WO2019149172A1 - Pik3ip1蛋白在调节t细胞反应和制备抗肿瘤药物中的应用 - Google Patents

Pik3ip1蛋白在调节t细胞反应和制备抗肿瘤药物中的应用 Download PDF

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WO2019149172A1
WO2019149172A1 PCT/CN2019/073451 CN2019073451W WO2019149172A1 WO 2019149172 A1 WO2019149172 A1 WO 2019149172A1 CN 2019073451 W CN2019073451 W CN 2019073451W WO 2019149172 A1 WO2019149172 A1 WO 2019149172A1
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pik3ip1
cells
antibody
tumor
ecd
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王智
王茜
房娟
文书琼
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4273—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to the application of PIK3IP1 protein in regulating T cell proliferation, regulating T cell activation and preparing antitumor drugs.
  • T cells are the most important element of the acquired immune system. Depending on the glycoprotein on the surface of T cells, T cells can be divided into CD4 + T cells and CD8 + T cells. While circulating CD4 + and CD8 + T cells patrol in vivo, they can distinguish whether the protein is a foreign body or a peptide of its own. Exogenous proteins release a signal that the immune system is at risk, leading to T cell activation and initiation of other immunity. The cells then kill the target cells. In the process of anti-tumor immunity, T cells act as core performers, first activated by T cell receptor-mediated antigen recognition signals, and numerous co-stimulatory signals and co-suppressive signals finely regulate the intensity and quality of T cell responses. The suppression signal is the immune checkpoint.
  • Tumor cells can evade immune damage by suppressing T cell activation through immune checkpoints. Therefore, enhancing the activation of T cells by different methods is of great significance for tumor immunotherapy, and blocking of immune checkpoints is one of the effective strategies for enhancing T cell activation.
  • the combination of tumor immunotherapy and other treatments represented by immunological checkpoint blockade is receiving increasing attention.
  • the immune system recognizes abnormal proteins expressed by tumor cells and kills tumor cells, but in cancer patients, this process is defective.
  • tumor cells can call other mechanisms to evade surveillance by the immune system, and some tumor cells induce T cells to limit cytotoxic T cells, making them unable to attack tumor cells.
  • the immune response of the body is regulated by various types of immune cells, cytokines, etc., to resist the attack of dangerous signals and foreign antigens; T cells play an important role in the immune response process. Regulating the body's immune response and maintaining immune tolerance by autoantigens depends on the involvement of a complex network of co-stimulatory and co-suppressor molecules. T cell activation is regulated by a synergistic signaling network that runs throughout the entire T cell response. Some members of the Ig superfamily and the TNF receptor superfamily represented by B7/CD28 constitute the main body of T cell costimulatory molecules. The importance of these synergistic signaling pathways has been demonstrated in a variety of human diseases, including graft versus host response, autoimmune diseases, infections and tumors.
  • checkpoint regulatory molecules and signaling pathways such as the programmed death molecule (PD)-1 and B7-H4 pathways
  • PD programmed death molecule
  • B7-H4 pathways the programmed death molecule
  • new checkpoint regulatory molecules such as T-cell-activated V-region Ig inhibitory molecules (VISTA) and B7-H6 have been discovered and confirmed.
  • VISTA T-cell-activated V-region Ig inhibitory molecules
  • Immunological checkpoint monoclonal antibody (CTLA-4 monoclonal antibody, PD1/PDL1 monoclonal antibody) is the latest achievement of tumor immunotherapy, especially in hematological tumors and melanoma, and has made significant progress due to its good anti-tumor activity.
  • Ipilimumab was approved in 2011 for anti-CTLA-4 monoclonal antibody against metastatic melanoma and has been used to treat advanced melanoma, but with a low response rate, anti-CTLA-4 treatment is immunotoxic to patients.
  • MDX-1106 as an anti-PD-1 monoclonal antibody, is also highly promising in clinical practice. Studies have shown that MDX-1106 is less toxic than Ipilimumab, but does not inhibit tumor growth in cancers such as prostate cancer because these tumors do not have a target for PD-L1. This suggests that many cancers still have some key negative regulatory factors to destroy the protective anti-tumor immune system.
  • Phosphatidylinositol 3-kinase interacting protein 1 (PI3K interacting protein1, PIK3IP1) is a recently-reported protein that binds to the p110 subunit of phosphatidylinositol-3-kinase (PI3K) and downregulates PI3K Activity, inhibition of activation of serine threonine kinase (AKT, RAC-alphaserine/threonine-proteinkinase).
  • AKT serine threonine kinase
  • RAC-alphaserine/threonine-proteinkinase serine threonine-proteinkinase
  • PIK3IP1 By overexpressing PIK3IP1 protein on human leukemia cell line Jurkat and mouse Th2 cell line D10 cells, activation of transcriptional signaling molecules NFAT and AP-1 associated with T cell activation was inhibited. In contrast, silencing of the PIK3IP1 gene in the above cell lines with SiRNA enhanced Akt phosphorylation, promoted T cell activation, and IL-2 secretion. This suggests that the new PIK3IP1 signaling pathway regulator PIK3IP1 may inhibit T cell activation.
  • the inventors of the present invention examined and analyzed the expression of PIK3IP1 on common tissues, immune cells, tumor cell lines and the like. It was found to be highly expressed on immune cells, and the intensity of expression was related to the immune state of the body. Especially in T cells there is a relatively specific expression. The body's immune response is regulated by a series of molecules to ensure that the body resists dangerous signals and foreign antigen attacks. Among them, T cells play an important role in maintaining immune homeostasis and immune defense in vivo.
  • the present invention comparatively studied the proliferation of T cells in Pik3ip1 knockout mice and wild type mice after OVA antigen-specific immune reaction, and found that the proliferation of T cells of Pik3ip1 knockout mice was stronger than that of wild type mice. This indicates that under the acute stimulation of new antigen, the expression of Pik3ip1 on mouse T cells has an inhibitory effect on the proliferation of T cells.
  • the present invention compares the effects of Pik3ip1 knockout mice on T cell responses and tumor growth in the MC38 tumor model. It was found that Pik3ip1-deficient T cells were more potent than wild-type mice in the proliferation of tumor cells and the secretion of IFN- ⁇ and other effector molecules. This indicates that under the stimulation of tumor antigen, the expression of Pik3ip1 on mouse T cells can significantly inhibit the T cell response, and the decrease of Pik3ip1 expression can effectively inhibit the growth of tumor.
  • the present invention further studies peripheral blood lymphocytes of patients with oral squamous cell carcinoma, and also finds that the fluorescence intensity of PIK3IP1 on peripheral blood CD4 + and CD8 + T cells of patients with oral squamous cell carcinoma is negatively correlated with their proliferative ability, ie, CD4 + and CD8 + T The higher the expression level of PIK3IP1 on the cells, the weaker the proliferation ability of the corresponding cells.
  • the present inventors have specifically studied that the PIK3IP1 ECD-Fc fusion molecule can promote the proliferation of mouse CD8 + T cells in vivo, and the PIK3IP1 ECD-Fc fusion molecule alone or with the 4-1BB activating antibody 2A (Anti- m4-1BB(2A)) combined immunotherapy effect on tumor formation in mice. It was found that the combination of the PIK3IP1 ECD-Fc fusion molecule and the 4-1BB activating antibody 2A significantly inhibited tumor growth.
  • PIK3IP1 inhibitor in the manufacture of a medicament for modulating an immune response in a subject.
  • the PIK3IP1 inhibitor is selected from at least one of a PIK3IP1, PIK3IP1 ECD, and PIK3IP1 ECD fusion molecule.
  • the PIK3IP1 inhibitor is an antibody that binds to PIK3IP1, such as a polyclonal or monoclonal antibody to PIK3IP1.
  • PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or antibody of PIK3IP1 in the manufacture of a medicament for modulating an immune response in a subject.
  • modulating a subject's immune response comprises enhancing an immune response in the subject, for example, enhancing proliferation of a subject's CD8 + T cells and/or CD4 + T cells.
  • a PIK3IP1 inhibitor for example, a PIK3IP1, a PIK3IP1ECD, a PIK3IP1 ECD fusion molecule or an antibody of PIK3IP1, together with an antigen, is used in the preparation of a medicament for enhancing an immune response of a subject to an antigen
  • Enhancements include administration of an antigen and a PIK3IP1 inhibitor to a subject, eg, a PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecule, or an antibody to PIK3IP1, such that the subject's immune response to the antigen is enhanced.
  • the antigen may be a cancerous cell, a tumor antigen, a viral antigen, a bacterial antigen or the like.
  • antigens that may be used include antigenic peptides of melanoma, such as peptide gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase.
  • the antigen is gp100, more preferably hgp100 25-33 .
  • the antigen is OVA, more preferably OVA 257-264 .
  • the antigen is a cancerous cell, such as MC38 cells, B16 cells, B16-F10 cells, and the like.
  • a second aspect of the invention provides the use of a PIK3IP1 inhibitor for the preparation of an immunotherapeutic tumor drug.
  • the PIK3IP1 inhibitor is selected from at least one of a PIK3IP1, PIK3IP1 ECD, and PIK3IP1 ECD fusion molecule.
  • the PIK3IP1 inhibitor is an antibody that binds to PIK3IP1, such as a polyclonal or monoclonal antibody to PIK3IP1.
  • PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or PIK3IP1 antibody for the preparation of immunotherapeutic tumor drugs.
  • a PIK3IP1 inhibitor for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody of PIK3IP1 achieves an effect of treating a tumor by enhancing an immune response of a drug user.
  • a PIK3IP1 inhibitor for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody against PIK3IP1 can inhibit the growth of tumor cells.
  • PIK3IP1, PIK3IP1ECD or PIK3IP1 ECD fusion molecules may block the inhibition of T cell proliferation or activity by PIK3IP1 activation as a competitive inhibitor of PIK3IP1.
  • PIK3IP1 inhibitors for example, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or antibodies to PIK3IP1, can also be used in combination with other standard tumor treatments for the treatment of tumors.
  • PIK3IP1 inhibitors for example, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or antibodies to PIK3IP1, can be effective in combination with chemotherapy regimens and have the potential to reduce the dose of chemotherapeutic agents administered.
  • An example of such a combination is the treatment of melanoma with PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody in combination with decarbazine.
  • PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or PIK3IP1 antibody combined with interleukin-2 (IL-2) to treat melanoma.
  • IL-2 interleukin-2
  • PIK3IP1 inhibitors for example, PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecules or PIK3IP1 antibodies, in combination with chemotherapeutic agents, is that the cytotoxic effects of most chemotherapeutic agents cause tumor cell death, tumor cell death leads to tumors in the antigen presentation pathway The antigen level is elevated.
  • Other combination therapies that act synergistically with PIK3IP1 inhibitors, eg, PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecules, or antibodies to PIK3IP1 by cell death may also include radiation therapy, surgery, and the like.
  • angiogenesis inhibitors can also be combined with PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or PIK3IP1 antibodies, inhibition of angiogenesis can also lead to tumor cell death, allowing tumor antigens to enter the antigen presentation pathway.
  • Tumors circumvent host immune surveillance through a variety of mechanisms. These mechanisms can be abolished by inhibiting the activity of tumor-expressing immunosuppressive proteins including, for example, TGF- ⁇ and Fas ligands and the like. Antibodies against each of these proteins can also be used in combination with PIK3IP1 inhibitors, for example, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or antibodies to PIK3IP1 to counteract the immunosuppressive effects of tumors and promote the tumor immune response of the host.
  • immunosuppressive proteins including, for example, TGF- ⁇ and Fas ligands and the like.
  • Antibodies against each of these proteins can also be used in combination with PIK3IP1 inhibitors, for example, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or antibodies to PIK3IP1 to counteract the immunosuppressive effects of tumors and promote the tumor immune response of the host.
  • PIK3IP1 inhibitors capable of activating host immune responsiveness
  • PIK3IP1 inhibitors for example, PIK3IP1, PIK3IP1ECDPIK3IP1 ECD fusion molecules or antibodies to PIK3IP1.
  • These antibodies include molecules that activate DC function and antigen presentation on the surface of dendritic cells.
  • an anti-CD40 antibody is effective for increasing T helper cell activity and can be used in combination with a PIK3IP1 inhibitor, for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody to PIK3IP1.
  • T cell activity can also be increased against activating antibodies to T cell costimulatory molecules such as CTLA-4, OX-40, 4-1BB, and ICOS.
  • one embodiment of the invention provides the use of a PIK3IP1 inhibitor, eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule, or an antibody to PIK3IP1, alone or in combination with another therapeutic agent, in the manufacture of a medicament for treating a tumor.
  • a PIK3IP1 inhibitor eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule, or an antibody to PIK3IP1 alone or in combination with another therapeutic agent, in the manufacture of a medicament for treating a tumor.
  • the other therapeutic agent or agents include, but are not limited to, a tumor chemotherapeutic agent, a compound capable of activating T cells, an angiogenesis inhibitor, and the like.
  • the additional therapeutic agent is an activating antibody of 4-1BB, such as 4-1BB activating antibody 2A.
  • One embodiment of the present invention provides the use of a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody to PIK3IP1 and an activating antibody of 4-1BB, such as 2A, in combination with a medicament for the preparation of a tumor.
  • antibodies such as PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or PIK3IP1 are administered at subtherapeutic doses.
  • an activating antibody of 4-1BB such as 2A is administered at a subtherapeutic dose.
  • an antibody in which PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 and 4-1BB activation antibody, such as 2A are each administered at a subtherapeutic dose.
  • a therapeutic combination of a PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecule or antibody of PIK3IP1 and Antibody 2A can be administered as a single composition in admixture with a pharmaceutically acceptable carrier, or each active ingredient can be separately administered
  • the pharmaceutically acceptable carriers are mixed and administered simultaneously as separate compositions.
  • a combination of PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 fusion molecule or antibody 2A can be administered sequentially, eg, antibody 2A is administered followed by administration of an antibody to PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1, or An antibody against PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 was administered after administration of antibody 2A.
  • the order of sequential administration may be reversed or maintained in the same order at each administration, and sequential administration may be combined with simultaneous administration or any combination thereof.
  • the combination of the first administration of PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody and antibody 2A may be simultaneous, and the second administration may be sequential, first administration of PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody
  • Antibody 2A is administered post-administration, and the third administration can be sequential, followed by administration of antibody 2A followed by administration of PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecule or antibody to PIK3IP1, and the like.
  • Another representative dosing regimen can include sequential administration of the first administration, followed by administration of antibody 2A followed by administration of antibodies to PIK3IP1, PIK3IP1 ECD, PIK3IP1 ECD fusion molecule or PIK3IP1, while subsequent administration can be simultaneous.
  • a combination of PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or PIK3IP1 antibody and 4-1BB activation antibody, eg, antibody 2A can be further combined with other immunogenic agents, such as cancerous cells, purified tumor antigens, and the like.
  • immunogenic agents include peptides of melanoma antigens, such as peptide gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase.
  • combinations of PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecules or antibodies to PIK3IP1 and activating antibodies to 4-1BB, such as antibody 2A can be further combined with standard tumor treatment methods.
  • a combination of PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or PIK3IP1 antibody and antibody 2A in combination with dacarbazine for treatment of melanoma PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or combination of PIK3IP1 antibody and antibody 2A in combination with interleukin-2 (IL-2) to treat melanoma.
  • IL-2 interleukin-2
  • a third aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a PIK3IP1 inhibitor, for example, a PIK3IP1, PIK3IP1ECD, a PIK3IP1 ECD fusion molecule or an antibody against PIK3IP1, and a pharmaceutically acceptable carrier.
  • a PIK3IP1 inhibitor for example, a PIK3IP1, PIK3IP1ECD, a PIK3IP1 ECD fusion molecule or an antibody against PIK3IP1, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for use in modulating an immune response in a subject.
  • the modulation of the subject's immune response comprises enhancing the subject's immune response, for example, enhancing proliferation of the subject's CD8 + T cells and/or CD4 + T cells.
  • the pharmaceutical composition is for the treatment of a tumor.
  • the treatment of the tumor achieves a therapeutic effect of inhibiting tumor growth by enhancing the immune response of the drug user.
  • the pharmaceutical composition may also be administered in a combination therapy, i.e. in combination with other therapeutic agents, for example, other immunomodulatory agents or tumor therapeutic agents.
  • the pharmaceutical composition may further comprise another therapeutic agent or agents.
  • therapeutic agents include, but are not limited to, tumor chemotherapeutic agents, compounds capable of activating T cells, angiogenesis inhibitors, and the like.
  • the additional therapeutic agent is an activating antibody of 4-1BB, such as antibody 2A.
  • the amount of active ingredient in a single dosage form of the pharmaceutical composition may vary depending on the subject and the particular route of administration.
  • the weight percentage of a PIK3IP1 inhibitor for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 is from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably About 1% to about 30%.
  • the dosage range is from about 0.0001 to 100 mg/kg, more typically from 0.01 to 5 mg/kg body weight.
  • the dose may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or in the range of 1-10 mg/kg.
  • An exemplary treatment regimen can be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.
  • a preferred dosage regimen comprises intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight using one of the following dosing regimens Administration: (i) up to six doses per week, then one dose every three months; (ii) every three weeks of dosing; (iii) one dose of 3 mg/kg body weight followed by one mg/kg body weight every three weeks.
  • the actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to provide an amount of active ingredient that is effective to achieve a therapeutic response to a particular patient and route of administration without being toxic to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the route of administration, time of administration, drug metabolism, age, sex, weight, general health status, and past medical history of the patient being treated.
  • One embodiment of the present invention also provides a pharmaceutical kit comprising an antibody against PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or PIK3IP1 and an activation antibody of 4-1BB, such as Antibody 2A.
  • the kit may further comprise instructions for treating the tumor.
  • the PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody and 4-1BB activating antibody, such as antibody 2A are co-packaged in unit dosage form in the pharmaceutical kit.
  • the PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or antibody of PIK3IP1 and 4-1BB activation antibody, such as antibody 2A are separately packaged in the respective unit dosage forms in the pharmaceutical kit. .
  • a fourth aspect of the invention provides a method of modulating an immune response in a subject, comprising administering to the subject a PIK3IP1 inhibitor, for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or antibody of PIK3IP1, such that the subject The immune response is regulated.
  • a PIK3IP1 inhibitor eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody to PIK3IP1, enhances, stimulates or increases the immune response of the subject.
  • the enhancing, stimulating or increasing the immune response of the subject includes, but is not limited to, promoting proliferation of CD4 + T cells and/or CD8 + T cells.
  • a method of enhancing a subject's immune response to an antigen comprising administering to the subject an antigen and a PIK3IP1 inhibitor, eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule, or an antibody to PIK3IP1,
  • a PIK3IP1 inhibitor eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule, or an antibody to PIK3IP1
  • the subject's immune response to the antigen is enhanced.
  • the antigen may be a cancerous cell, a tumor antigen, a viral antigen, a bacterial antigen or the like.
  • antigens that may be used include antigenic peptides of melanoma, such as peptide gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase.
  • the antigen is gp100, more preferably hgp100 25-33 .
  • the antigen is OVA, more preferably OVA 257-264 .
  • the antigen is a cancerous cell, such as MC38 cells, B16 cells, B16-F10 cells, and the like.
  • a fifth aspect of the invention provides a method of treating a tumor or delaying tumor progression in an individual, the method comprising administering to the individual a therapeutically effective amount of a PIK3IP1 inhibitor, eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody.
  • a PIK3IP1 inhibitor eg, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody.
  • the method further comprises administering to the individual another therapeutic agent or agents.
  • the other therapeutic agent or agents include, but are not limited to, other compounds that activate T cells, tumor chemotherapeutic agents, angiogenesis inhibitors, and the like.
  • the PIK3IP1 inhibitor for example, a PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or an antibody to PIK3IP1, and another therapeutic agent or agents are each administered simultaneously in a separate composition, such as a PIK3IP1 inhibitor, for example , PIK3IP1, PIK3IP1ECD, PIK3IP1ECD fusion molecule or antibody of PIK3IP1, and another therapeutic agent are administered simultaneously in the same composition, or the PIK3IP1 inhibitor, for example, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or PIK3IP1 antibody, Each of the other therapeutic agents is administered sequentially or sequentially in separate compositions in a separate period of time.
  • the additional therapeutic agent is an activating antibody of 4-1BB, such as antibody 2A.
  • the administration of the PIK3IP1 inhibitor increases T cells compared to an untreated individual or an individual treated with monotherapy of another therapeutic agent proliferation.
  • the average ratio of proliferation of T cells is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. .
  • the PIK3IP1 inhibitor eg, PIK3IP1, PIK3IP1ECD, PIK3IP1 ECD fusion molecule or antibody to PIK3IP1 and 2A are administered, compared to an untreated individual or with the PIK3IP1 inhibitor, eg, Tumor volume is reduced in individuals treated with PIK3IP1, PIK3IP1ECDPIK3IP1ECD fusion molecules or antibodies to PIK3IP1, 2A or other therapeutic agents.
  • PIK3IP1 comprises a sequence selected from the group consisting of SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, and/or 17; in some embodiments, PIK3IP1 consists of A sequence consisting of one of SEQ ID NOS. 1 to 8 is selected.
  • the PIK3IP1 ECD comprises a sequence selected from the group consisting of SEQ ID NO. 9 and/or 10; in some embodiments, the PIK3IP1 ECD consists of a sequence selected from SEQ ID NO. 9 or 10.
  • the PIK3IP1 ECD fusion molecule comprises a sequence selected from the group consisting of SEQ ID NO. 9 and/or 10. In some embodiments, at least one fusion partner of the PIK3IP1 ECD fusion molecule is selected from the group consisting of Fc, albumin, and polyethylene glycol. In some embodiments, at least one fusion partner of the PIK3IP1 ECD fusion molecule is Fc. In some embodiments, at least one fusion partner of the PIK3IP1 ECD fusion molecule is an IgG Fc. In some embodiments, the base sequence of the IgG Fc consists of a sequence selected from SEQ ID NO. 15 or 16. In some embodiments, the base sequence of the PIK3IP1 ECD fusion molecule consists of a sequence selected from one of SEQ ID NO. 11-14.
  • the antibody to PIK3IP1 may be a polyclonal antibody to PIK3IP1, a monoclonal antibody to PIK3IP1.
  • the tumor includes a tumor that is generally responsive to immunotherapy, including but not limited to melanoma, leukemia, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast. Cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer; preferably melanoma, leukemia, lung cancer, liver cancer, colon cancer, breast cancer, glioma, stomach cancer, oral scale Cellular cancer.
  • immunotherapy including but not limited to melanoma, leukemia, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast.
  • Cancer glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer
  • melanoma leukemia, lung cancer, liver cancer, colon cancer, breast cancer, glioma, stomach cancer, oral scale Cellular cancer.
  • the tumor is metastatic.
  • the tumor is preferably colon cancer, melanoma, oral squamous cell carcinoma, hepatocellular carcinoma.
  • the tumor chemotherapeutic agent includes, but is not limited to, an alkylating agent, a nitrogen mustard, a thiotepa, a nitrosourea, a methanesulfonate, a platinum compound, a mitochondrial
  • an alkylating agent a nitrogen mustard, a thiotepa, a nitrosourea, a methanesulfonate, a platinum compound, a mitochondrial
  • drugs that affect nucleic acid synthesis such as dihydrofolate reductase inhibitors, thymidine synthase inhibitors, purine nucleoside synthase inhibitors, ribonucleo
  • the compound capable of activating T cells includes, but is not limited to, an activating antibody of a T cell costimulatory molecule, for example, activation of CTLA-4, OX-40, 4-1BB or ICOS.
  • an activating antibody of a T cell costimulatory molecule for example, activation of CTLA-4, OX-40, 4-1BB or ICOS.
  • the T cell competent compound is a 4-1BB activating antibody, such as Antibody 2A.
  • the angiogenesis inhibitor includes, but is not limited to, those inhibitors that inhibit the action of vascular endothelial growth factor, such as lenalidomide, thalidomide, an anti-vascular endothelial growth factor antibody, for example.
  • Bevacizumab Bevacizumab, VEGF receptor tyrosine kinase inhibitor vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SU11248), axitinib (AG) -013736), pazopanib (GW786034) and 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-yl Propoxy)quinazoline and the like.
  • PIK3IP1 refers to phosphatidylinositol 3-kinase interacting protein 1 (PI3K interacting protein1, PIK3IP1), which binds to the p110 subunit of phosphatidylinositol-3-kinase (PI3K) It can down-regulate PI3K activity, inhibit the activation of serine threonine kinase, including its naturally occurring allelic forms and processed isoforms.
  • PIK3IP1 also refers to PIK3IP1 from human species and non-human species such as mice, rats or primates.
  • PIK3IP1 from a particular species is sometimes indicated by terms such as h PIK3IP1 for human PIK3IP1, m PIK3IP1 for murine PIK3IP1, and the like.
  • the PIK3IP1 protein of the mouse is also referred to by Pik3ip1.
  • Exemplary PIK3IP1 includes, but is not limited to, consisting of an amino acid sequence selected from the group consisting of SED IDs NO. 1, 2, 3, 4, 5, 6, 7, 8, and 17.
  • PIK3IP1 inhibitor refers to a chemical substance capable of blocking the activation or activity of PIK3IP1, thereby inhibiting the inhibition of T cell proliferation or activity caused by PIK3IP1 activation.
  • the chemical substance may be a small molecule compound, a protein, a polypeptide, DNA, RNA or the like.
  • the protein or polypeptide may be a PIK3IP1 extracellular domain as described in the present invention, or a fusion protein of the PIK3IP1 extracellular domain, or may be an antibody to PIK3IP1, such as a polyclonal antibody and a monoclonal antibody.
  • PIK3IP1 extracellular domain includes full length PIK3IP1 ECD, PIK3IP1 ECD fragment and PIK3IP1 ECD variant.
  • PIK3IP1 ECD refers to a PIK3IP1 polypeptide that lacks an intracellular domain and a transmembrane domain, with or without a signal peptide.
  • full length PIK3IP1 ECD refers to a PIK3IP1 ECD that extends to the last amino acid of the extracellular domain, and may or may not include an N-terminal signal peptide.
  • the PIK3IP1 ECD is a human PIK3IP1 ECD, consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. In some embodiments, the PIK3IP1 ECD is a murine PIK3IP1 ECD, consisting of an amino acid sequence selected from the group consisting of SEQ ID NO.
  • the PIK3IP1ECD When the PIK3IP1ECD is "consisted" from a sequence selected from SEQ ID NOS. 9 and 10, the PIK3IP1 ECD may or may not contain a variety of post-translational modifications, such as glycosylation and sialylation. In other words, when PIK3IP1ECD consists of a specific amino acid sequence, it does not contain additional amino acids in the contiguous amino acid sequence, but may contain modifications to the amino acid side chain, the N-terminal amino group, and/or the C-terminal carboxyl group.
  • PIK3IP1 ECD fragment refers to a PIK3IP1 ECD that lacks one or more residues from the N and/or C terminus of a full length ECD and maintains the same binding ability as the full length PIK3IP1 ECD.
  • the PIK3IP1 ECD fragment may or may not include an N-terminal signal peptide.
  • PIK3IP1 ECD variant refers to a PIK3IP1 ECD containing amino acid additions, deletions and substitutions and maintaining the same binding ability as the parental PIK3IP1 ECD. Such variants may have at least 90%, 92%, 95%, 97%, 98% or 99% identity to the parent PIK3IP1 ECD.
  • identity of two polypeptides can be measured by a similarity score, which is known in the art, for example, using the Bestfit program with default settings to determine similarity to compare the identity of the amino acid sequences of two polypeptides or Similarity.
  • PIK3IP1 ECD fusion molecule refers to a molecule comprising a PIK3IP1 ECD and one or more "fusion partners".
  • the PIK3IP1 ECD and the fusion partner are covalently linked.
  • the fusion partner is also a polypeptide ("fusion partner polypeptide")
  • the PIK3IP1 ECD and fusion partner polypeptide can be part of a contiguous amino acid sequence, and the fusion partner polypeptide can be linked to the N-terminus or C-terminus of the PIK3IP1 ECD.
  • the PIK3IP1 ECD and fusion partner polypeptide can be translated into a single polypeptide ("PIK3IP1 ECD fusion protein") from the coding sequence encoding both the PIK3IP1 ECD and the fusion partner polypeptide.
  • the PIK3IP1 ECD and the fusion partner are covalently linked by other means, such as chemical bonds other than peptide bonds.
  • the PIK3IP1 ECD and fusion partner can be fused by a "linker" consisting of at least one amino acid or chemical moiety.
  • the PIK3IP1 ECD polypeptide and fusion partner are non-covalently linked, eg, joined using a binding pair.
  • binding pairs include, but are not limited to, biotin and avidin or streptavidin, antibodies and antigens thereof, and the like.
  • Exemplary fusion partners include, but are not limited to, immunoglobulin Fc domains, albumin, and polyethylene glycol.
  • the base sequences of some exemplary Fc domains are shown in SEQ ID NOs. 15 and 16.
  • the Fc domain is selected from the group consisting of IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc.
  • the PIK3IP1 ECD fusion molecule comprises a PIK3IP1 ECD and a fusion partner, the fusion partner is Fc.
  • the base sequence of the PIK3IP1 ECD fusion molecule consists of a base sequence selected from the group consisting of SEQ ID NO. 11, 12, 13 and 14.
  • the PIK3IP1 ECD fusion molecule comprises a signal peptide. In some embodiments, the PIK3IP1 ECD fusion molecule lacks a signal peptide. In some embodiments, the PIK3IP1 ECD portion of the PIK3IP1 ECD fusion molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 9 and 10. In some embodiments, the PIK3IP1 ECD portion of the PIK3IP1 ECD fusion molecule consists of a sequence selected from the group consisting of SEQ ID NOs: 9 and 10.
  • additional amino acids may be present at the N-terminus and/or C-terminus of the PIK3IP1 ECD, but those amino acids are not derived from the PIK3IP1 sequence, but may be derived, for example, from a linker sequence or a fusion partner sequence.
  • a PIK3IP1 ECD can be combined with at least one fusion partner to generate a PIK3IP1 ECD fusion molecule.
  • These fusion partners can facilitate purification or extend the in vivo half-life of the PIK3IP1 ECD fusion molecule.
  • Suitable fusion partners for PIK3IP1 ECD include, for example, polymers such as water soluble polymers, constant domains of immunoglobulins; all or part of human serum albumin (HSA); fetuin A; fetuin B; leucine Acid zipper domain; tetranectin trimerization domain; mannose binding protein (also known as mannose-binding lectin), for example, mannose binding protein 1; and Fc region.
  • the fusion partner does not produce a neutralizing antigenic or other untoward reaction.
  • Polymers for example, water soluble polymers, are used as fusion partners to reduce precipitation of PIK3IP1 ECD fusion molecules in aqueous environments, such as in physiological environments.
  • the polymers employed in the present invention are pharmaceutically acceptable polymers including, but not limited to, polyethylene glycol (PEG), polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy- Polyethylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane Hexane, ethylene/maleic anhydride copolymer, poly( ⁇ -amino acid) (whether homopolymer or random copolymer), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer (PPG) And other polyoxynitrene (polyakylene oxide), poly
  • chemical derivatization can be carried out under any suitable conditions that allow the protein to react with the activated polymer molecules.
  • Activating groups that can be used to attach the polymer to the active moiety include sulfones, maleimides, sulfhydryls, thiols, trifluoromethanesulfonates, trifluoroethylsulfonates, aziridines. , ethylene oxide and 5-pyridyl.
  • the polymer of the invention is typically linked to the PIK3IP1 ECD on the alpha or epsilon amino or reactive thiol group of the amino acid.
  • the PIK3IP1 ECD of the invention can be fused to a marker sequence.
  • the marker amino acid sequence may be a hexahistidine peptide, and hexahistidine facilitates purification of the fusion protein.
  • oligomerization provides some functional advantages to the fusion protein including, but not limited to, multivalent, increased binding strength, and combined function of different domains.
  • the fusion partner comprises an oligomerization domain, eg, a dimerization domain.
  • oligomerization domains include, but are not limited to, coiled-coil domains, including alpha-helical coiled-coil domains; collagen domains; collagen-like domains; and certain immunoglobulin domains.
  • Exemplary coiled-coil polypeptide fusion partners include, but are not limited to, a tetranectin coiled-coil domain; a coiled-coil domain of a cartilage oligomeric matrix protein; an angiopoietin coiled-coil domain; and a leucine zipper domain.
  • the fusion partner is an Fc immunoglobulin domain.
  • An Fc fusion partner can be a wild-type Fc found in a naturally occurring antibody, a variant thereof, or a fragment thereof.
  • Non-limiting exemplary Fc fusion partners include Fc comprising a hinge domain of human IgG (eg, human IgGl, IgG2, IgG3, or IgG4) and a CH2 and CH3 constant domain. Additional exemplary Fc fusion partners include, but are not limited to, human IgA and IgM.
  • the fusion partner is albumin.
  • albumins include, but are not limited to, human serum albumin (HSA) and HSA fragments that are capable of increasing the serum half-life or bioavailability of the polypeptide to which it is fused.
  • HSA human serum albumin
  • HSA fragments that are capable of increasing the serum half-life or bioavailability of the polypeptide to which it is fused.
  • the fusion partner can be covalently or non-covalently linked to the N-terminus or C-terminus of PIK3IP1 ECD.
  • Linkage can also occur in the PIK3IP1 ECD at positions other than the N-terminus or C-terminus, for example, by amino acid side chains (eg, side chains of cysteine, lysine, serine, or threonine).
  • the linker can be contained between the fusion partner and the PIK3IP1 ECD.
  • Such linkers can be composed of at least one amino acid or chemical moiety.
  • Exemplary methods of covalently linking a fusion partner to a PIK3IP1 ECD include, but are not limited to, translation of the fusion partner and PIK3IP1 ECD as a single amino acid sequence and chemical ligation of the fusion partner to the PIK3IP1 ECD. When the fusion partner and PIK3IP1 ECD are translated as a single amino acid sequence, additional amino acids can be included as a linker between the fusion partner and the PIK3IP1 ECD.
  • a linker is selected based on a polynucleotide sequence encoding a linker to facilitate cloning of the fusion partner and/or PIK3IP1 ECD into a single expression construct (eg, a polynucleotide containing a particular restriction site can be included) Between a polynucleotide encoding a fusion partner and a polynucleotide encoding PIK3IP1 ECD, the polynucleotide containing the restriction site encodes a short amino acid linker sequence).
  • the fusion partner and the PIK3IP1 ECD are covalently coupled by chemical means, different sizes of linkers can typically be included during the coupling reaction.
  • a vector comprising a polynucleotide encoding PIK3IP1ECD is provided.
  • Vectors comprising a polynucleotide encoding a PIK3IP1 ECD fusion molecule are also provided.
  • Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like.
  • the selected vector is optimized for expression of the polypeptide in CHO or CHO derived cells.
  • the PIK3IP1 ECD or PIK3IP1 ECD fusion molecule can be expressed in a prokaryotic cell, such as a bacterial cell; or expressed in a eukaryotic cell, such as a fungal cell, a plant cell, an insect cell, and a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a eukaryotic cell such as a fungal cell, a plant cell, an insect cell, and a mammalian cell.
  • eukaryotic cells that can be used to express a polypeptide include, but are not limited to, COS cells, 293 cells, CHO cells, and NSO cells.
  • nucleic acid into a desired host cell can be achieved by any method known in the art including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated Transfection, electroporation, transduction, infection, etc.
  • the PIK3IP1 ECD or PIK3IP1 ECD fusion molecule can be purified by a variety of methods known in the art. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include any ligand of PIK3IP1 ECD or a fusion partner. For example, Protein A, Protein G, Protein A/G or antibody affinity columns can be used to bind to an Fc fusion partner to purify a PIK3IP1 ECD fusion molecule.
  • signal peptide refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that facilitates secretion of the polypeptide from mammalian cells.
  • the signal peptide can be cleaved after the polypeptide is removed from the mammalian cell to form a mature protein.
  • the signal peptides can be natural or synthetic and they can be heterologous or homologous to the protein to which they are attached.
  • antibody is used in its broadest sense and specifically covers synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, humans Antibodies, humanized antibodies, chimeric antibodies, primatized antibodies, Fab fragments, F(ab') fragments, single chain FvFc (scFvFc), single chain Fv (scFv), anti-idiotypic (anti-Id) antibodies and Any other immunologically active antibody fragments are provided as long as they exhibit the desired biological activity (i.e., tag related or binding).
  • the antibodies of the invention comprise immunologically active fragments of immunoglobulin molecules and immunoglobulin molecules (ie, molecules containing antigen binding sites), wherein these fragments may or may not be associated with another immunoglobulin structure. Domains (including but not limited to Fc regions or fragments thereof) are fused.
  • antibody and various antibodies specifically include Fc variants or fragments thereof, including full length antibodies and variant Fc-fusions comprising an Fc region, optionally comprising at least one Amino acid residues are modified and fused to immunologically active fragments of immunoglobulins.
  • “Pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, preservatives, isotonic agents, sustained release agents, and the like.
  • the carrier is suitable for intravenous, subcutaneous, intramuscular, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the pharmaceutical compositions of the present invention may include one or more pharmaceutically acceptable salts, antioxidants, water and non-aqueous vehicles, and/or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Immuno response refers to a biological response in a vertebrate to a foreign agent of action that protects the organism from the damage caused by or by the agents of the action medium.
  • the immune response is made up of cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer NK cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and by these cells.
  • soluble macromolecules including antibodies, cytokines, and complements
  • the liver which cause the vertebrate organism to invade pathogens, cells, or tissues infected with pathogens, cancer cells, or other abnormal cells, or In the autoimmune or pathological inflammation, selective targeting, binding, damage, destruction, and/or elimination of normal human cells or tissues.
  • Immunotherapy refers to the treatment of a subject having a disease, having a risk of developing a disease, or a relapse of a disease, using a method comprising inducing, potentiating, inhibiting, or modifying an immune response.
  • Enhancing an endogenous immune response means enhancing the effectiveness or strength of an existing immune response in a subject. Such potentiation of efficiency and potential can be achieved, for example, by overcoming the mechanisms that inhibit the endogenous host immune response, or by stimulating mechanisms that potentiate the endogenous host immune response.
  • a “therapeutically effective amount” or “therapeutically effective amount” of a drug or therapeutic agent is any amount of a drug described below, when used alone or in combination with another therapeutic agent. It can promote the regression of the disease, which is manifested by a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of the symptom-free period, or prevention of a disorder or disability caused by the disease.
  • a therapeutically effective amount or dose of a drug includes a "prophylactically effective amount” or “prophylactically effective amount", “prophylactically effective amount” or “prophylactically effective amount” is any amount of a drug as described below, when the amount of the drug is administered alone or When administered in combination with another therapeutic agent to a subject having a risk of developing a disease or suffering from a disease recurrence, the occurrence or recurrence of the disease can be inhibited.
  • the ability of a therapeutic agent to promote disease regression or inhibit disease progression or relapse can be assessed by various methods known to the skilled artisan, such as in animal models of human subjects, in animal model systems that predict efficacy in humans. Or by measuring the activity of the reagent in an in vitro assay system.
  • Sub-therapeutic dose means a therapeutic compound such as a sub-therapeutic dose of a CTLA-4 antibody that is less than about 3 mg/kg (i.e., a known dose of anti-CTLA-4 antibody).
  • Cancer refers to a wide variety of diseases characterized by uncontrolled growth of abnormal cells in the body, which can be used interchangeably with “tumors” herein. Uncontrolled cell division and growth division and growth lead to the formation of malignant tumors or cells that invade adjacent tissues and can also be transferred to the distal part of the body through the lymphatic system or blood flow.
  • Subject means a living body that is subjected to the action of the active substance of the present invention, and can be used interchangeably with the “medicalist” in the present application.
  • the subject can be a human or an animal, such as a monkey, rat, mouse, dog, rabbit, and the like.
  • Figure 1 shows the results of PIK3IP1 and Pik3ip1 electrophoresis.
  • Figure 2a - Figure 2d shows the results of fusion protein profiling.
  • Figure 3 shows the specificity of the anti-PIK3IP1 polyclonal antibody specificity, using mouse anti-human PIK3IP1 antibody; diluting polyclonal antibodies in different proportions, and staining with CHO cell transfectants stably expressing human PIK3IP1, flow cytometry to antibody The result of the titer identification.
  • Fig. 4 Identification results of anti-Pik3ip1 polyclonal antibody specificity, using rat anti-mouse Pik3ip1 antibody; diluting polyclonal antibodies in different proportions, and staining with CHO cell transfectants stably expressing mouse Pik3ip1, flow cytometry The results of antibody titer identification.
  • FIG. 5 Flow cytometric detection of PIK3IP1 expression in human peripheral blood.
  • PIK3IP1 has higher expression of CD8 + T cells, CD4 + T cells and B cells in human peripheral blood PBMC, and lower expression in CD56 + NK cells.
  • Pik3ip1 Detection of Pik3ip1 expression in mouse tissue mRNA levels. Pik3ip1 is expressed at higher levels in mouse muscle, PBMC, lymph nodes and spleen.
  • FIG. 7 Flow cytometry detection of Pik3ip1 expression in mouse spleen lymphocytes. Pik3ip1 was expressed on spleen CD8 + T cells, CD4 + T cells, B cells, CD11b + cells and CD11c + cells, among which T cells, B cells and CD11b + cells were highly expressed.
  • FIG. 8 Flow cytometry analysis of Pik3ip1 expression in common mouse cell lines, B16 is a mouse melanoma cell line, P338D1 is a mouse macrophage cell line, DC2.4 is a mouse dendritic cell line, and CT26 is small.
  • FIG. 9 Flow cytometric detection of Pik3ip1 expression on lymphocytes during immune activation in mice.
  • Pmel-1 TCR transgenic mice were immunized with 100 ⁇ g of hgp100 25-33 and CFA or CFA alone. After 24 hours, P83ip1 was detected in mouse draining lymph nodes, CD8 + T cells, CD11b + monocytes, CD11c + DC cells, Expression of CD19 + B cells.
  • FIG. 10 Flow cytometry was used to detect the proliferation of CD8 + T cells in PIK3ip1 knockout mice and wild-type mice in OVA antigen-specific immune responses. After antigen-specific immunization, CD8 + T of Pik3ip1 knockout mice The proliferation of cells is higher than that of wild type mice.
  • the left picture shows the flow cytometry map, and the right picture shows the flow cytometry fluorescence value.
  • KO represents a Pik3ip1 knockout mouse
  • WT represents a wild type mouse.
  • FIG. 11 Tumor model was established using MC38 tumor cells in Pik3ip1 knockout mice and wild-type mice, and proliferation of CD4 + and CD8 + T cells in mice was detected by flow cytometry at 24 hours, 48 hours and 72 hours. The proliferation of CD4 + and CD8 + T cells in Pik3ip1 knockout mice was higher than that in wild type mice, and the difference between 48 hours and 72 hours was significant.
  • KO represents a Pik3ip1 knockout mouse
  • WT represents a wild type mouse.
  • Fig. 12 The relationship between the expression of PIK3IP1 in peripheral blood lymphocytes and the inhibition state of T cells in patients with oral squamous cell carcinoma.
  • the fluorescence intensity of PIK3IP1 on CD4 + T cells and CD8 + T cells is negatively correlated with the proliferation ability of corresponding T cells. The correlations are all significant.
  • Pik3ip1-Ig fusion protein plays a competitive inhibitory role in mice, promoting the proliferation of CD8 + T cells; the left picture shows the proliferation of CD8 + T cells under the action of Pik3ip1-Ig fusion protein; the right picture shows the CD8 + T cells Percent proliferation statistics (*p ⁇ 0.05).
  • Figure 14pik3ip1-Ig fusion protein combined with 2A inhibits the growth of mouse B16-F10 tumor; A is treated with Pik3ip1-Ig fusion protein alone, B16-F10 tumor growth inhibition is not obvious (nsp>0.05); B is Pik3ip1-Ig fusion Protein combined with 2A treatment, the growth of B16-F10 tumor was significantly inhibited (*p ⁇ 0.05).
  • Fig. 15 is a view of the tumor-bearing C57BL/6 mice, and the tumor is in vitro; A is a general view of the tumor-bearing mice in each group; B is an isolated view of each group of tumors.
  • PIK3IP1 represents the human PIK3IP1 full-length base sequence and its protein product
  • Pik3ip1 represents the murine Pik3ip1 full-length base sequence and its protein product
  • PIK3IP1-mIg represents the human PIK3IP1 extracellular domain and mouse
  • Pik3ip1-mIg represents a fusion protein consisting of the extracellular domain of mouse Pik3ip1 and mouse IgG Fc
  • PIK3IP1-hIg represents a fusion protein composed of human PIK3IP1 extracellular domain and human IgG Fc
  • Pik3ip1 -hIg represents a fusion protein consisting of the extracellular domain of mouse Pik3ip1 and human IgG Fc
  • Flag-mIg is a fusion protein of a disordered control sequence and murine IgG Fc
  • Flag-hIg is a fusion of a disordere
  • SPF-class C57BL/6 mice, BALB/c mice, and Wistar rats were purchased from the Experimental Animal Center of Sun Yat-sen University (University City) and raised at the Experimental Animal Center of Sun Yat-sen University North Campus;
  • Pmel-1 TCR transgenic mice 293T cells, mouse melanoma cell line (B16-F10), mouse colon cancer cell line CT26 cells, mouse bladder cancer cell line MB49 cells, CHO cell line, hybridoma 2A, small Mouse macrophage cell line P338D1, mouse dendritic cell line DC2.4, mouse T cell lymphoma cell line EL4, MC38 cell line, plasmid pMIgV, pHIgV were donated by the research group Professor Jian Ping of Zhongshan University of Sun Yat-sen University.
  • Hgp100-specific Pmel-1 splenocytes and lymphocytes were obtained from the spleen and lymph nodes of Pmel-1 TCR transgenic mice.
  • OT1 mice were purchased from the Institute of Model Animals, Nanjing University.
  • Pik3ip1-/- mice were prepared by knockout using the TALENS method at Cyagen Biosciences Inc.
  • DMEM high glucose medium RPMI 1640 medium, Ham's F10 medium, trypsin (0.25% Trypsin, 0.02% EDTA): Gibco, USA; 2-acetyl-2-deamide derivative (ADT), penicillin, Streptomyces , L-glutamate, hydroxyfluorescein diacetate succinimide lipid (CFSE): Invitrogen, USA.
  • RNAiso Plus (9108Q), PrimeScript TM RT reagent Kit Kit, SYBR Green qPCR Master Mix-SYBR Advantage (638320Clontech) Kit: by Takara Company; All-In-One one-step Reverse Transcription Kit: U.S.
  • Cytofix/Cytoperm immobilization/rupture kit Anti-human CD4 Percp-cy5.5, Anti-human CD4BV421, Anti-human CD8 APC: US BD; Anti-human CD69 Pe-cy7, Anti-human CD95FITC, Anti- Human HLA-DR Pe-cy7, Anti-human CD3 (clone: OKT3): American Biolegend; Anti-human CD28, Anti-human CD25 APC, Anti-human Foxp3 PE, Anti-human CD25PE: American eBioscience; CD4 + T cell magnetic bead positive sorting kit, human CD8 + T cell magnetic bead positive sorting kit, human CD3 + T cell magnetic bead negative sorting kit: German Meitian company; recombinant human TGF-beta1 Recombinant human IL-2: Peprotech, USA.
  • Axio observer Z1 inverted microscope, laser scanning confocal microscope (LSM780): Germany ZEISS company; MACS sorter, MACS sorting column: Germany Mei Tianni company; 480 automatic fluorescence quantitative PCR instrument: Roche, Germany; BD FACSVerse flow cytometer: American BD company; M205FA stereo fluorescence microscope: Lycra Instrument Co., Ltd.; ABI 9700 PCR instrument: American ABI company; automatic microplate reader: USA Thermo Electron; ScanScope Pathology Slice Scanning Imaging System: Apero, USA.
  • Peripheral blood mononuclear cells were isolated according to the kit instructions of Stemcell No. 07811, and the peripheral blood mononuclear cells were extracted using the corresponding kit.
  • RNA extraction was performed using the Takara RNAiso Plus (9108Q) kit according to the instructions: cells were digested or lysed by tissue with about 0.5-1 ml of RNAiso Plus; chloroform-isopropanol- The RNA was extracted with 75% ethanol, and the extracted RNA was dissolved in DEPC water. The RNA concentration was measured by a spectrophotometer, and the RNA concentration was adjusted to about 500 ng/ ⁇ l.
  • RNA-Primer Mix total volume 13 ⁇ l
  • denatured RNA as follows:
  • the reverse transcription reaction conditions were as follows: reverse transcription reaction at 37 ° C for 10 min, followed by heating to 85 ° C for 5 min to inactivate the reverse transcriptase and terminate the reaction.
  • the reaction system is as follows:
  • the reaction was carried out on a Roche LightCycler 480 Real-Time PCR machine with amplification conditions: 95 ° C for 5 min; 95 ° C for 10 sec; 58 ° C for 20 sec; 72 ° C for 30 sec, 40 cycles; dissolution curve analysis: 95 ° C for 5 sec; 65 ° C for 1 min; 5 min; 40 ° C for 10 sec to cool.
  • the plasmid vector was pMIgV (PIK3IP1-mIg, Pik3ip1-mIg) and pHIgV (PIK3IP1-hIg, Pik3ip1-hIg), and the restriction sites were selected as Bgl II and EcoR I.
  • the plasmid vector was PCDNA3.1 (PIK3IP1 and murine Pik3ip1 full-length sequence), and Xhol I and Ecor I were selected for the restriction sites.
  • the human reversed human cDNA and the mouse cDNA were separately mixed and used as a template for PCR to amplify the corresponding sequence;
  • the PCR reaction system is as follows:
  • the PCR reaction conditions are as follows:
  • the PCR product was recovered using a Qiagen gel recovery kit.
  • the gel recovery product and the vector plasmid are selected for the corresponding digestion enzymes for double digestion.
  • the reaction system used is as follows:
  • Enzyme digestion conditions the rubber recovered product was 37 ° C, and the enzyme was cut for 40 min.
  • the plasmid was cleaved at 37 ° C for 50-55 min;
  • the digested product is subjected to agarose gel electrophoresis, and the gel is recovered to obtain a target band for connection:
  • Ligation reaction conditions 2 hours at 37 ° C, or 2 hours at 16 ° C, overnight at 4 ° C;
  • plasmids of PIK3IP1-hIg, PIK3IP1-mIg, Pik3ip1-hIg, and Pik3ip1-mIg were also constructed.
  • the constructed plasmid is transformed into a bacterium, cultured to obtain a cloned bacterium of interest, and the positive clone having normal sequence after sequencing is expanded and cultured.
  • Plasmid extraction and purification were performed using the NucleoBond Xtra Maxi Plus Plasmid Extraction and Purification Kit according to the instructions in the instructions.
  • the supernatant of the cell culture supernatant was collected to obtain a supernatant, and the high affinity protein A column of GE was used, and the fusion protein was extracted and purified according to the instruction manual.
  • the collected proteins were then transferred to a protein dialysis bag and dialyzed overnight at 4° C. in pH 7.2-7.4 in 1 ⁇ PBS. The dialyzed protein was used in subsequent experiments.
  • the fusion protein was run on an SDS-PAGE gel, and the gelatin was stained with Coomassie blue and sent to mass spectrometry for identification.
  • the purified fusion proteins were determined to be PIK3IP1-hIg, PIK3IP1-mIg, Pik3ip1-hIg, Pik3ip1-mIg, respectively.
  • the preparation method of Ham's F10 whole medium for culturing CHO cell line is as follows: 500ml Ham's F10 medium containing 10% FBS, 1% Hepes, 1% streptomycin and penicillin mixture, 1 ⁇ ADT.
  • the resuscitated CHO cell line was cultured for 2 passages, and after the cells were in good condition, 1 ⁇ 10 5 cells were added to one well of a 24-well plate, and the culture medium was adjusted to 1 ml, and the cells in the well plate were grown to fusion. Approximately 90%, the medium was aspirated. Wash with an appropriate amount of PBS, and then change to 200 ⁇ l of antibiotic-free medium.
  • Transfection was performed using the Invitrogen LipofectaminTM 2000 kit according to the instructions: 0.8-1 ⁇ g of plasmid DNA expressing the full length of PIK3IP1 and the vector was PCDAN3.1 was added to 50 ⁇ l of Opti-MEM and mixed well. 2 ⁇ l of the transfection reagent Lipo2000 was added to 50 ⁇ l of Opti-MEM and mixed well. The diluted transfection reagent was added dropwise to the diluted plasmid DNA, and the DNA-Lipo2000 complex was added to the cell-coated wells.
  • the 24-well plate was placed in a 5% CO 2 37 ° C cell culture incubator; after 6-8 hours of transfection, the liquid in the well was removed and replaced with Ham's F10 whole medium. The well plates were placed in a 5% CO 2 37 ° C cell incubator for 48 hours.
  • the cells After mixing the cells, they were cultured in a 5% CO 2 37 ° C cell culture incubator; after 5-7 days in a cell culture dish, the culture dishes were taken out, the original medium was aspirated, and the whole culture was performed with Ham's F10 containing 1000 ⁇ g/ml G418. Base 20ml. The culture was continued in a 5% CO 2 37 ° C cell culture incubator; cell monoclonal formation was generally observed after 10 days in the culture dish. CHO cells transfected with the PIK3IP1 plasmid were subcloned for selection when all cells in the untransfected screening control dish died.
  • One-third or half of the cells were subjected to flow cytometry or WB identification, and 2-3 clones with the highest expression intensity were selected, cultured, and stored frozen.
  • Pik3ip1 stably transformed strain PIK3IP1-hIg stably transformed strain, PIK3IP1-mIg stably transformed strain, Pik3ip1-hIg stably transformed strain, and Pik3ip1-mIg stably transformed strain were also prepared.
  • mice of SPF grade 6-8 weeks old were selected for the preparation of human PIK3IP1 polyclonal antibody; female Wistar rats of SPF grade of 200 g were selected for the preparation of mouse Pik3ip1 polyclonal antibody.
  • First immunization Before immunization, take appropriate amount of blood in the tail vein of immunized animals, extract serum, and store frozen as a negative control serum; each mouse is emulsified with 100 ⁇ l of CFA and an equal volume of 100 ⁇ g of human PIK3IP1-mIg fusion protein. Injected into Balb/c mice bilaterally underarm and inguinal, not less than 4 points; each rat was emulsified by 500 ⁇ l of CFA and an equal volume of 500 ⁇ g of mouse Pik3ip1-mIg fusion protein, and injected into the bilateral armpits of rats. And the inguinal subcutaneous, no less than 4 points; 2 weeks after immunization, blood samples were taken from the tail vein of immunized animals to prepare serum, and antibody titer was detected by ELISA or flow cytometry.
  • 2nd and 3rd immunization Each mouse was immunized with 100 ⁇ l of IFA and an equal volume of 50 ⁇ g of human PIK3IP1-mIg fusion protein; each rat was immunized with 500 ⁇ l of IFA and an equal volume of 250 ⁇ g of mouse Pik3ip1- mIg fusion protein, fully emulsified and immunized rats; antibody titer was detected 2 weeks after each immunization.
  • the second or third sera after immunization are diluted 1:100, ELISA, the OD450 value is about 1.5, or the flow test 1:1000-fold dilution has a positive peak
  • 50 ⁇ g of human PIK3IP1-mIg fusion protein can be injected intraperitoneally. Or 250 ⁇ g murine Pik3ip1-mIg fusion protein to enhance immunity; 3-5 days after boosting immunization, the animals were sacrificed, and blood was taken from the inferior vena cava to prepare polyclonal antibody serum.
  • 293T cells transfected with PIK3IP1 were seeded in glass bottom culture dishes, and cultured in DMEM medium supplemented with 10% FBS overnight; the medium was aspirated, washed twice with PBS, and fixed in 4% paraformaldehyde for 15 min at room temperature.
  • the fixed solution was aspirated, rinsed with PBS for 3 min, washed twice; 10% normal goat serum was blocked at room temperature for 30 min; the blocked serum was aspirated, and the mouse anti-human PIK3IP1 polyclonal serum was added 1:200 diluted and incubated at 4 ° C overnight; PBS rinsed for 3 min, Wash 3 times; goat anti-mouse Alexa Fluor 647 fluorescent secondary antibody 1:1000 dilution, incubate at room temperature for 1 hour in the dark; PBS rinse for 3 min, wash 3 times; 10 ⁇ g / ml WGA incubation at room temperature for 10 min; PBS rinse for 3 min, wash 3 times; The DAPI staining solution was stained for 3 min; the PBS was rinsed for 3 min, washed 3 times; the anti-quenching seal tablets were added to the Petri dish, and the coverslips were mounted. Confocal microscopy observation, photographing.
  • mice 100 ⁇ g of hgp100 25-33 polypeptide and CFA were emulsified in equal volume, and the same amount of CFA was emulsified with equal volume of PBS.
  • 100 ⁇ l of hgp100 25-33 polypeptide and CFA were injected subcutaneously into the right side of female 6-8 week old Pmel-1 TCR transgenic mice. Emulsifier.
  • Another group of mice was immunized by subcutaneous injection of 100 ⁇ l of CFA and PBS emulsifier; after 24 hours of immunization, the mice were sacrificed. Drainage lymph nodes were taken to prepare single cell suspension, and the expression of Pik3ip1 on CD8 + T cells, CD11b + monocytes, CD11c + DC cells and CD19 + B cells was detected during the immunization.
  • the primary antibody was stained by adding about 2-3 ml of the flow buffer. Centrifuge at 2000 rpm for 3 min. The supernatant was discarded so that the total volume of the cell suspension in the flow tube was about 100 ⁇ l. Add a suitable dose of fluorescent secondary antibody, mix, place in a refrigerator at 4 ° C, incubate for 30 min in the dark; add about 2-3 ml of flow buffer to terminate the fluorescent secondary antibody staining. The cells were washed, centrifuged at 2000 rpm for 3 min, and the supernatant was discarded. Depending on the number of cells, add about 300-400 ⁇ l of flow buffer. After mixing, the flow is detected on the machine.
  • the inventors of the present invention isolated healthy human PBMC and detected the expression of major lymphocyte subsets of PIK3IP1 in PBMC by flow cytometry. As a result, as shown in Fig. 5, it was found that PIK3IP1 was highly expressed in human peripheral blood CD8 + T cells, CD4 + T cells, and B cells.
  • the inventors of the present invention examined the expression levels of mouse Pik3ip1 mRNA in PBMC, eyes, spleen, brain, ovary, lung, lymph node, bladder, pancreas, kidney, liver, small intestine, thymus, heart, muscle, uterus and testis. As a result, as shown in Fig. 6, it was found to have a high expression level in muscle, PBMC, lymph nodes and spleen.
  • the inventors of the present invention isolated and prepared a mouse spleen single cell suspension, and detected the expression of Pik3ip1 in the main lymphocyte subsets of the spleen by flow cytometry. The results are shown in Figure 7. It was found that Pik3ip1 was able to detect expression on CD8 + T cells, CD4 + T cells, B cells, CD11b + cells and CD11c + cells, and expression in T cells, B cells and CD11b + cells. high.
  • the inventors of the present invention examined Pik3ip1 in mouse melanoma cell line B16, mouse macrophage cell line P338D1, mouse dendritic cell line DC2.4, mouse colon cancer cell line CT26, mouse T cell lymphoma.
  • the expression of the cell line EL4 and the mouse bladder cancer cell line MB49 showed that Pik3ip1 was highly expressed in P338D1, DC2.4, and EL4 as shown in Fig. 8 .
  • the inventors of the present invention specifically immunized with 100 ⁇ g of hgp100 25-33 polypeptide and CFA, or non-specifically immunized Pmel-1 TCR transgenic mice after emulsification with CFA alone.
  • Pik3ip1 was detected for expression of CD8 + T cells, CD11b + monocytes, CD11c + DC cells, and CD19 + B cells in the draining lymph nodes of Pmel-1 TCR transgenic mice.
  • Fig. 9 it was found that the expression of Pik3ip1 on the above lymphocytes varies depending on the intensity of immune activation.
  • KO and WT were intraperitoneally immunized with OVA 257–264 (100 ⁇ g) with CFA as adjuvant.
  • anti-mCD3, anti-mCD28 gusset anti-mCD3, anti-mCD28 monoclonal antibody, each formulated to a final concentration of 0.125 ⁇ g / ml, each 50 ⁇ l / well into a 96-well plate, sealing plate, overnight at 4 ° C; The unbound protein solution in the well was removed, and cold PBS was added at 100 ⁇ l/well, and the plate was repeatedly washed at least 3 times.
  • mice 3 after two weeks, splenocytes were removed KO and WT mice, prepared single cell suspensions, stem cell company with EasySep TM mouseCD4 + T cel isolation kit and EasySep TM mouseCD8 + T cel isolation kit , according to the kit instructions , CD4 + and CD8 + T cells were sorted. Add anti-mCD3, anti-mCD28 gusset plate to the plate at 3.5 ⁇ 10 5 cells/well.
  • Example 8 PIK3IP1 is related to the inhibition state of T cells in cancer patients
  • anti-hCD3, anti-hCD28 gusset anti-hCD3, anti-hCD28 monoclonal antibody, each formulated to a final concentration of 0.125 ⁇ g / ml, each 50 ⁇ l / well into a 96-well plate, sealed, overnight at 4 ° C; The unbound protein solution in the well was aspirated, and cold PBS was added at 100 ⁇ l/well, and the plate was repeatedly washed at least 3 times.
  • Fig. 12 The results are shown in Fig. 12: the fluorescence intensity of PIK3IP1 in CD4 + and CD8 + T cells of peripheral blood of OSCC patients was inversely correlated with the proliferation intensity, and the correlation was significant. It indicated that the expression intensity of PIK3IP1 in CD4 + and CD8 + T cells of peripheral blood of OSCC patients was negatively correlated with the proliferation ability of corresponding cells, and the expression of PIK3IP1 inhibited the proliferation of corresponding cells.
  • the cell concentration was adjusted to 1 ⁇ 10 7 /ml with PBS, and each C57BL/6 mouse was injected with 250-300 ⁇ l cell suspension in the tail vein, and 10 female C57 mice of 6-8 weeks old were injected.
  • the specificity of gp100 was transferred.
  • 100 ⁇ g of each mouse hgp100 25-33 polypeptide was fully emulsified with CFA in a total volume of 100 ⁇ l, and then each mouse was administered subcutaneously to the right side, and 3 mice were separately administered.
  • Pik3ip1-mIg was injected intraperitoneally, and 7 mice were intraperitoneally injected with Flag-mIg fusion protein at a dose of 200 ⁇ g/300 ⁇ l/day. On the 2nd day after adoptive transfer, 1 day of intraperitoneal administration of Flag-mIg fusion protein was performed. In rats, draining lymph nodes were taken to make a single cell suspension. The flow-through antibody Anti-mouse CD90.1APC, Anti-mouse CD8BV421 stained the labeled cells; the ratio of proliferating peaks of CFSE and CD8 double positive cells to CFSE was detected by flow cytometry. When an obvious and regular proliferation peak was detected, 3 mice in each of the experimental group and the control group were sacrificed, and the proliferation level of CD8 + T cells was examined.
  • the average ratio of CD8 + T cell proliferation in the Pik3ip1-mIg fusion protein group was 88.32%, while the average proliferation ratio of CD8 + T cells in the Flag-mIg group was 53.34%, which was significantly lower than that in the Pik3ip1-mIg fusion protein group.
  • Example 10 Pik3ip1-mIg fusion protein enhances the immunotherapeutic effect of 2A on mouse B16-F10 tumor formation - Pik3ip1-mIg combined with 2A treatment inhibits the growth of B16-F10 tumor
  • Hybridoma 2A was resuscitated and cultured. The medium was centrifuged, the supernatant was taken, the supernatant was filtered through a 0.45 ⁇ m filter, and the filtrate was collected on ice or stored at 4 ° C; Anti-m4-1BB (2A) was carried out with an affinity protein G column. Purification, purification of the antibody was transferred to a protein dialysis card, and dialyzed at 4 ° C for 24 to 48 hours in 1 x PBS at pH 7.2-7.4.
  • DC2.4 cells were resuscitated and cultured. The cells were resuspended in 1 ml of RPMI 1640 medium containing 10% FBS, DC2.4 cells were labeled with gp100 25-33 polypeptide at a working concentration of 5-8 ⁇ M, and the cells were resuspended in an appropriate amount of PBS. Formulated to 3.5 ⁇ 10 6 cells / ml.
  • mice Prepare a single cell suspension of spleen and/or lymph node cells of Pmel-1 TCR transgenic mice as described above, and resuspend the cells at a ratio of at least 1 ⁇ 10 7 /cell per 300 ⁇ l of cell suspension; SPF grades of similar body weight 6-8 weeks
  • Female C57BL/6 mice were set up in the experimental mice group: Flag-mIg control group, Pik3ip1-mIg treatment group, 2A treatment group, Pik3ip1-mIg combined with 2A treatment group.
  • mice per group were subcutaneously injected with 100 ⁇ l/mouse of DC2.4 cell suspension labeled with hgp100 25-33 polypeptide, and 300 ⁇ l of gp100-specific Pmel-1 spleen cell suspension was injected into the tail vein.
  • B16-F10 cell suspension with a cell concentration of 3 ⁇ 10 6 /ml was injected subcutaneously into each tumor with 100 ⁇ l of each mouse; 300 ⁇ l of PBS was administered intraperitoneally every 3 days after subcutaneous tumor formation.
  • the prepared protein solution contained 200 ⁇ g of Flag-mIg, 200 ⁇ g of Pik3ip1-mIg, 200 ⁇ g of 2A, and 200 ⁇ g of Pik3ip1-mIg+200 ⁇ g of 2A; the tumor growth was observed, and the tumor was started under the skin, and every other day was measured with an electronic vernier caliper. Tumor size was recorded until the tumor grew to an average diameter of about 1.5 cm. The average diameter of the tumor is calculated as: (long diameter + wide diameter) / 2.
  • the inventors of the present invention constructed a model in which Pik3ip1-mIg fusion protein alone and in combination with 2A was used to treat mouse B16-F10 tumor growth.
  • Fig. 15A in general, the Pik3ip1-mIg fusion protein was combined with the 2A treatment group, the B16-F10 tumor formation was smaller than the Flag-mIg group, the Pik3ip1-mIg fusion protein group was administered alone, and the average diameter of the 2A group was small; The group 2A was administered alone; the average diameter of the tumors of the Pik3ip1-mIg fusion protein group alone was slightly smaller than that of the Flag-mIg group. The same results can be seen from the images of the tumor isolated from Figure 15B.

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Abstract

本发明提供一种PIK3IP1蛋白在调节T细胞反应和用于个体癌症治疗的方法及其相关应用，以及由此获得的组合物和试剂盒产品。

Description

PIK3IP1蛋白在调节T细胞反应和制备抗肿瘤药物中的应用
本申请要求2018年2月5日向中国国家知识产权局提交的专利申请号为201810112922.X，发明名称为“PIK3IP1蛋白在调节T细胞反应和制备抗肿瘤药物中的应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医药领域，具体涉及PIK3IP1蛋白在调节T细胞增殖、调控T细胞活化和制备抗肿瘤药物中的应用。
背景技术
T细胞是获得性免疫系统最核心的要素。根据T细胞表面的糖蛋白的不同，T细胞可以分为CD4 +T细胞和CD8 +T细胞。而循环性的CD4 +和CD8 +T细胞在体内巡逻时可以区分这种蛋白是异物还是自身的多肽，外源性的蛋白会释放免疫系统处于危险的信号，导致T细胞激活，同时启动其他免疫细胞，然后杀死靶细胞。在对抗肿瘤免疫过程中，T细胞作为核心的执行者，首先被T细胞受体介导的抗原识别信号激活，同时众多的共刺激信号和共抑制信号精细调节T细胞反应的强度和质量，这些抑制信号即为免疫检查点。
在生理情况下，一方面参与维持对自身抗原的免疫耐受，避免自身免疫性疾病，另一方面避免免疫反应的过度激活对组织造成的损伤。肿瘤细胞可以通过免疫检查点，抑制T细胞激活，从而逃避免疫损伤。因此，通过不同方法增强T细胞的激活对肿瘤免疫治疗具有重要意义，其中针对免疫检查点的阻断是增强T细胞激活的有效策略之一。以免疫检查点阻断为代表的肿瘤免疫治疗与其它治疗的联合正在被日益关注。免疫系统可以识别肿瘤细胞表达的异常蛋白并将肿瘤细胞杀灭，但是在癌症患者身上，这个过程是有缺陷的。另外，肿瘤细胞可以调用其他多种机制逃避免疫系统的监视，也有一些肿瘤细胞会诱导调控T细胞限制细胞毒T细胞，导致其无法攻击肿瘤细胞。
机体的免疫反应受到多种类型免疫细胞、细胞因子等的调控，以抵御危险信号和外来抗原的攻击；T细胞在免疫应答过程中发挥了重要作用。调节机体免疫反应并维持自身抗原引起的免疫耐受，有赖于一个由共刺激和共抑制分子组成的复杂网络的参与。T细胞活化受协同信号网络的调控，这种调控贯穿于T细胞反应的全程。B7/CD28为代表的Ig超家族和TNF受体超家族的一些成员构成了T细胞共刺激分子的主体。这些协同信号通路的重要性在人类多种疾病中得到了证实，包括移植物抗宿主反应、自身免疫性疾病、感染和肿瘤。从数目众多的临床试验中可以看出，除了大家熟知的CTLA-4，越来越多的检查点调控分子及信号通路，如程序 性死亡分子(PD)-1和B7-H4通路被发现在多种疾病的治疗中发挥作用。近年来，新的检查点调控分子，如T细胞活化的V-区Ig抑制分子(VISTA)和B7-H6被陆续发现和证实。
负向关键调控因子近来成为肿瘤研究的前沿。近年来，抗体药物发展的另外一个新的方向是免疫检查点单抗，利用免疫检查点，肿瘤细胞可以抑制、削弱机体对肿瘤的免疫反应，可以通过抑制T细胞的活化来逃避免疫系统的攻击，而这种抗体可以激活T细胞、缩小肿瘤、提高病人生存率。这类受体包括CTLA-4和PD-1，当用单抗来阻断CTLA-4，PD-1或PD-L1，具有保护性的抗肿瘤免疫系统功能将会加强。2011年第一个免疫检查点抑制剂Yervoy获得FDA批准上市，用于治疗黑色素瘤；2014年9月，FDA通过加速批准程序批准了首个PD-1抑制剂Keytruda(pembrolizumab)上市，用于治疗不再对其他药物应答的晚期或无法切除的黑色素瘤。免疫检查点单抗(CTLA-4单抗、PD1/PDL1单抗)是肿瘤免疫治疗的最新成果，特别是在血液肿瘤和黑色素瘤方面，因具有良好的抗肿瘤活性，取得了重大的进展。Ipilimumab于2011年获批，针对转移性黑色素瘤的抗CTLA-4单抗，已被用于治疗晚期黑色素瘤，但是反应率低，抗CTLA-4治疗对患者有免疫毒性。MDX-1106，作为抗PD-1单抗，在临床上也是极具应用潜力的。研究表明MDX-1106的毒性比Ipilimumab低，但是在前列腺癌等癌症没有抑制肿瘤生长的能力，因为这些肿瘤并没有PD-L1的靶点。这说明，很多癌症仍存在一些关键的负向调控因子来破坏具有保护性的抗肿瘤免疫系统。
磷脂酰肌醇3激酶相互作用蛋白1(PI3K interacting protein1,PIK3IP1)是一个新近报道的能与磷脂酰肌醇3激酶(phosphatidylinositol-3-kinase，PI3K)的p110亚基结合的蛋白,可以下调PI3K活性，抑制丝氨酸苏氨酸激酶(AKT，RAC-alphaserine/threonine-proteinkinase)的活化。2012年，有研究报道了PIK3IP1蛋白可在T细胞上表达。通过在人白血病细胞系Jurkat和小鼠Th2细胞系D10细胞上过表达PIK3IP1蛋白，抑制了T细胞活化相关的转录信号分子NFAT、AP-1的活化。相反，用SiRNA在上述细胞系沉默掉PIK3IP1基因，能够增强Akt的磷酸化、促进T细胞的活化和IL-2的分泌。说明，新的PIK3IP1信号通路调控因子PIK3IP1可能抑制T细胞的活化。
发明内容
本发明发明人对PIK3IP1在常见的各组织、免疫细胞、肿瘤细胞系上等的表达进行了检测和分析。发现其在免疫细胞上表达较高，且表达强度与机体的免疫状态相关。尤其在T细胞有相对较特异的表达。机体的免疫反应受到一系列分子的调控，以保证机体抵御危险信号和外来抗原的攻击。其中，T细胞在维持体内免疫内稳态和免疫防御方面发挥了重要作用。
本发明对比研究了Pik3ip1敲除小鼠和野生型小鼠在经OVA抗原特异性免疫反应后T细胞的增殖情况，发现Pik3ip1敲除小鼠的T细胞的增殖能力强于野生型小鼠。说明在新抗原的急性刺激下，小鼠T细胞上Pik3ip1的表达对T细胞的增殖有抑制作用。
本发明对比研究了Pik3ip1敲除小鼠在MC38肿瘤模型中T细胞的反应和对肿瘤生长的影响。发现Pik3ip1缺失的T细胞在肿瘤抗原刺激下其增殖能力和IFN-γ等效应分子分泌均强于野 生型小鼠。由此表明，在肿瘤抗原刺激下，小鼠T细胞上Pik3ip1的表达对T细胞反应有明显抑制作用，Pik3ip1表达降低可有效抑制肿瘤的生长。
本发明进一步研究了口腔鳞癌患者外周血淋巴细胞，同样发现，口腔鳞癌患者外周血CD4 +和CD8 +T细胞上PIK3IP1的荧光强度与其增殖能力呈负相关，即，CD4 +和CD8 +T细胞上PIK3IP1表达水平越高，相应细胞的增殖能力越弱。
此外，本发明发明人还具体研究了PIK3IP1ECD-Fc融合分子在体内可促进小鼠CD8 +T细胞的增殖，并研究了PIK3IP1ECD-Fc融合分子单独使用或与4-1BB激活性抗体2A(Anti-m4-1BB(2A))联合应用对小鼠成瘤的免疫治疗效果。结果发现，PIK3IP1ECD-Fc融合分子和4-1BB激活性抗体2A联合，能显著抑制肿瘤的生长。
在此基础上，本发明提出如下技术方案：
本发明的一个方面提供，PIK3IP1抑制剂在制备用于调控受试者免疫应答的药物中的用途。在本发明的一些实施方式中，所述PIK3IP1抑制剂选自PIK3IP1、PIK3IP1ECD和PIK3IP1ECD融合分子中的至少一个。在本发明的一些实施方式中，所述PIK3IP1抑制剂是能结合到PIK3IP1上的抗体，例如PIK3IP1的多克隆抗体或单克隆抗体。
PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体在制备用于调控受试者免疫应答的药物中的用途。
根据本发明，调控受试者免疫应答包括增强受试者免疫应答，例如增强受试者CD8 +T细胞和/或CD4 +T细胞的增殖。
作为本发明的一种实施方式，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和抗原一同在制备用于增强受试者对抗原的免疫应答的药物中的用途，所述增强包括给受试者施用抗原和PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，使得受试者对抗原的免疫应答得到增强。
根据本发明，所述抗原可以是癌性细胞、肿瘤抗原、病毒抗原、细菌抗原等。可以使用的抗原非限制性实例包括黑素瘤的抗原肽，诸如肽gp100、MAGE抗原、Trp-2、MART1和/或酪氨酸酶。在本发明的一个实施方式中，所述抗原是gp100，更优选为hgp100 25-33。在本发明的另一个实施方式中，所述抗原是OVA，更优选为OVA 257–264。在本发明的再一个实施方式中，所述抗原是癌性细胞，例如MC38细胞，B16细胞，B16-F10细胞等。
本发明的第二个方面提供，PIK3IP1抑制剂在制备免疫治疗肿瘤药物中的用途。在本发明的一些实施方式中，所述PIK3IP1抑制剂选自PIK3IP1、PIK3IP1ECD和PIK3IP1ECD融合分子中的至少一个。在本发明的一些实施方式中，所述PIK3IP1抑制剂是能结合到PIK3IP1上的抗体，例如PIK3IP1的多克隆抗体或单克隆抗体。
PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体在制备免疫治疗肿瘤药物中的用途。
根据本发明，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或 PIK3IP1的抗体通过增强用药者的免疫应答实现治疗肿瘤的效果。
根据本发明，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，可以抑制肿瘤细胞的生长。
根据本发明，不受特殊理论的限制，发明人推测在体内，PIK3IP1、PIK3IP1ECD或PIK3IP1ECD融合分子可能作为PIK3IP1的竞争性抑制剂阻断了PIK3IP1活化带来的T细胞增殖或活性的抑制作用。
PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，还可以联合其他的肿瘤标准治疗方法，用于治疗肿瘤。
PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，可以有效联合化疗方案，并有可能减少所施用的化疗剂的剂量。这种联合的一个例子是，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体联合达卡巴嗪(decarbazine)来治疗黑素瘤。另一个例子是，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体联合白介素-2(IL-2)来治疗黑素瘤。
PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和化疗剂联合使用的科学原理是，大多数化疗剂的细胞毒性作用使肿瘤细胞死亡，肿瘤细胞死亡导致抗原呈递途径中肿瘤抗原水平升高。通过细胞死亡带来与PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体协同作用的其它联合疗法还可以包括放射治疗、手术等。此外，血管发生抑制剂也可以与PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体联合，对血管发生的抑制同样能导致肿瘤细胞死亡，使肿瘤抗原进入抗原呈递途径。
肿瘤通过多种机制来规避宿主的免疫监视。可以通过抑制肿瘤表达的具有免疫抑制性的蛋白质的活性来解除这些机制，所述蛋白质包括例如TGF-β和Fas配体等。针对这些蛋白质中的每一种的抗体也可以与PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体联合使用，以抵消肿瘤的免疫抑制效果并促进宿主的肿瘤免疫应答。
此外，能够激活宿主免疫响应性的其它抗体也可以与PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECDPIK3IP1ECD融合分子或PIK3IP1的抗体联合使用。这些抗体包括树突细胞表面上激活DC功能和抗原呈递的分子。例如，抗CD40抗体能够有效提高T辅助细胞活性，可以与PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体联合使用。针对T细胞共刺激性分子诸如CTLA-4、OX-40、4-1BB和ICOS等的激活性抗体，也可以提高T细胞活性。
因此，本发明的一个实施方式提供，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，单独或者和另一种或多种治疗剂联合在制备治疗肿瘤药物中的用途。
根据本发明，所述另一种或多种治疗剂包括但不限于肿瘤化疗剂，能够活化T细胞的化合物，血管生成抑制剂等。
在本发明的一个具体实施方式中，所述另一种治疗剂是4-1BB的激活性抗体，例如4-1BB激活性抗体2A。
本发明的一个实施方式是提供，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如2A，联合在制备治疗肿瘤的药物中的用途。
根据本发明，其中PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体以亚治疗剂量施用。
根据本发明，其中4-1BB的激活性抗体，例如2A，以亚治疗剂量施用。
根据本发明，其中PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如2A，各自以亚治疗剂量施用。
在某些实施方案中，可以PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和抗体2A的治疗性组合与药学上可接受的载体混合作为单一组合物同时施用，或者可以将每种活性成分分别与药学上可接受的载体混合，作为分开的组合物同时施用。在另一个实施方案中，可以序贯施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和抗体2A的组合，例如，先施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体后施用抗体2A，或者先施用抗体2A后施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。另外，如果序贯施用超过一个剂量的联合疗法，可以在每次施用时颠倒序贯施用的次序或保持相同次序，序贯施用可以与同时施用或其任意组合联合。例如，第一次施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和抗体2A的组合可以是同时的，第二次施用可以是序贯的，先施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体后施用抗体2A，而第三次施用可以是序贯的，先施用抗体2A后施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体等。另一种代表性的剂量给药方案可以包括第一次施用是序贯的，先施用抗体2A后施用PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，而后续施用可以是同时的。
任选的是，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如抗体2A的组合可以进一步联合其他免疫原剂，诸如癌性细胞、纯化的肿瘤抗原等。可以使用的免疫原剂的非限制性实例包括黑素瘤抗原的肽，诸如肽gp100、MAGE抗原、Trp-2、MART1和/或酪氨酸酶。
同样，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如抗体2A的组合可以进一步联合标准的肿瘤治疗方法。例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和抗体2A的组合联合达卡巴嗪(decarbazine)治疗黑素瘤，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和抗体2A的组合联合白介素-2(IL-2)来治疗黑素瘤。
本发明的第三个方面是提供，一种药物组合物，其包含PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和药学上可接受的载体。
根据本发明，该药物组合物用于调控受试者的免疫应答。所述调控受试者免疫应答包括增强受试者免疫应答，例如增强受试者CD8 +T细胞和/或CD4 +T细胞的增殖。
根据本发明，该药物组合物用于治疗肿瘤。所述治疗肿瘤通过增强用药者的免疫应答，来实现抑制肿瘤生长的治疗效果。
根据本发明，所述药物组合物还可以以联合疗法的方式施用，即联合其他治疗剂，例如，其他的免疫调节剂或肿瘤治疗药物。
根据本发明，所述药物组合物还可以进一步含有另一种或多种治疗剂。所述治疗剂包括但不限于肿瘤化疗剂，能够活化T细胞的化合物，血管生成抑制剂等。
在本发明的一个具体实施方式中，所述另一种治疗剂是4-1BB的激活性抗体，例如抗体2A。
单剂量形式的药物组合物中活性成分的量，可以根据受试者及具体的给药途径而变化。一般而言，在药物组合物中，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体的重量百分比为大约0.01％到大约99％，优选大约0.1％到大约70％，最优选大约1％到大约30％。
对于PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，其剂量范围为大约0.0001-100mg/kg，更通常为0.01-5mg/kg体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重或10mg/kg体重或在1-10mg/kg的范围内。示例性的治疗方案可以是每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。对于PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体而言，优选的剂量方案包括经静脉内施用1mg/kg体重或3mg/kg体重，其利用下列剂量给药方案之一来给予：(i)每四周一剂达六个剂量，然后每三个月一剂；(ii)每三周一剂；(iii)给予一次3mg/kg体重，随后每三周给予1mg/kg体重。
本发明药用组合物中活性成分的实际剂量水平可以变化，从而获得对于特定患者和给药途径能有效实现治疗性应答而对患者无毒的活性成分用量。选定的剂量水平将取决于多种药动学因素，包括给药途径，用药时间，药物代谢情况，所治疗患者的年龄、性别、体重、大体健康状态和既往病史等。
本发明的一个实施方式还提供，包含PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如抗体2A的药物试剂盒。
该试剂盒还可以进一步包含用于治疗肿瘤的说明书。
在本发明的一个具体实施方案中，所述药物试剂盒中将PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如抗体2A以单位剂量形式共同包装。
在本发明的另一个具体实施方案中，所述药物试剂盒中分别将PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和4-1BB的激活性抗体，例如抗体2A以各自的单位剂量形式分别包装。
本发明的第四个方面是提供，一种调控受试者免疫应答的方法，其包括给受试者施用PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，使得受试者的免疫应答得到调控。优选的是，PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，增强、刺激或增加受试者的免疫应答。
根据本发明，所述增强、刺激或增加受试者的免疫应答包括但不限于，促进CD4 +T细胞和/或CD8 +T细胞的增殖。
本发明的一个实施方式中提供，一种增强受试者对抗原的免疫应答的方法，其包括给受试者施用抗原和PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，使得受试者对抗原的免疫应答得到增强。
根据本发明，所述抗原可以是癌性细胞，肿瘤抗原，病毒抗原，细菌抗原等。可以使用的抗原非限制性实例包括黑素瘤的抗原肽，诸如肽gp100、MAGE抗原、Trp-2、MART1和/或酪氨酸酶。在本发明的一个实施方式中，所述抗原是gp100，更优选为hgp100 25-33。在本发明的另一个实施方式中，所述抗原是OVA，更优选为OVA 257–264。在本发明的再一个实施方式中，所述抗原是癌性细胞，例如MC38细胞，B16细胞，B16-F10细胞等。
本发明的第五个方面是提供，一种在个体中治疗肿瘤或延迟肿瘤进展的方法，所述方法是给所述个体治疗有效量的PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
根据本发明，所述方法还包括给予所述个体另一种或多种治疗剂。
根据本发明，所述另一种或多种治疗剂包括但不限于其他的活化T细胞的化合物，肿瘤化疗剂，血管生成抑制剂等。
根据本发明，所述PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和另一种或多种治疗剂各自在单独的组合物中同时施用，所述PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和另一种治疗剂在同一个组合物中同时施用，或者，所述PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，和另一种治疗剂各自在单独的组合物中间隔一段时间先后施用或顺序施用。在本发明的一个实施方式中，所述另一种治疗剂是4-1BB的激活性抗体，例如抗体2A。
根据本发明，其中施用所述PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体，相比于未经治疗的个体或用其他治疗剂的单一疗法治疗的个体，增加T细胞的增殖。T细胞的增殖平均比例至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％或至少约90％。
在本发明的一个实施方式中，施用所述PIK3IP1抑制剂，例如，PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体和2A，相比于未经治疗的个体或用所述PIK3IP1抑制 剂，例如，PIK3IP1、PIK3IP1ECDPIK3IP1ECD融合分子或PIK3IP1的抗体、2A或其他治疗剂的单一疗法治疗的个体，肿瘤体积减少。
在以上描述的技术方案的一些实施方式中，PIK3IP1包含选自SEQ ID NO.1、2、3、4、5、6、7、8和/或17的序列；在一些实施方式中，PIK3IP1由选自SEQ ID NO.1至8之一的序列组成。
在以上描述的技术方案的一些实施方式中，PIK3IP1ECD包含选自SEQ ID NO.9和/或10的序列；在一些实施方式中，PIK3IP1ECD由选自SEQ ID NO.9或10的序列组成。
在以上描述的技术方案的一些实施方式中，PIK3IP1ECD融合分子包含选自SEQ ID NO.9和/或10的序列。在一些实施方案中，PIK3IP1ECD融合分子的至少一个融合伴侣选自Fc、白蛋白和聚乙二醇。在一些实施方案中，PIK3IP1ECD融合分子的至少一个融合伴侣是Fc。在一些实施方案中，PIK3IP1ECD融合分子的至少一个融合伴侣是IgG Fc。在一些实施方案中，所述IgG Fc的碱基序列由选自SEQ ID NO.15或16的序列组成。在一些实施方案中，PIK3IP1ECD融合分子的碱基序列由选自SEQ ID NO.11至14之一的序列组成。
在以上描述的技术方案的一些实施方式中，PIK3IP1的抗体可以是PIK3IP1的多克隆抗体、PIK3IP1的单克隆抗体。
在以上描述的技术方案中，所述肿瘤包括通常对免疫疗法有响应的肿瘤，包括但不限于黑色素瘤、白血病、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌、头颈癌；优选为黑色素瘤、白血病、肺癌、肝癌、结肠癌、乳腺癌、神经胶质瘤、胃癌、口腔鳞状细胞癌。
在一些实施方案中，所述肿瘤是转移性的。
在一些实施方案中，所述肿瘤优选为结肠癌、黑色素瘤、口腔鳞状细胞癌、肝细胞癌。
在以上描述的技术方案中，所述肿瘤化疗剂包括但不限于：烷化剂、氮芥类、塞替派类、亚硝脲类、甲基磺酸酯类、铂类化合物、丝裂霉素等，具体如：氮芥、苯丁酸氮芥、环磷酰胺、异环磷酰胺、塞替派、卡莫司汀、司莫司汀、白消安、顺铂、奥沙利铂、卡铂、草酸铂、丝裂霉素等；影响核酸合成的药物，例如二氢叶酸还原酶抑制剂、胸腺核苷合成酶抑制剂、嘌呤核苷合成酶抑制剂、核苷酸还原酶抑制剂、DNA多聚酶抑制剂，具体如：甲氨蝶呤、5-FU、FT-207、卡培他滨、6-巯基嘌呤、6-TG、羟基脲、阿糖胞苷、吉西他滨、培美曲塞等；作用于核酸转录的药物，例如放线菌素D、柔红霉素、阿霉素、表阿霉素、阿克拉霉素、光辉霉素等；作用于DNA复制的拓扑异构酶I抑制剂，例如伊立替康、拓扑替康、羟基喜树碱等；作用于有丝分裂M期干扰微管蛋白合成的药物，例如紫杉醇、多西他赛、长春花碱、长春新碱、长春瑞滨、鬼臼碱类、高三尖杉酯碱等。
在以上描述的技术方案中，所述能够活化T细胞的化合物包括但不限于T细胞共刺激性分子的激活性抗体，例如：CTLA-4、OX-40、4-1BB或ICOS等的激活性抗体，抗CD40抗体等。在一些实施方案中，所述能够活化T细胞的化合物为4-1BB的激活性抗体，例如抗体2A。
在以上描述的技术方案中，所述血管生成抑制剂包括但不限于：抑制血管内皮生长因子 的作用的那些抑制剂，例如来那度胺、沙利度胺、抗血管内皮细胞生长因子抗体例如贝伐单抗，VEGF受体酪氨酸激酶抑制剂凡德他尼(vandetanib)(ZD6474)、瓦他拉尼(vatalanib)(PTK787)、舒尼替尼(SU11248)、阿西替尼(AG-013736)、帕唑帕尼(GW786034)和4-(4-氟-2-甲基吲哚-5-基氧基)-6-甲氧基-7-(3-吡咯烷-1-基丙氧基)喹唑啉等。
本申请中的序列和描述
Figure PCTCN2019073451-appb-000001
Figure PCTCN2019073451-appb-000002
Figure PCTCN2019073451-appb-000003
Figure PCTCN2019073451-appb-000004
术语
为了使本发明更加容易理解，首先对一些术语进行定义。如本申请中使用的，除非在本文中有明确描述，否则下列的每一个术语应当具有下文所述的含义。其它定义在本申请全文中给出。
本文所用的术语“PIK3IP1”是指磷脂酰肌醇3激酶相互作用蛋白1(PI3K interacting protein1，PIK3IP1)，其能与磷脂酰肌醇3激酶(phosphatidylinositol-3-kinase，PI3K)的p110 亚基结合，可以下调PI3K活性，抑制丝氨酸苏氨酸激酶的活化，包含其天然存在的等位基因形式和经加工的各种同工型。术语“PIK3IP1”还指来自人物种和非人物种诸如小鼠、大鼠或灵长类的PIK3IP1。有时通过诸如用于人PIK3IP1的h PIK3IP1、用于鼠PIK3IP1的m PIK3IP1等术语来指示来自特定物种的PIK3IP1。在本发明的一些实施例中，还用Pik3ip1指代小鼠的PIK3IP1蛋白。
示例性的PIK3IP1包括但不限于，由选自SED ID NO.1、2、3、4、5、6、7、8和17的氨基酸序列组成。
术语“PIK3IP1抑制剂”是指能够阻断PIK3IP1活化或活性的化学物质，从而能够抑制PIK3IP1活化造成的T细胞增殖或活性的抑制。所述化学物质可以是小分子化合物，蛋白质，多肽，DNA，RNA等。所述的蛋白质或多肽可以是如本发明中所述的PIK3IP1胞外域，或PIK3IP1胞外域的融合蛋白，也可以是PIK3IP1的抗体，例如多克隆抗体和单克隆抗体。
术语“PIK3IP1胞外域”(PIK3IP1Extracellular domain“PIK3IP1ECD”)包括全长PIK3IP1ECD、PIK3IP1ECD片段和PIK3IP1ECD变体。如本文所用的，术语“PIK3IP1ECD”指PIK3IP1多肽，其缺少胞内域和跨膜域，具有或没有信号肽。如本文所用的，术语“全长PIK3IP1ECD”指延伸至胞外域的最后氨基酸的PIK3IP1ECD，并且可以包括或不包括N-末端信号肽。在一些实施方案中，PIK3IP1ECD是人PIK3IP1ECD，由选自SEQ ID NO.9的氨基酸序列组成。在一些实施方案中，PIK3IP1ECD是鼠PIK3IP1ECD，由选自SEQ ID NO.10的氨基酸序列组成。
当PIK3IP1ECD“由”选自SEQ ID NO.9和10的序列“组成”时，PIK3IP1ECD可含有或不含有多种翻译后修饰，诸如糖基化和唾液酸化。换言之，当PIK3IP1ECD由特定的氨基酸序列组成时，其在连续的氨基酸序列中不含有另外的氨基酸，但可能含有对氨基酸侧链、N末端氨基和/或C-末端羧基的修饰。
如本文所用的，术语“PIK3IP1ECD片段”指从全长ECD的N和/或C末端缺失一个或多个残基并保持和全长PIK3IP1ECD相同结合能力的PIK3IP1ECD。PIK3IP1ECD片段可以包括或不包括N-末端信号肽。
如本文所用的，术语“PIK3IP1ECD变体”指含有氨基酸添加、缺失和取代并保持和亲代PIK3IP1ECD相同结合能力的PIK3IP1ECD。此类变体可与亲代PIK3IP1ECD具有至少90％、92％、95％、97％、98％或99％同一性。两个多肽的同一性可通过相似性得分来测量，所述测量方法是本领域已知的，例如使用确定相似性的具有缺省设置的Bestfit程序来比较两个多肽的氨基酸序列的同一性或相似性。
术语“PIK3IP1ECD融合分子”指包含PIK3IP1ECD和一个或多个“融合伴侣”的分子。在一些实施方案中，PIK3IP1ECD和融合伴侣是共价连接的。若融合伴侣也是多肽(“融合伴侣多肽”)，则PIK3IP1ECD和融合伴侣多肽可以是连续的氨基酸序列的部分，且融合伴侣多肽可与PIK3IP1ECD的N末端或C末端相连接。在这样的情况下，PIK3IP1ECD和融合伴侣多肽可自编码PIK3IP1ECD和融合伴侣多肽两者的编码序列被翻译为单一的多肽(“PIK3IP1ECD融合蛋 白”)。在一些实施方案中，PIK3IP1ECD和融合伴侣通过其他方式共价连接，诸如，除肽键外的化学键。在另外的实施方案中，PIK3IP1ECD和融合伴侣可通过“接头”来融合，所述接头由至少一个氨基酸或化学部分组成。
在一些实施方案中，PIK3IP1ECD多肽和融合伴侣是非共价连接的，例如使用结合对将它们连接。示例性的结合对包括但不限于，生物素和亲和素或链霉亲和素、抗体和其抗原等。
示例性的融合伴侣包括但不限于，免疫球蛋白Fc结构域、白蛋白和聚乙二醇。一些示例性的Fc结构域的碱基序列示于SEQ ID NO.15和16。在一些实施方案中，Fc结构域选自IgG1Fc、IgG2Fc、IgG3Fc和IgG4Fc。
在一些实施方式中，PIK3IP1ECD融合分子包含PIK3IP1ECD和一个融合伴侣，所述融合伴侣为Fc。在一个具体实施方式中，PIK3IP1ECD融合分子的碱基序列由选自SEQ ID NO.11、12、13和14的碱基序列组成。
在一些实施方案中，PIK3IP1ECD融合分子包含信号肽。在一些实施方案中，PIK3IP1ECD融合分子缺少信号肽。在一些实施方案中，PIK3IP1ECD融合分子的PIK3IP1ECD部分包括选自SEQ ID NO:9和10的序列。在一些实施方案中，PIK3IP1ECD融合分子的PIK3IP1ECD部分由选自SEQ ID NO:9和10的序列组成。并且，因为PIK3IP1ECD与融合分子相连接，在PIK3IP1ECD的N末端和/或C末端可能存在另外的氨基酸，但那些氨基酸并非来自PIK3IP1序列，但可来自，例如，接头序列或融合伴侣序列。
以下是可用于本发明的非限制性的示例性融合伴侣。
如本文所讨论的，可将PIK3IP1ECD与至少一种融合伴侣组合，产生PIK3IP1ECD融合分子。这些融合伴侣可促进纯化，或者使PIK3IP1ECD融合分子的体内半衰期延长。PIK3IP1ECD的合适的融合伴侣包括例如，聚合物，诸如水溶性聚合物，免疫球蛋白的恒定结构域；人血清白蛋白(HSA)的全部或部分；胎球蛋白A；胎球蛋白B；亮氨酸拉链结构域；四连接素三聚化结构域；甘露糖结合蛋白(也称作甘露糖结合凝集素)，例如，甘露糖结合蛋白1；和Fc区。
为了本发明的治疗目的，所述的融合伴侣不会产生中和抗原性反应或其他不利反应。
聚合物，例如，水溶性的聚合物，作为融合伴侣使用以降低PIK3IP1ECD融合分子在水性环境中(诸如生理环境中)的沉淀。本发明所采用的聚合物是药学上可接受的聚合物，包括但不限于，聚乙二醇(PEG)、聚乙二醇丙醛、乙二醇/丙二醇的共聚物、单甲氧基-聚乙二醇、羧甲基纤维素、葡聚糖、聚乙烯醇(PVA)、聚乙烯基吡咯烷酮、聚-1,3-二氧戊环、聚-1,3,6-三氧杂环己烷、乙烯/马来酸酐共聚物、聚(β-氨基酸)(无论是均聚物或无规共聚物)、聚(正乙烯基吡咯烷酮)聚乙二醇、聚丙二醇均聚物(PPG)和其他聚氧化烯(polyakylene oxide)、聚环氧丙烷/环氧乙烷共聚物、聚氧乙烯化的多元醇(POG)(例如，甘油)和其他聚氧乙烯化的多元醇、聚氧乙烯化山梨糖醇或聚氧乙烯化葡萄糖、结肠酸或其他碳水化合物聚合物，聚蔗糖(Ficoll)或葡聚糖及其混合物。
通常，可在使蛋白质与活化的聚合物分子反应的任何合适的条件下进行化学衍生。可用 于将聚合物与活性部分连接的活化的基团包括砜、马来酰亚胺、巯基、硫醇、三氟甲基磺酸盐、三氟乙基磺酸酯、氮丙啶(azidirine)、环氧乙烷和5-吡啶基。通常将本发明的聚合物在氨基酸的α或ε氨基或反应性硫醇基上与PIK3IP1ECD连接。
此外，可将本发明的PIK3IP1ECD与标记物序列融合。标记物氨基酸序列可以是六聚组氨酸肽，六聚组氨酸为融合蛋白的纯化提供了便利。
在不同的实施方案中，寡聚化对融合蛋白提供了一些功能优点，包括但不限于，多价、增加的结合强度和不同的结构域的组合功能。因此，在一些实施方案中，融合伴侣包含寡聚化结构域，例如，二聚化结构域。示例性的寡聚化结构域包括但不限于，卷曲螺旋结构域，包括α-螺旋卷曲螺旋结构域；胶原蛋白结构域；胶原蛋白样结构域；和某些免疫球蛋白结构域。示例性的卷曲螺旋多肽融合伴侣包括但不限于，四连接素卷曲螺旋结构域；软骨寡聚基质蛋白的卷曲螺旋结构域；血管生成素卷曲螺旋结构域；和亮氨酸拉链结构域。
可用作融合伴侣的许多Fc结构域是本领域中已知的。在一些实施方案中，融合伴侣是Fc免疫球蛋白结构域。Fc融合伴侣可以是在天然存在的抗体中发现的野生型Fc、其变体或其片段。非限制性的示例性Fc融合伴侣包括包含人IgG(例如，人IgG1、IgG2、IgG3或IgG4)的铰链结构域以及CH2和CH3恒定结构域的Fc。另外的示例性Fc融合伴侣包括但不限于，人IgA和IgM。
在一些实施方案中，融合伴侣是白蛋白。示例性的白蛋白包括但不限于，人血清白蛋白(HSA)和能够增加与其融合的多肽的血清半衰期或生物利用率的HSA片段。
融合伴侣的示例性的连接
可将融合伴侣共价地或非共价地与PIK3IP1ECD的N末端或C末端连接。连接还可发生在PIK3IP1ECD中除N末端或C末端的位置，例如，通过氨基酸侧链(例如，半胱氨酸、赖氨酸、丝氨酸或苏氨酸的侧链)来连接。
在共价的或非共价的连接的实施方案中，接头可含于融合伴侣和PIK3IP1ECD之间。此类接头可以由至少一个氨基酸或化学部分组成。将融合伴侣与PIK3IP1ECD共价连接的示例性方法包括但不限于，将融合伴侣和PIK3IP1ECD作为单一的氨基酸序列来翻译和将融合伴侣与PIK3IP1ECD化学连接。当融合伴侣和PIK3IP1ECD作为单一的氨基酸序列来翻译时，在融合伴侣和PIK3IP1ECD之间可包括另外的氨基酸作为接头。在一些实施方案中，基于编码接头的多核苷酸序列来选择接头，以有利于将融合伴侣和/或PIK3IP1ECD克隆到单个表达构建体中(例如，可将含有特定的限制位点的多核苷酸置于编码融合伴侣的多核苷酸和编码PIK3IP1ECD的多核苷酸之间，其中含有限制位点的多核苷酸编码短的氨基酸接头序列)。当通过化学方式将融合伴侣和PIK3IP1ECD共价偶联时，在偶联反应过程中通常可包括不同大小的接头。
PIK3IP1ECD和PIK3IP1ECD融合分子表达和生产载体
提供了包含编码PIK3IP1ECD的多核苷酸的载体。还提供了包含编码PIK3IP1ECD融合分子的多核苷酸的载体。此类载体包括但不限于，DNA载体、噬菌体载体、病毒载体、逆转录 病毒载体等。
在一些实施方案中，选择的载体对于多肽在CHO或CHO来源的细胞中的表达是优化的。
宿主细胞
在不同的实施方案中，PIK3IP1ECD或PIK3IP1ECD融合分子可在原核细胞中表达，诸如细菌细胞；或在真核细胞中表达，诸如真菌细胞、植物细胞、昆虫细胞和哺乳动物细胞。可用于表达多肽的示例性真核细胞包括但不限于，COS细胞，293细胞，CHO细胞和NSO细胞。
将核酸导入到期望的宿主细胞中可通过本领域中已知的任何方法来实现，这些方法包括但不限于，磷酸钙转染、DEAE-葡聚糖介导的转染、阳离子脂质介导的转染、电穿孔、转导、感染等。
PIK3IP1ECD多肽的纯化
可通过本领域中已知的多种方法纯化PIK3IP1ECD或PIK3IP1ECD融合分子。此类方法包括但不限于，亲和基质或疏水作用色谱的使用。合适的亲和配体包括PIK3IP1ECD或融合伴侣的任何配体。例如，蛋白A、蛋白G、蛋白A/G或抗体亲和柱可用于与Fc融合伴侣结合以纯化PIK3IP1ECD融合分子。
术语“信号肽”指位于多肽的N末端的氨基酸残基的序列，其促进多肽从哺乳动物细胞中分泌。在将多肽从哺乳动物细胞中运出之后可将信号肽裂解，形成成熟蛋白质。信号肽可以是天然的或合成的，并且它们对于其所连接的蛋白质可以是异源的或同源的。
术语“抗体”以最宽泛的意义使用，具体地涵盖合成抗体、单克隆抗体、多克隆抗体、重组抗体、胞内抗体、多特异性抗体、双特异性抗体、单价抗体、多价抗体、人抗体、人源化抗体、嵌合抗体、灵长类化抗体、Fab片段、F(ab')片段、单链FvFc(scFvFc)、单链Fv(scFv)、抗独特型(抗Id)抗体和任何其他免疫活性抗体片段，只要它们展示出所希望的生物活性(即，标记相关或结合)即可。在更广的意义上，本发明的抗体包括免疫球蛋白分子和免疫球蛋白分子(即，含有抗原结合部位的分子)的免疫活性片段，其中这些片段可以或可以不与另一个免疫球蛋白结构域(包括但不限于Fc区或其片段)融合。此外，如在此更详细地概述的，术语抗体和多种抗体具体地包括Fc变体或其片段，包括全长抗体和包含Fc区的变体Fc-融合物，其任选地包含至少一个氨基酸残基修饰并且与免疫球蛋白的免疫活性片段融合。
“药学上可接受的载体”包括任何和全部的生理上相容的溶剂、分散介质、包衣、防腐剂、等渗剂、缓释剂等等。优选地，载体适合于静脉内、皮下、肌内、肠胃外、脊柱或表皮施用(例如，通过注射或输注)。本发明的药物组合物可以包括一种或多种药学上可接受的盐、抗氧化剂、水和非水性载体，和/或佐剂，如防腐剂、润湿剂、乳化剂和分散剂。
“免疫应答”是指脊椎动物体内针对外来作用介质的生物学应答，该应答可保护生物体免于被这些作用介质或者由其造成的疾病的伤害。免疫应答是由免疫系统的细胞(例如，T淋巴细胞，B淋巴细胞，自然杀伤NK细胞，巨噬细胞，嗜酸性粒细胞，肥大细胞，树突细胞或嗜中性粒细胞)和由这些细胞的任一种或肝脏产生的可溶性大分子(包括抗体，细胞因子和补 体)的作用介导的，其导致脊椎动物机体入侵病原体、细胞或被病原体感染的组织、癌细胞或其它异常细胞，或者，在自身免疫性或病理性炎症中，针对正常的人细胞或组织的选择性靶向、结合、损害、破坏，和/或消除。
“免疫疗法”是指用包括诱导、强化、抑制或者修饰免疫应答的方法对患有疾病、具有发生疾病风险、或者疾病复发的受试者进行治疗。
“增强内源性免疫应答”意思是强化受试者体内现有的免疫应答的有效性或强度。此类效率和潜力的强化可以通过，例如，克服抑制内源性宿主免疫应答的机制，或者通过刺激强化内源性宿主免疫应答的机制来实现。
药物或治疗剂(例如本发明的PIK3IP1)的“治疗有效量”或“治疗有效剂量”是药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。药物的治疗有效量或剂量包括“预防有效量”或“预防有效剂量”，“预防有效量”或“预防有效剂量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的受试者时，可抑制疾病的发生或复发。治疗剂促进疾病消退或抑制疾病发展或复发的能力可以用技术人员已知的各种方法进行评估，例如在人类受试者的临床试验中，在可预测在人类中的效力的动物模型系统中，或者通过在体外测定系统中测定试剂的活性。
“亚治疗剂量”表示治疗化合物例如，亚治疗剂量的CTLA-4抗体为少于约3mg/kg(即抗CTLA-4抗体的已知剂量)的单剂抗体。
“癌症”是指一大类以异常细胞在体内不受控制地生长为特征的各种疾病，在本文中可与“肿瘤”互换使用。不受控制的细胞分裂和生长分裂和生长导致形成恶性肿瘤或细胞，它们侵入邻近组织，还可以通过淋巴系统或血流转移到身体的远端部分。
可以使用本发明免疫疗法治疗的癌症实例包括但不限于：骨癌症、胰腺癌症、皮肤癌症、头或颈部的癌症、乳腺癌症、肺癌症、皮肤或眼内恶性黑素瘤、肾癌症、子宫癌症、卵巢癌症、结肠直肠癌症、结肠癌症、直肠癌症、肛门区域的癌症、胃癌症、睾丸癌症、子宫癌症、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、食道的癌症、小肠的癌症、内分泌系统的癌症、甲状腺的癌症、甲状旁腺的癌症、肾上腺的癌症、软组织肉瘤、尿道的癌症、阴茎的癌症、血液恶性肿瘤、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱的癌症、肾或输尿管的癌症、肾盂癌、中枢神经系统(CNS)的肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊椎轴肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮样癌症、鳞状细胞癌症、环境诱导的癌症包括被石棉诱导的癌症，转移性癌症，和所述癌症的任意组合。在其它优选的实施方案中，癌症是黑色素瘤。
“受试者”是指接受本发明活性物质作用的生命体，在本申请中可与“用药者”互换使用。受试者可以是人或者动物，例如猴、大鼠、小鼠、狗、兔等。
通过以下实施例进一步举例说明本发明，这些实施例不应当被解释为具有限制作用。本 申请明确地通过提述并入本申请全文中引用的所有图和所有参考文献、专利和被公开专利申请的内容。
附图说明
图1PIK3IP1和Pik3ip1电泳结果。
图2a-图2d融合蛋白质谱鉴定结果。
图3Anti-PIK3IP1多克隆抗体特异性的鉴定结果，使用小鼠抗人PIK3IP1抗体；按不同比例稀释多克隆抗体后，与稳定表达人PIK3IP1的CHO细胞转染株进行染色，流式细胞术对抗体效价鉴定结果。
图4Anti-Pik3ip1多克隆抗体特异性的鉴定结果，使用大鼠抗小鼠Pik3ip1抗体；按不同比例稀释多克隆抗体后，与稳定表达小鼠Pik3ip1的CHO细胞转染株进行染色，流式细胞术对抗体效价鉴定结果。
图5人外周血PIK3IP1表达的流式细胞术检测。PIK3IP1在人外周血PBMC中CD8 +T细胞、CD4 +T细胞、B细胞的表达较高，在CD56 +NK细胞的表达较低。
图6Pik3ip1在小鼠组织mRNA水平的表达情况检测。Pik3ip1在小鼠肌肉、PBMC、淋巴结和脾中表达水平较高。
图7Pik3ip1在小鼠脾淋巴细胞表达的流式细胞术检测。Pik3ip1在脾CD8 +T细胞、CD4 +T细胞、B细胞、CD11b +细胞和CD11c +细胞上均有表达，其中，在T细胞、B细胞和CD11b +细胞的表达较高。
图8Pik3ip1在常见小鼠细胞系中表达的流式细胞术检测，B16为小鼠黑色素瘤细胞系、P338D1为小鼠巨噬细胞系、DC2.4为小鼠树突状细胞系、CT26为小鼠结肠癌细胞系、EL4为小鼠T细胞淋巴瘤细胞系、MB49为小鼠膀胱癌细胞系。
图9Pik3ip1在小鼠免疫激活过程中淋巴细胞上表达的流式细胞术检测。用100μg hgp100 25-33和CFA或单独用CFA乳化后免疫Pmel-1TCR转基因小鼠，24小时后，检测Pik3ip1在小鼠引流淋巴结中CD8 +T细胞、CD11b +单核细胞、CD11c +DC细胞、CD19 +B细胞的表达。
图10用流式细胞术检测OVA抗原特异性免疫反应中Pik3ip1敲除小鼠和野生型小鼠的CD8 +T细胞的增殖情况，经过抗原特异性免疫后，Pik3ip1敲除小鼠的CD8 +T细胞的增殖高于野生型小鼠。左图为流式细胞检测图，右图为流式细胞检测荧光值统计图。图中KO代表Pik3ip1敲除小鼠，WT代表野生型小鼠。
图11在Pik3ip1敲除小鼠和野生型小鼠中采用MC38肿瘤细胞建立肿瘤模型，用流式细胞术检测小鼠中CD4 +和CD8 +T细胞的增殖情况，24小时、48小时和72小时，Pik3ip1敲除小鼠的CD4 +和CD8 +T细胞的增殖均高于野生型小鼠的，其中48小时和72小时的差异具有显著性。图中KO代表Pik3ip1敲除小鼠，WT代表野生型小鼠。
图12口腔鳞癌患者外周血淋巴细胞中PIK3IP1的表达与T细胞抑制状态之间的关系，CD4 +T细胞和CD8 +T细胞上的PIK3IP1的荧光强度与相应T细胞的增殖能力呈负相关，所述相关性均 具有显著性。
图13Pik3ip1-Ig融合蛋白在小鼠体内发挥竞争抑制作用，促进CD8 +T细胞的增殖；左图为Pik3ip1-Ig融合蛋白作用下，CD8 +T细胞的增殖情况；右图为CD8 +T细胞的增殖百分比统计(*p＜0.05)。
图14pik3ip1-Ig融合蛋白联合2A抑制小鼠B16-F10肿瘤的生长；A为Pik3ip1-Ig融合蛋白单独治疗，B16-F10肿瘤生长受抑制程度不明显(n.s.p＞0.05)；B为Pik3ip1-Ig融合蛋白联合2A治疗，B16-F10肿瘤的生长受到明显抑制(*p＜0.05)。
图15荷瘤C57BL/6小鼠大体观、肿瘤离体观；A为各组荷瘤小鼠大体观；B为各组肿瘤离体观。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，应理解，在阅读了本发明所记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的范围。
在以下实施例中，PIK3IP1代表人的PIK3IP1全长碱基序列及其蛋白产物，Pik3ip1代表鼠的Pik3ip1全长碱基序列及其蛋白产物，PIK3IP1-mIg代表由人的PIK3IP1胞外区和鼠的IgG Fc构成的融合蛋白，Pik3ip1-mIg代表由鼠的Pik3ip1胞外区和鼠的IgG Fc构成的融合蛋白，PIK3IP1-hIg代表由人的PIK3IP1胞外区和人的IgG Fc构成的融合蛋白，Pik3ip1-hIg代表由鼠的Pik3ip1胞外区和人的IgG Fc构成的融合蛋白，Flag-mIg为无序对照序列与鼠IgG Fc的融合蛋白，Flag-hIg为无序对照序列与人IgG Fc的融合蛋白。
以下实施例中：
1.细胞和动物材料
正常人单个核细胞，分离自健康志愿者的外周血；
SPF级C57BL/6小鼠、BALB/c小鼠、Wistar大鼠购买自中山大学(大学城)实验动物中心，饲养于中山大学北校区实验动物中心；
Pmel-1 TCR转基因小鼠，293T细胞、小鼠黑色素瘤细胞系(B16-F10)、小鼠结肠癌细胞系CT26细胞、小鼠膀胱癌细胞系MB49细胞、CHO细胞系、杂交瘤2A、小鼠巨噬细胞系P338D1、小鼠树突状细胞系DC2.4、小鼠T细胞淋巴瘤细胞系EL4、MC38细胞系、质粒pMIgV、pHIgV由中山大学中山医学院陈列平教授课题组赠予。
hgp100特异性Pmel-1脾细胞和淋巴细胞取自Pmel-1 TCR转基因小鼠脾脏和淋巴结。
OT1小鼠购自南京大学模式动物研究所。
Pik3ip1-/-小鼠，用TALENS法于赛亚生物公司(Cyagen Biosciences Inc.)进行敲除鼠的制备。
2.细胞培养试剂
DMEM高糖培养基，RPMI 1640培养基，Ham’s F10培养基，胰蛋白酶(0.25％Trypsin，0.02％EDTA)：美国Gibco公司；2-乙酰-2-去酰胺衍生物(ADT)，盘尼西林，链霉素，L- 谷氨酸盐，羟基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)：美国Invitrogen公司。
hgp100 25-33多肽，氨基酸序列为KVPRNQDWL，纯度大于95％：由上海英潍捷基公司合成。
3.质粒构建、细胞转染及融合蛋白纯化试剂
RNAiso Plus(9108Q)、PrimeScript TMRT reagent Kit试剂盒、SYBR Green qPCR Master Mix-SYBR Advantage(638320Clontech)试剂盒：Takara公司；All-In-One一步法逆转录试剂盒：美国GeneCopoeia公司；Lipofectamine 2000细胞转染试剂：上海英潍捷基公司；线性聚乙烯亚胺(Linear PEI)：美国Polysciences公司；NucleoBond Xtra Maxi Plus质粒提取和纯化试剂盒：德国Macherey-Nagel公司；SDS-PAGE凝胶配制试剂盒：上海碧云天公司；琼脂糖胶回收试剂盒：德国QIAGEN公司；蛋白质相对分子量Marker：美国Thermo Scientific。
4.细胞增殖和活化检测试剂
Cytofix/Cytoperm固定/破膜试剂盒、Anti-human CD4 Percp-cy5.5、Anti-human CD4BV421、Anti-human CD8 APC：美国BD公司；Anti-human CD69 Pe-cy7、Anti-human CD95FITC、Anti-human HLA-DR Pe-cy7、Anti-human CD3(克隆号：OKT3)：美国Biolegend公司；Anti-human CD28、Anti-human CD25 APC、Anti-human Foxp3 PE、Anti-human CD25PE：美国eBioscience公司；人CD4 +T细胞磁珠阳性分选试剂盒、人CD8 +T细胞磁珠阳性分选试剂盒、人CD3 +T细胞磁珠阴性分选试剂盒：德国美天旎公司；重组人TGF-beta1、重组人IL-2：美国Peprotech公司。
5.细胞凋亡检测实验材料和试剂
FITC Annexin V细胞凋亡检测、Anti-human CD4 BV421、Anti-human CD8 APC：美国BD公司。
6.其他材料和试剂
1Kb DNA ladder Marker：加拿大Fermentas公司；SYBR Green qPCR SuperMix：德国罗氏公司；弗氏完全佐剂(CFA)、弗氏不完全佐剂(IFA)：美国Sigma公司；蛋白A柱：美国GE公司；蛋白透析卡：美国Thermo Scientific；山羊抗小鼠Alexa Fluor 647荧光二抗：北京博奥森公司；DAPI染色剂：上海英潍捷基公司；美天妮gentleMACS组织处理M管：德国美天妮公司；中性树胶、枸橼酸钠抗原修复液pH6.0、苏木素染色液、EDTA抗原修复液pH9.0：北京中杉金桥公司；TO型生物制片透明剂：广州中南化工仪器公司；DAB显色剂、即用型兔抗人CD3免疫组化单克隆抗体(克隆号SP7)、正常兔血清封闭液、免疫组化染色检测试剂盒：中国迈新生物公司；两步法GK500705小鼠/兔通用型：丹麦DAKO公司；Anti-mouse CD3 FITC、Anti-mouse IFN-γAPC、Anti-mouse Ki67 PE：美国eBioscience公司；Anti-mouse CD8 BV421：美国BD公司；佛波醇酯(PMA)：比利时Acros公司；钙离子霉素(Ionomycin calcium salt)：美国Thermo Fisher公司；外周血单个核细胞(PBMC)分离试剂盒：stemcell公司。
7.主要实验仪器
Axio observer Z1倒置显微镜、激光扫描共聚焦显微镜(LSM780)：德国ZEISS公司； MACS分选器、MACS分选柱：德国美天妮公司；
Figure PCTCN2019073451-appb-000005
480全自动荧光定量PCR仪：德国罗氏公司；BD FACSVerse流式细胞仪：美国BD公司；M205FA体视荧光显微镜：莱卡仪器有限公司；ABI 9700 PCR仪：美国ABI公司；全自动酶标仪：美国Thermo Electron公司；ScanScope病理切片扫描成像系统：美国Apero公司。
实施例1质粒的构建
1、人外周血总RNA获取
参照stemcell公司07811号产品的试剂盒说明书进行人外周血单个核细胞的分离，采用对应试剂盒提取鼠外周血单个核细胞。
采用Takara公司RNAiso Plus(9108Q)试剂盒，按其说明书进行细胞总RNA提取：用约0.5-1ml RNAiso Plus将酶消化或从组织中分离的细胞进行充分破碎和溶解；采用氯仿-异丙醇-75％乙醇提取RNA，用DEPC水将提取的RNA溶解，利用分光光度计测定RNA浓度，调整RNA浓度为500ng/μl左右，备用。
2、RNA逆转录合成cDNA
采用Takara公司PrimeScript TMRT reagent Kit试剂盒，按其说明书进行cDNA合成：
(1)按如下比例配RNA-Primer Mix(总体积13μl)，变性RNA：
Figure PCTCN2019073451-appb-000006
(2)按如下比例配置逆转录反应体系，总体积25μl：
Figure PCTCN2019073451-appb-000007
逆转录反应条件如下：37℃逆转录反应10min，然后加热至85℃反应5min使逆转录酶失活，终止反应。
3、荧光定量PCR反应
采用Takara公司SYBR Green qPCR Master Mix-SYBR Advantage(638320 Clontech)试剂盒，按照其说明书进行操作：以扩增人PIK3IP1为例，
设计并合成QPCR引物，用于扩增PIK3IP1：F:ATGCTGTTGGCCTGGGTA；R: CGGGACTCCTGGGGCCTGA；
反应体系如下：
Figure PCTCN2019073451-appb-000008
在Roche LightCycler 480 Real-Time PCR仪上进行反应，扩增条件：95℃ 5min；95℃ 10sec；58℃ 20sec；72℃ 30sec，40 cycles；溶解曲线分析：95℃ 5sec；65℃ 1min；97℃ 5min；40℃10sec至冷却。
4、质粒构建
质粒载体为pMIgV(PIK3IP1-mIg、Pik3ip1-mIg)和pHIgV(PIK3IP1-hIg、Pik3ip1-hIg)者，酶切位点选择为Bgl Ⅱ和EcoR Ⅰ。质粒载体为PCDNA3.1者(PIK3IP1和鼠Pik3ip1全长序列)，酶切位点选择Xhol Ⅰ和Ecor Ⅰ。
设计正、反向PCR引物：
Figure PCTCN2019073451-appb-000009
分别混合之前逆转录的人的cDNA、鼠的cDNA，分别作为模板进行PCR，扩增相应序列；
PCR反应体系如下：
Figure PCTCN2019073451-appb-000010
PCR反应条件如下：
Figure PCTCN2019073451-appb-000011
Figure PCTCN2019073451-appb-000012
实验结果验证：
分别取上述3μl PCR产物，跑2％的琼脂糖凝胶电泳，观察电泳后条带大小，是否与目的基因相同。全长碱基序列的PCR结果如图1所示，人PIK3IP1全长碱基序列为792bp，鼠Pik3ip1全长碱基序列为795bp，证明获得了人和鼠的全长碱基序列，经测序，结果正确。
用Qiagen胶回收试剂盒回收得到PCR产物。胶回收产物和载体质粒，选择对应的酶切反应酶分别进行双酶切。
使用的反应体系，如下：
Figure PCTCN2019073451-appb-000013
酶切反应条件：胶回收产物37℃，酶切40min
质粒37℃，酶切50-55min；
酶切产物经琼脂糖凝胶电泳，胶回收得到目的条带进行连接：
Figure PCTCN2019073451-appb-000014
连接反应条件：37℃连接2小时，或16℃连接2小时，4℃过夜；
用所述方法，还分别构建了PIK3IP1-hIg，PIK3IP1-mIg，Pik3ip1-hIg，Pik3ip1-mIg(对应的碱基序列分别为SEQ ID NO.12、11、13、14)的质粒。
将构建好的质粒转化入细菌，培养获得目的克隆菌，将测序后序列正常的阳性克隆菌进行扩大培养。
采用NucleoBond Xtra Maxi Plus质粒提取和纯化试剂盒，按照说明书的指引进行质粒抽提和纯化。
实施例2 PIK3IP1-Ig、Flag-Ig融合蛋白质粒分别转染293T细胞和CHO稳转株的构建
1、采用线性聚乙烯亚胺(PEI)进行质粒转染。
以PIK3IP1-hIg融合蛋白质粒为例，方法如下：
培养293T细胞，以PEI(μg)：DNA总量(μg)＝3:1配置PEI和DNA的混合溶液，加入细 胞培养皿中转染6-8小时后，换含1％FBS和2mM丙戊酸的新鲜DMEM培养基，继续培养细胞，5天后，收集细胞培养皿上清，融合蛋白已分泌至上清中。
2、融合蛋白的提取、纯化和鉴定
细胞转染后5天，收集细胞培养上清离心获得上清液，采用GE公司高亲和力蛋白A柱，按照其使用说明书进行融合蛋白的提取和纯化。之后将收集到的蛋白，移入蛋白透析袋中，在pH7.2-7.4的1×PBS中4℃透析过夜。透析后的蛋白用于后续实验。
融合蛋白跑SDS-PAGE凝胶，考马斯亮蓝染色后切胶，送质谱鉴定。
10％SDS-PAGE分离胶的配制(5ml)
Figure PCTCN2019073451-appb-000015
5％SDS-PAGE浓缩胶的配制(2ml)
Figure PCTCN2019073451-appb-000016
鉴定结果如图2a-图2d所示。根据质谱鉴定结果，确定所纯化融合蛋白分别为PIK3IP1-hIg，PIK3IP1-mIg，Pik3ip1-hIg，Pik3ip1-mIg。
3、CHO稳转株的构建和鉴定
以人PIK3IP1稳转株构建为例：培养CHO细胞系的Ham’s F10全培养基配制方法如下：500ml Ham’s F10培养基含10％FBS、1％Hepes、1％链霉素和青霉素混合液、1×ADT。
将复苏后的CHO细胞系培养至传代2次，细胞状态良好后，取1×10 5细胞加入24孔板的一个孔内，将该孔培养基调整至1ml，待孔板内细胞长至融合度约90％，吸去培养基。加适量PBS洗一遍，后换为200μl无抗生素培养基。
用invitrogen公司LipofectaminTM 2000试剂盒，按说明书操作转染：将0.8-1μg表达PIK3IP1全长序列、载体为PCDAN3.1的质粒DNA加入50μl Opti-MEM中混合均匀。将2μl转染试剂Lipo2000加入50μl Opti-MEM中混合均匀。将稀释后的转染试剂逐滴加入稀释后的质粒DNA中，将DNA-Lipo2000复合物加入铺有细胞的孔中。混匀后，将24孔板放置于5％CO 2 37℃ 细胞培养箱；转染后6-8小时，吸去孔内液体，换为Ham’s F10全培养基。继续将孔板放置于5％CO 2 37℃细胞培养箱48小时。
取100mm×20mm细胞培养皿9个，每个培养皿加入含1000μg/ml G418的Ham’s F10全培养基20ml，将24孔板内用500μl培养基重悬的CHO细胞按体积比1:100(1个细胞培养皿)、1:5000(3个细胞培养皿)、1:10000(2个细胞培养皿)、1:20000(1个细胞培养皿)，即分别加入细胞悬液200μl、4μl、2μl、1μl至细胞培养皿中，将剩下的细胞悬液全部加入一个培养皿中，另将未做过转染正常培养的CHO细胞加入一个细胞培养皿作为筛选对照。混匀细胞后，于5％CO 2 37℃细胞培养箱中培养；在细胞培养皿中培养5-7天后，取出培养皿，吸去原培养基，换含1000μg/ml G418的Ham’s F10全培养基20ml。于5％CO 2 37℃细胞培养箱中继续培养；一般转至培养皿中10天后可以观察到细胞单克隆形成。待未经转染的筛选对照培养皿中的细胞全部死亡时，进行转染PIK3IP1质粒的CHO细胞亚克隆挑选。
取1/3或一半细胞进行流式细胞术或WB鉴定，挑选出表达强度最高的2-3个克隆，进行细胞培养，冻存备用。
按照如上方法还制备了Pik3ip1稳转株、PIK3IP1-hIg稳转株，PIK3IP1-mIg稳转株，Pik3ip1-hIg稳转株，Pik3ip1-mIg稳转株。
实施例3多克隆抗体的制备
选择SPF级6-8周龄雌性Balb/c小鼠2-3只，用于人PIK3IP1多抗的制备；选择SPF级体重200g左右雌性Wista大鼠，用于小鼠Pik3ip1多抗的制备。
初次免疫：免疫前，于免疫动物尾静脉取适量血液，提取血清，分装冻存，作为阴性对照的血清；每只小鼠按100μl CFA和等体积的100μg人PIK3IP1-mIg融合蛋白乳化后，注射于Balb/c小鼠双侧腋下及腹股沟，不少于4个点；每只大鼠按500μl CFA和等体积的500μg鼠Pik3ip1-mIg融合蛋白乳化后，注射于大鼠双侧腋下及腹股沟皮下，不少于4个点；免疫后2周，取免疫动物尾静脉血，制备血清，用ELISA或流式细胞术检测抗体效价。
第2次和第3次免疫：每只小鼠按100μl IFA和等体积的50μg人PIK3IP1-mIg融合蛋白，充分乳化后免疫小鼠；每只大鼠按500μl IFA和等体积的250μg鼠Pik3ip1-mIg融合蛋白，充分乳化后免疫大鼠；每次免疫后2周，检测抗体效价。
若免疫后第2或3次血清按1:10万倍稀释后，ELISA检测OD450值约1.5，或流式检测1:1000倍稀释有明显阳性峰，则可腹腔注射50μg人PIK3IP1-mIg融合蛋白或250μg鼠Pik3ip1-mIg融合蛋白，增强免疫；增强免疫后3-5天，处死动物，下腔静脉取血，制备多克隆抗体血清。
实施例4多克隆抗体特异性的检测(以小鼠抗人PIK3IP1多克隆抗体为例)
转染PIK3IP1的293T细胞接种于玻璃底培养皿，加10％FBS的DMEM培养基培养过夜；吸去培养基，用PBS清洗2遍，4％多聚甲醛室温固定15min。吸去固定液，PBS漂洗3min，洗2遍；10％正常山羊血清室温封闭30min；吸去封闭血清，加入小鼠抗人PIK3IP1多克隆血清1： 200稀释后4℃孵育过夜；PBS漂洗3min，洗3遍；山羊抗小鼠Alexa Fluor 647荧光二抗1：1000稀释，常温避光孵育1小时；PBS漂洗3min，洗3遍；10μg/ml WGA常温孵育10min；PBS漂洗3min，洗3遍；DAPI染色液染核3min；PBS漂洗3min，洗3遍；培养皿滴加抗淬灭封片剂，盖玻片封片。共聚焦显微镜观察，拍照。
采用免疫荧光和流式细胞染色对血清进行特异性和结合强度的检测。免疫荧光结果发现anti-PIK3IP1和anti-Pik3ip1多克隆抗体能分别与瞬时转染后的293T细胞膜上表达的PIK3IP1和Pik3ip1分子特异性结合。
流式细胞染色显示，实施例2制备的稳定表达PIK3IP1和Pik3ip1分子的CHO细胞株，能与不同稀释比例的anti-PIK3IP1和anti-Pik3ip1多克隆抗体呈现不同强度的结合，且多克隆抗体1:1000倍稀释后仍然能出现阳性染色(结果参见图3和图4)。
实施例5 Pik3ip1在细胞和组织中的定位表达研究
1、Pik3ip1在Pmel-1TCR转基因小鼠免疫激活过程中淋巴细胞上的表达
取hgp100 25-33多肽100μg和CFA等体积乳化，另取等量CFA与等体积PBS乳化；于雌性6-8周龄Pmel-1TCR转基因小鼠右侧皮下广泛注射100μl hgp100 25-33多肽和CFA的乳化剂。另一组小鼠则皮下注射CFA与PBS的乳化剂100μl进行免疫；免疫24小时后，处死小鼠。取引流淋巴结，制备单细胞悬液，检测免疫过程中CD8 +T细胞、CD11b +单核细胞、CD11c +DC细胞、CD19 +B细胞上Pik3ip1的表达。
2、流式细胞术检测
收集待染色的细胞，用1％FBS的流式缓冲液洗1-2遍，2000rpm离心3min；标记好待用的流式管，若为多色染色，则每一个荧光需进行单独染色，以供调节荧光补偿；将离心后的细胞用流式缓冲液重悬，调整细胞浓度为10 6-10 8/ml，取100μl细胞悬液加入流式管中。按产品的说明向每个流式管中加入human-Ig或mouse-Ig等混匀，封闭细胞上的Fc受体。向封闭后的流式管内，加入一抗。混匀后，置于4℃冰箱，孵育30min。加入约2-3ml流式缓冲液，终止一抗染色。2000rpm离心3min。弃去上清，使流式管中细胞悬液的总体积约100μl。加入合适剂量的荧光二抗，混匀后，置于4℃冰箱，避光孵育30min；加入约2-3ml流式缓冲液，终止荧光二抗染色。洗涤细胞，2000rpm离心3min，弃上清。视细胞的数量，加入约300-400μl流式缓冲液。混匀后，流式上机检测。
3、PIK3IP1在人外周血主要淋巴细胞亚群的表达
本发明的发明人分离了健康人PBMC，采用流式细胞术对PIK3IP1在PBMC中主要淋巴细胞亚群的表达情况进行了检测。结果如图5所示，发现PIK3IP1在人外周血CD8 +T细胞、CD4 +T细胞、B细胞的表达较高。
4、Pik3ip1在小鼠组织、脾淋巴细胞亚群和常见细胞系的表达
本发明的发明人检测了小鼠Pik3ip1在PBMC、眼、脾、脑、卵巢、肺、淋巴结、膀胱、胰腺、肾、肝、小肠、胸腺、心脏、肌肉、子宫和睾丸中mRNA水平的表达情况，结果如图6所 示，发现其在肌肉、PBMC、淋巴结和脾中表达水平较高。
5、Pik3ip1在小鼠脾淋巴细胞表达的流式细胞术检测
本发明的发明人分离并制备了小鼠脾单细胞悬液，采用流式细胞术对Pik3ip1在脾主要淋巴细胞亚群的表达情况进行了检测。结果如图7所示，发现Pik3ip1在CD8 +T细胞、CD4 +T细胞、B细胞、CD11b +细胞和CD11c +细胞上均能检测到表达，在T细胞、B细胞和CD11b +细胞的表达较高。
6、Pik3ip1在小鼠常见细胞系的表达
本发明的发明人检测了Pik3ip1在小鼠黑色素瘤细胞系B16、小鼠巨噬细胞系P338D1、小鼠树突状细胞系DC2.4、小鼠结肠癌细胞系CT26、小鼠T细胞淋巴瘤细胞系EL4、小鼠膀胱癌细胞系MB49的表达，结果如图8所示，发现Pik3ip1在P338D1、DC2.4、EL4高表达。
7、Pik3ip1在小鼠免疫激活过程中淋巴细胞上的表达
本发明的发明人用100μg hgp100 25-33多肽和CFA乳化后特异性免疫，或单独用CFA乳化后非特异性免疫Pmel-1TCR转基因小鼠。免疫激活24小时后，检测Pik3ip1在Pmel-1TCR转基因小鼠引流淋巴结中CD8 +T细胞、CD11b +单核细胞、CD11c +DC细胞、CD19 +B细胞的表达。结果如图9所示，发现Pik3ip1在上述淋巴细胞上的表达，随免疫激活强度的不同呈现变化。
实施例6抗原特异性免疫反应中Pik3ip1缺失增强了T细胞增殖
1、day0，CFSE-标记的3×10 5个OT1小鼠脾细胞尾静脉转输入Pik3ip1-/-(Pik3ip1knock-out,KO)和野生型小鼠(wild-type,WT)中。
2、day1，KO和WT鼠均腹腔免疫OVA 257–264(100μg)，其中以CFA作为佐剂。
3、day2，小鼠牺牲，淋巴细胞进行流式分析，观察CD8 +T细胞的增殖情况。
结果如图10所示：KO鼠的CD8 +T细胞增殖能力比WT鼠的强，表明Pik3ip1的缺失有利于T细胞增殖。
实施例7肿瘤模型中Pik3ip1缺失增强了T细胞增殖
1、anti-mCD3，anti-mCD28扣板：anti-mCD3，anti-mCD28单克隆抗体，各配成终浓度0.125μg/ml，各50μl/孔加入96孔板，封板，4℃过夜；吸去孔内未结合的蛋白溶液，按100μl/孔加入冷的PBS，重复洗板至少3遍。
2、2×10 5个MC38肿瘤细胞皮下接种在KO和WT鼠的侧翼。
3、两周后，取出KO和WT鼠的脾细胞，制成单细胞悬液，用stem cell公司的EasySep TMmouseCD4 +T cel isolation kit和EasySep TM mouseCD8 +T cel isolation kit，按照试剂盒说明书操作，分选出CD4 +和CD8 +T细胞。按3.5×10 5细胞/孔加入anti-mCD3，anti-mCD28扣板的孔板中。
4、在24小时、48小时和72小时检测T细胞增殖情况。
结果如图11所示：24小时、48小时和72小时，Pik3ip1敲除小鼠的CD4 +和CD8 +T细胞的增 殖均高于野生型小鼠的，其中48小时和72小时的差异具有显著性。
实施例8 PIK3IP1与癌症患者T细胞的抑制状态有关
1、从中山大学附属口腔医院随机选取口腔鳞癌(Oral Squamous Cell Carcinoma,OSCC)患者5例，抽取每个患者10ml外周血，参照stemcell公司07811号产品的试剂盒说明书进行人外周血单个核细胞的分离，得到每个患者的PBMC。
2、流式分析每个样品PIK3IP1的表达强度，得到平均荧光强度值(Mean Fluorescence Index，MFI)值。
3、anti-hCD3,，anti-hCD28扣板：anti-hCD3，anti-hCD28单克隆抗体，各配成终浓度0.125μg/ml，各50μl/孔加入96孔板，封板，4℃过夜；吸去孔内未结合的蛋白溶液，按100μl/孔加入冷的PBS，重复洗板至少3遍。
4、CFSE标记每个样品，加入扣有anti-hCD3，anti-hCD28的孔板中。
5、两天后收集样品，通过流式分析OSCC和HCC患者CD4 +与CD8 +T细胞的CFSE比例确定增殖强度。
结果如图12所示：OSCC患者外周血的CD4 +和CD8 +T细胞中PIK3IP1的荧光强度与其增殖强度呈负相关，且所述相关性均具有显著性。表明OSCC患者外周血的CD4 +和CD8 +T细胞中PIK3IP1的表达强度与相应细胞的增殖能力负相关，PIK3IP1的表达会抑制相应细胞的增殖。
实施例9 Pik3ip1-mIg在体内促进gp100特异性Pmel-1CD8 +T细胞的增殖
1、Pik3ip1-mIg融合蛋白对CD8 +T细胞体内增殖影响的研究方法
取雌性6-8周龄Pmel-1TCR转基因小鼠2只，用CO 2处死后，取脾，研磨制成细胞悬液，用ACK裂解红细胞，0.22μm无菌滤网过滤离心，PBS重悬细胞，制备成单细胞悬液；用CFSE标记细胞。
用PBS调整细胞浓度为1×10 7/毫升，每只C57BL/6小鼠尾静脉注射250-300μl细胞悬液，共注射10只6-8周龄雌性C57小鼠；过继转输gp100特异性Pmel-1脾细胞后第1天，按每只小鼠hgp100 25-33多肽100μg，与CFA配成100μl总体积充分乳化后，给予每只实验鼠右侧皮下注射，同时分别对3只小鼠腹腔注射Pik3ip1-mIg，7只小鼠腹腔注射Flag-mIg融合蛋白，剂量均为200μg，300μl/只；于过继转输后第2天开始，每天分别处死1只腹腔给予Flag-mIg融合蛋白小鼠，取引流淋巴结制成单细胞悬液。流式抗体Anti-mouse CD90.1APC，Anti-mouse CD8BV421染色标记细胞；流式上机检测CD90.1、CD8双阳性细胞CFSE的增殖峰比例。待检测到明显而规则的增殖峰出现时，则处死实验组和对照组各3只小鼠，检测CD8 +T细胞的增殖水平。
2、结果
为了研究Pik3ip1-mIg融合蛋白在体内对CD8 +T细胞增殖的影响，本研究检测了gp100特异性Pmel-1脾细胞过继转输C57BL/6后，hgp100 25-33多肽和CFA乳化免疫小鼠，腹腔给予Pik3ip1-mIg融合蛋白和Flag-mIg 4天后，gp100特异性Pmel-1CD8 +T细胞体内增殖的情况。由 图13可以看出：与给予Flag-mIg对照蛋白相比，Pik3ip1-mIg融合蛋白促进了gp100特异性Pmel-1CD8 +T的增殖。Pik3ip1-mIg融合蛋白组CD8 +T细胞增殖平均比例为88.32％，而Flag-mIg组CD8 +T细胞的增殖平均比例为53.34％，明显低于Pik3ip1-mIg融合蛋白组。
实施例10 Pik3ip1-mIg融合蛋白增强2A对小鼠B16-F10成瘤的免疫治疗效果——Pik3ip1-mIg联合2A治疗抑制B16-F10肿瘤的生长
1、4-1BB激活抗体Anti-m4-1BB(2A)的提取和纯化
复苏杂交瘤2A并培养，将培养基连同细胞离心后取上清，0.45μm过滤器过滤上清，收集滤液置于冰上或4℃保存；用亲和力蛋白G柱进行Anti-m4-1BB(2A)的纯化，提纯后的抗体移入蛋白透析卡中，在pH7.2-7.4的1×PBS中，4℃透析24-48小时。
2、Pik3ip1-mIg融合蛋白联合2A治疗小鼠恶性黑色素瘤实验方法
复苏DC2.4细胞并培养，用1ml含10％FBS的RPMI 1640培养基重悬细胞，按工作浓度为5-8μM的gp100 25-33多肽标记DC2.4细胞后，用适量PBS重悬细胞，配成3.5×10 6细胞/毫升。
按前述方法制备Pmel-1TCR转基因小鼠脾和(或)淋巴结细胞单细胞悬液，按每300μl细胞悬液至少1×10 7/细胞的比例重悬细胞；体重相近的SPF级6-8周龄雌性C57BL/6小鼠，设置实验小鼠组别为：Flag-mIg对照组、Pik3ip1-mIg治疗组、2A治疗组、Pik3ip1-mIg联合2A治疗组。每组至少5只小鼠，皮下广泛注射标记了hgp100 25-33多肽的DC2.4细胞悬液100μl/小鼠，同时尾静脉注射gp100特异性Pmel-1脾细胞悬液300μl。
小鼠免疫一周后，将细胞浓度为3×10 6/ml的B16-F10细胞悬液按每只小鼠100μl皮下注射成瘤；自皮下成瘤后每3天，经腹腔给予对应组300μl PBS配制的蛋白溶液，分别含200μg Flag-mIg、200μg Pik3ip1-mIg、200μg 2A、200μg Pik3ip1-mIg+200μg 2A；观察肿瘤生长情况，待可于皮下摸及肿瘤开始，每隔一天用电子游标卡尺测量并记录肿瘤大小，直至肿瘤长至平均直径约1.5cm。肿瘤平均直径计算方法为：(长径+宽径)/2。
3、结果
通过研究Pik3ip1-mIg融合蛋白对小鼠黑色素瘤的治疗，明确该蛋白对CD8 +T杀伤肿瘤功能的影响。本发明发明人构建了Pik3ip1-mIg融合蛋白单独和联合2A治疗小鼠B16-F10肿瘤生长的模型。结果发现：如图14A所示，Pik3ip1-mIg融合蛋白对B16-F10肿瘤的生长有抑制作用，但作用不显著(p＝0.062)。如图14B所示，Pik3ip1-mIg融合蛋白联合2A治疗B16-F10成瘤，能明显抑制肿瘤的生长(p＝0.029)。
于成瘤后20天，采集实验各组荷瘤小鼠的大体观、离体肿瘤的照片。由图15A可以看出：大体看来，Pik3ip1-mIg融合蛋白联合2A治疗组，B16-F10成瘤较Flag-mIg组、单独给予Pik3ip1-mIg融合蛋白组、单独给予2A组平均直径小；其次为单独给予2A组；单独给予Pik3ip1-mIg融合蛋白组瘤体平均直径稍小于Flag-mIg组。由图15B肿瘤离体后的影像也能看出相同的结果。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. PIK3IP1抑制剂在制备用于调控受试者免疫应答的药物中的用途；优选，所述PIK3IP1抑制剂是PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
  2. PIK3IP1抑制剂和抗原一同在制备用于增强受试者对抗原的免疫应答的药物中的用途，所述增强包括给受试者施用抗原和PIK3IP1抑制剂，使得受试者对抗原的免疫应答得到增强；优选，所述PIK3IP1抑制剂是PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
  3. 如权利要求1或2所述的用途，其特征在于，所述调控受试者免疫应答或增强受试者免疫应答为增强受试者CD8 +T细胞和/或CD4 +T细胞的增殖。
  4. 如权利要求2或3所述的用途，其特征在于，所述抗原是癌性细胞、肿瘤抗原、病毒抗原、细菌抗原；
    优选所述抗原为黑素瘤的抗原肽；更优选为肽gp100、MAGE抗原、Trp-2、MART1和/或酪氨酸酶；更优选为hgp100 25-33；
    优选所述抗原是OVA，更优选为OVA 257–264；
    优选所述抗原是癌性细胞，更优选为MC38细胞，B16细胞，和/或B16-F10细胞。
  5. PIK3IP1抑制剂单独或者和另一种或多种治疗剂联合在制备治疗肿瘤药物中的用途；优选，所述PIK3IP1抑制剂是PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
  6. PIK3IP1抑制剂和4-1BB的激活性抗体联合在制备治疗肿瘤的药物中的用途；优选所述4-1BB的激活性抗体是抗体2A；优选，所述PIK3IP1抑制剂是PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
  7. 如权利要求5或6所述的用途，其特征在于，所述肿瘤是对免疫疗法有响应的肿瘤；
    优选所述肿瘤选自黑色素瘤、白血病、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌、头颈癌；
    优选，所述肿瘤是转移性的；
    优选，所述肿瘤为结肠癌、黑色素瘤、口腔鳞状细胞癌、肝细胞癌。
  8. 一种用于调控受试者免疫应答或治疗肿瘤的药物组合物，其包含PIK3IP1抑制剂，和药学上可接受的载体；
    优选，所述药物组合物进一步含有另一种或多种治疗剂；
    优选，所述PIK3IP1抑制剂是PIK3IP1、PIK3IP1ECD、PIK3IP1ECD融合分子或PIK3IP1的抗体。
  9. 如权利要求1-7任一项所述的用途或权利要求8所述的药物组合物，其特征在于，所述PIK3IP1包含选自SEQ ID NO.1、2、3、4、5、6、7和/或8的序列；所述PIK3IP1ECD包含选自SEQ ID NO.9和/或10的序列；PIK3IP1ECD融合分子包含选自SEQ ID NO.9和/或10的序列；
    优选，所述PIK3IP1由选自SEQ ID NO.1、2、3、4、5、6、7或8的序列组成；
    优选，所述PIK3IP1ECD由选自SEQ ID NO.9或10的序列组成；
    优选，所述PIK3IP1ECD融合分子的至少一个融合伴侣选自Fc、白蛋白和聚乙二醇；
    优选，所述PIK3IP1ECD融合分子的至少一个融合伴侣是Fc；
    优选，所述PIK3IP1ECD融合分子的至少一个融合伴侣是IgGFc；更优选所述IgGFc的碱基序列由选自SEQ ID NO.15或16的序列组成；
    优选，所述PIK3IP1ECD融合分子的碱基序列由选自SEQ ID NO.11、12、13或14的序列组成。
  10. 如权利要求5所述的用途或权利要求8所述的药物组合物，其特征在于，所述另一种或多种治疗剂选自肿瘤化疗剂，能够活化T细胞的化合物，和/或血管生成抑制剂；
    优选，所述肿瘤化疗剂选自氮芥、苯丁酸氮芥、环磷酰胺、异环磷酰胺、塞替派、卡莫司汀、司莫司汀、白消安、顺铂、奥沙利铂、卡铂、草酸铂、丝裂霉素、甲氨蝶呤、5-FU、FT-207、卡培他滨、6-巯基嘌呤、6-TG、羟基脲、阿糖胞苷、吉西他滨、培美曲塞、放线菌素D、柔红霉素、阿霉素、表阿霉素、阿克拉霉素、光辉霉素、伊立替康、拓扑替康、羟基喜树碱、紫杉醇、多西他赛、长春花碱、长春新碱、长春瑞滨、鬼臼碱类和高三尖杉酯碱组成的组；
    优选，所述能够活化T细胞的化合物选自T细胞共刺激性分子的激活性抗体；更优选为CTLA-4、OX-40、4-1BB或ICOS的激活性抗体，抗CD40抗体；更优选为4-1BB激活性抗体2A；
    优选，所述血管生成抑制剂选自来那度胺、沙利度胺、贝伐单抗、凡德他尼、瓦他拉尼、舒尼替尼、阿西替尼、帕唑帕尼和4-(4-氟-2-甲基吲哚-5-基氧基)-6-甲氧基-7-(3-吡咯烷-1-基丙氧基)喹唑啉组成的组。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314476A (zh) * 2000-03-17 2001-09-26 上海博德基因开发有限公司 一种新的多肽——人磷脂酰肌醇3(PtdIns 3)－激酶36和编码这种多肽的多核苷酸
US20160025734A1 (en) * 2014-07-23 2016-01-28 Children's Medical Center Corporation Use of urinary protein biomarkers to distinguish between neoplastic and non-neoplastic disease of the prostate
WO2016092508A1 (en) * 2014-12-12 2016-06-16 Massachusetts General Hospital Treatment of breast cancer brain metastases
CN107223163A (zh) * 2014-12-24 2017-09-29 豪夫迈·罗氏有限公司 用于膀胱癌症的治疗，诊断和预后方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314476A (zh) * 2000-03-17 2001-09-26 上海博德基因开发有限公司 一种新的多肽——人磷脂酰肌醇3(PtdIns 3)－激酶36和编码这种多肽的多核苷酸
US20160025734A1 (en) * 2014-07-23 2016-01-28 Children's Medical Center Corporation Use of urinary protein biomarkers to distinguish between neoplastic and non-neoplastic disease of the prostate
WO2016092508A1 (en) * 2014-12-12 2016-06-16 Massachusetts General Hospital Treatment of breast cancer brain metastases
CN107223163A (zh) * 2014-12-24 2017-09-29 豪夫迈·罗氏有限公司 用于膀胱癌症的治疗，诊断和预后方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN121046386A (zh) * 2025-10-30 2025-12-02 苏州系统医学研究所 一种Pik3ip1基因敲除小鼠模型的构建方法及其应用

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