153] Transwell is used to achieve physical isolation of tumor cells and immune cells. Transwell chambers are cup-shaped devices with a permeable membrane at the bottom of the chamber. Different types of cells are inoculated at the bottom of the chamber and the culture plate. During culture, the chamber is placed in the culture plate to establish co-culture of cells. Transwell chambers for co-culture use PET material membranes (Corning) with a pore size of 0.4μm or 3μm.

[0154] 1) Select Jurkat cells with good growth status and density of 80-90%, count them and add PRMI-1640 complete medium at a concentration of 5×10 5 Pieces / mL;

[0155] 2) Add PMA (200 ng / mL) and Iono (300 ng / mL) and mix thoroughly by pipetting, place in a 5% CO2, 37°C cell culture incubator for 24 h, centrifuge after 24 h and discard the supernatant, and add new 1640 medium containing 1% serum;

[0156] 3) The culture chamber was placed on a well plate, and the counted Jurkat cells were added to the upper layer and the tumor cells were added to the lower layer. The ratio of tumor cells to Jurkat cells was 5:1. The two types of cells were evenly distributed in each layer and cultured for 24 hours before conducting subsequent experiments.

[0157] 3. Detection of the effect of sVASN on the activation of Jurkat cells after blocking / inhibiting CD71

[0158] (1) Jurkat cells were cultured normally. Cells were counted in the logarithmic growth phase and 2×10 cells were seeded in a T25 cell culture flask. 5 Cells were stimulated with PMA (200 ng / mL) and Iono (300 ng / mL) for 24 h;

[0159] (2) CD71 blocking peptide (transferrin receptor peptide, abcam, ab101219) was added to the cells in the control group and the activation group at a concentration ratio of 10:1. After 6 h of culture, sVASN was added and cultured for another 24 h.

[0160] (3) The CD71 inhibitor Ferristatin II (MCE, HY-D0256, which promotes the degradation of CD71 in vitro and in vivo) was added to the control group and the activated group, respectively, and the cells were cultured for 24 h before adding sVASN and culturing for another 24 h;

[0161] (4) Collect the cell supernatant, centrifuge at 1000 rpm for 10 min, and take the supernatant for ELISA detection;

[0162] (5) ELISA test is the same as before.

[0163] 4. Knockdown or overexpression of VASN in tumor cells followed by co-culture with Jurkat

[0164] (1) Knockdown: Tumor cells HCT116, HepG2, Ishikawa, U251, etc. were cultured in 12-well plates (direct co-culture) or 6-cm dishes (indirect co-culture). After 24 hours, transfection was performed and siRNA was used to knock down the expression of VASN in tumor cells. The siRNA was purchased from RiboBio (stB0014993B, whose target sequence was CCAACAGGCTGCATGAAAT (SEQ ID NO: 3)). Lipofectamine TM RNAiMAX Transfection Reagent was used for transfection in the conventional manner, and 48 hours later, the cells were co-cultured with activated Jurkat for another 24 hours (direct co-culture). 48 hours later, the cell culture supernatant was collected and co-cultured with activated Jurkat for another 24 hours (indirect co-culture).

[0165] (2) Overexpression: Tumor cells HCT116, HepG2, Ishikawa, U251, etc. were cultured in 12-well plates (direct co-culture) or 6-cm dishes (indirect co-culture) and transfected 24 hours later using pcDNA3.1-sVASN-His plasmid or pCMV-VASN-His plasmid. TM Transfection was performed using 3000 Transfection Reagent in a conventional manner, and 48 hours later, the cells were co-cultured with activated Jurkat for another 24 hours (direct co-culture). 48 hours later, the cell culture supernatant was collected and co-cultured with activated Jurkat for another 24 hours (indirect co-culture).

[0166] (3) After the co-culture, the cell culture supernatant was collected for ELISA or Western blot analysis.

[0167] 5. Western blot analysis of changes in key proteins after sVASN inhibited Jurkat cell activation

[0168] (1) Jurkat cell activation and sVASN treatment were the same as before;

[0169] (2) Collect cells and centrifuge at 1000 rpm for 5 min;

[0170] (3) Cell lysis: Add an appropriate volume of M-PER to the collected cells. TM Mammalian Protein Extraction Reagent and Protease inhibitor cocktail (used at 1:1000) were mixed thoroughly, lysed on ice for 30 min, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was taken for protein quantification;

[0171] (4) Determine the protein concentration using the BCA method: dilute the standard (2 mg / mL) in multiples to obtain the following concentrations: 0, 0.125, 0.25, 0.5, 1, 2 mg / mL; add 20 μL of the diluted standard to each well of a 96-well plate in order; dilute the sample to be tested 10 times, i.e., take 18 μL of deionized water, add 2 μL of the sample to be tested, and pat gently to mix; prepare the colorimetric solution, prepare the colorimetric solution according to the ratio of A and B solutions at 50:1, and each well of the standard and sample requires 200 μL of the colorimetric solution, react at 37°C for 60 min, remove the sample, and measure the absorbance at 570 nm with an enzyme reader to obtain the results and draw a standard curve;

[0172] (5) Prepare samples according to the measured protein concentration. The total protein concentration is generally set at 2-5 μg / mL;

[0173] (6) Take the calculated protein volume, add appropriate amount of water and protein loading buffer, and cook at 100℃ for 15 min. The sample is now ready and can be stored at -20℃ or used directly for electrophoresis.

[0174] (7) SDS-PAGE electrophoresis operation method is as follows:

[0175] 1) Gel preparation: Take equal volumes of separation gel buffer and separation gel solution and mix them evenly. Then add an appropriate volume of modified ammonium persulfate according to the instructions, mix evenly, pour into the gel plate, add an appropriate amount of ethanol along the glass plate wall to cover and separate. After 20 minutes, the separation gel can solidify. Pour off the residual ethanol, prepare the concentrated gel solution according to the instructions, add the corresponding volume of modified ammonium persulfate solution, mix evenly, pour into the gel plate, and carefully insert the comb teeth. After 20 minutes of concentrated gel solidification, gently pull out the comb teeth and electrophoresis can be performed.

[0176] 2) Load 20-40 μg of sample and perform electrophoresis at 80 V for about 30 min (based on the entry of bromophenol blue into the separation gel). Then adjust the voltage to 120 V and perform electrophoresis for 60-90 min (based on the position of the target protein band).

[0177] (8) Transfer: Use GenScript electroporator to transfer the membrane, and the rapid mode can be used;

[0178] (9) After transfer, rinse once with 1× TBST, add blocking solution, and block at room temperature for 1 h;

[0179] (10) Mark the membrane, cut the membrane according to the required band size, put it into a hybridization bag, add an appropriate amount of diluted antibody, seal the bag to remove bubbles, and incubate at 4°C overnight;

[0180] (11) Wash the membrane with 1× TBST for 3 times with shaking, 10 min each time;

[0181] (12) Secondary antibody blocking: transfer the membrane to an incubation box with secondary antibody blocking solution, incubate at room temperature with shaking for 1 h, and dilute the secondary antibody at 1:10000;

[0182] (13) Wash the membrane with 1× TBST for 3 times with shaking, 10 min each time;

[0183] (14) ECL colorimetric analysis.

[0184] 3. Experimental Results

[0185] In this example, phorbol ester and ionomycin were used to activate Jurkat cells, a human T-cell leukemia cell line. Successfully activated Jurkat cells secreted IL-2. The activation of Jurkat can be determined by detecting the IL-2 content in the cell culture supernatant by ELISA. HepG2 cells highly express VASN, which is released as a soluble form (sVASN) after being cleaved by ADAM17. The sVASN content in the HepG2 culture supernatant detected by ELISA increased significantly with the increase of culture time.

[0186] The HepG2 culture supernatant was collected and concentrated, and the concentrated supernatant was added while activating Jurkat cells. The culture was continued for 24 hours, and the cell culture supernatant was collected to detect the IL-2 level. The percentage change in IL-2 level relative to that of unactivated cells was calculated to determine the effect of adding the concentrated supernatant on the activation of Jurkat cells.

[0187] The results showed that the expression of IL-2 in Jurkat cells increased after the addition of PMA and ionomycin, while the expression of IL-2 decreased after the addition of HepG2 culture supernatant. With the increase of concentration times, the expression of IL-2 gradually decreased in a dose-dependent manner ( Figure 1 ). This indicates that HepG2 culture supernatant can inhibit the activation of Jurkat, and the active ingredient in the culture supernatant may be sVASN.

[0188] In order to further determine whether sVASN in the HepG2 culture supernatant inhibits the activation of Jurkat, in this example, HepG2 cells were treated with BB94 (bastama) and TAPI, inhibitors of ADAM17 metalloproteinases, and the treated culture supernatants were collected. It was found that when the treated HepG2 culture supernatant was added to the activated Jurkat cells, the level of IL-2 was significantly increased, indicating that after inhibiting the metalloenzyme activity of ADAM17, the sVASN released by cleavage decreased, and the inhibitory effect on the activation of Jurkat cells was weakened ( Figure 2 ). The above results further illustrate the immunosuppressive ability of sVASN.

[0189] In order to prove that the immunosuppressive effect is caused by sVASN rather than other components of the cell culture supernatant, different concentrations of soluble recombinant VASN protein (rhsVASN, 1-575aa, the corresponding amino acid sequence is shown in SEQ ID NO:1, expressed in CHO cells, GenScript Biotech Co., Ltd.) were added to activated Jurkat cells in this example. The results were consistent with expectations. After adding sVASN, the expression of IL-2 was reduced, that is, the ability to suppress immune cells was increased ( Figure 3 ).

[0190] At the same time, this example also uses flow cytometry to detect the changes in T cell surface molecules after adding different concentrations of soluble recombinant VASN protein (rhsVASN). The results show that after adding different concentrations of sVASN, the expression of CD26 molecules on the surface of Jurkat cells decreased and was negatively correlated with the concentration of sVASN, showing a dose-dependent manner ( Figure 4 ). CD26 is a marker for T cell activation, which means that the higher the concentration of sVASN, the more obvious the inhibitory effect on T cell activation.

[0191] Since most current studies believe that the main function of VASN depends on TGF-β, the present invention further explores the role of TGF-β in the immunosuppression process. By co-culturing tumor cells with Jurkat cells, the activation inhibition of Jurkat was detected after knocking down VASN or TGF-β in tumor cells. The results showed that in human brain glioma cells U118, U251 and U87, after gradient knockdown of VASN, the IL-2 level of Jurkat cells increased in a dose-dependent manner; while there was no such trend change after gradient knockdown of TGF-β ( Figure 5 ), indicating that in the co-culture model of U118, U251 and U87, the immunosuppressive effect is mainly caused by sVASN directly rather than sVASN through TGF-β.

[0192] The previous inventors proved that sVASN functions in tumor cells through the transferrin receptor CD71, so we asked whether the immunosuppressive ability of sVASN is also through CD71. After treating Jurkat cells with Ferristatin II (MCE, HY-D0256, which promotes the degradation of CD71 in vivo and in vitro), the supernatant of HepG2 culture still showed the ability to inhibit activation, but it did not change with the dose ( Figure 6 ), indicating that the ability of sVASN to inhibit the activation of Jurkat cells was not obvious after inhibiting CD71, that is, the inhibitory effect of sVASN on T cell activation may be mediated by CD71.

[0193] This example further verified the inhibition of CD71 expression by adding different concentrations of soluble recombinant VASN protein (rhsVASN) and treating Jurkat cells with Ferristatin. The results showed that after inhibiting CD71, different concentrations of sVASN had no inhibitory ability on T cells ( Figure 7 ), indicating that when CD71 is inhibited, the overall immunosuppressive ability of sVASN is reduced, and high-concentration sVASN still has a certain immunosuppressive ability. Ferristatin promotes the degradation of existing CD71, that is, sVASN may suppress immunity through CD71.

[0194] Since sVASN exerts its immunosuppressive effect extracellularly, we chose to block CD71 extracellularly to further visually determine whether CD71 is involved in the process. By adding different concentrations of sVASN and CD71 blocking peptide, the results showed that high concentrations of sVASN had a significant inhibitory effect on immune activation, while after blocking peptide blocked CD71, sVASN had no immunosuppressive ability ( Figure 8 ), the results indicate that CD71 is involved in the inhibitory process of sVASN on immune activation, and this process mainly depends on the function of CD71 as a receptor of sVASN.

[0195] To determine whether the immunosuppressive ability of sVASN is related to tumor type, we selected different tumor cells and detected Jurkat activation under co-culture conditions by knocking down or overexpressing VASN in tumor cells. The results showed that under different tumor cell co-culture conditions, knocking down VASN in tumor cells could lead to more IL-2 secretion by Jurkat, i.e., T cell activation; overexpression of VASN led to less IL-2 secretion by Jurkat, i.e., T cell inhibition ( Fig. 9 ). The above results indicate that sVASN secreted by tumor cells can inhibit T cell activation, that is, sVASN has immunosuppressive ability.

[0196] Finally, we analyzed the reasons why sVASN caused the inhibition of T cell activation. We used Western blot to analyze the expression changes of key proteins in Jurkat cells after adding different concentrations of sVASN. The results showed that after adding sVASN, the total amount of mTOR remained unchanged, but the phosphorylated mTOR (p-mTOR) was significantly reduced ( Fig.10 ), which again shows that sVASN has an inhibitory effect on activated T cells. sVASN inhibits T cell activation by inhibiting mTOR phosphorylation.

Claims

1. Use of sVASN in inhibiting T cell activation in vitro, characterized in that: The amino acid sequence of the sVASN is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The sVASN inhibits T cell activation by inhibiting the mTOR signaling pathway.

3. The use according to claim 1, characterized in that: The functional receptors of sVASN that inhibit T cell activation include CD71.

4. The use according to claim 1, characterized in that: The sVASN includes exogenously expressed sVASN or sVASN endogenously expressed and secreted by tumor cells.

5. Use of a sVASN promoter in inhibiting T cell activation in vitro, characterized in that: The sVASN promoter is recombinantly expressed sVASN, and the amino acid sequence of the sVASN is shown in SEQ ID NO:

1.

6. Use of a VASN promoter in inhibiting T cell activation in vitro, characterized in that: The VASN promoter is a vector for overexpressing VASN, and the amino acid sequence of VASN is shown in SEQ ID NO:

2.

7. Use of a VASN inhibitor in promoting T cell activation in vitro, characterized in that: The VASN inhibitor is siRNA, and the target sequence of the siRNA is shown in SEQ ID NO:

3.

8. Use of a sVASN inhibitor and / or a CD71 inhibitor for attenuating or eliminating the inhibitory effect of sVASN on T cell activation in vitro, characterized in that: The amino acid sequence of the sVASN is shown in SEQ ID NO: 1; The sVASN inhibitor includes BB94, an inhibitor of ADAM17 metalloproteinase, and / or TAPI, an inhibitor of ADAM17 metalloproteinase; The CD71 inhibitors include Ferristatin II and / or CD71 blocking peptides.

9. The use according to claim 8, characterized in that: The sVASN includes exogenously expressed sVASN or sVASN endogenously expressed and secreted by tumor cells.

10. A method for inhibiting T cell activation in vitro, characterized in that: The method comprises administering any one or more of sVASN, sVASN promoter, and VASN promoter to a system comprising T cells in need thereof; The amino acid sequence of the sVASN is shown in SEQ ID NO: 1, the sVASN promoter is recombinantly expressed sVASN, the VASN promoter is a vector that overexpresses VASN, and the amino acid sequence of the VASN is shown in SEQ ID NO:

2.

11. A method for promoting T cell activation in vitro, characterized in that: The method comprises administering a VASN inhibitor to a system comprising T cells in need thereof; The VASN inhibitor is siRNA, and the target sequence of the siRNA is shown in SEQ ID NO:

3.

12. Use of any one or more of sVASN, sVASN promoter, and VASN promoter in the preparation of a reagent for inhibiting excessive activation of T cells; The amino acid sequence of the sVASN is shown in SEQ ID NO: 1, the sVASN promoter is recombinantly expressed sVASN, the VASN promoter is a vector that overexpresses VASN, and the amino acid sequence of the VASN is shown in SEQ ID NO:

2.

13. Use of VASN inhibitors in the preparation of drugs for treating and / or preventing tumors; The VASN inhibitor is siRNA, and the target sequence of the siRNA is shown in SEQ ID NO: 3; The tumor is liver cancer, glioma, colorectal cancer or endometrial cancer.

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