WO2022083694A1 - 叶酸和叶酸修饰在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途 - Google Patents

叶酸和叶酸修饰在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途 Download PDF

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WO2022083694A1
WO2022083694A1 PCT/CN2021/125346 CN2021125346W WO2022083694A1 WO 2022083694 A1 WO2022083694 A1 WO 2022083694A1 CN 2021125346 W CN2021125346 W CN 2021125346W WO 2022083694 A1 WO2022083694 A1 WO 2022083694A1
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folic acid
modified
drugs
mabs
immunogenic
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PCT/CN2021/125346
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English (en)
French (fr)
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占昌友
王欢
姜铸轩
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复旦大学
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Priority to JP2023524381A priority Critical patent/JP2023549464A/ja
Priority to EP21882109.8A priority patent/EP4233868A1/en
Publication of WO2022083694A1 publication Critical patent/WO2022083694A1/zh
Priority to US18/304,987 priority patent/US20230381183A1/en

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Definitions

  • the present invention relates to the use of folic acid and folic acid modifications in inducing B cell immune tolerance and targeting mIgM positive expressing B cell lymphomas, in particular for reducing the production of antibodies against immunogenic substances, for use in Prevention or treatment of diseases or conditions that may be mediated by B-cell immune tolerance (eg, hypersensitivity, autoimmune disease, or transplant rejection), and/or for targeted diagnosis, treatment, or prevention of mIgM-positive B-cell lymphocytes tumor.
  • B-cell immune tolerance eg, hypersensitivity, autoimmune disease, or transplant rejection
  • the present invention also relates to methods, pharmaceutical compositions and combination products for inducing B-cell immune tolerance and targeted therapy, diagnosing or preventing mIgM-positive B-cell lymphomas.
  • Biological macromolecular drugs such as peptides and proteins have significant effects in the treatment of immune-related diseases.
  • these drugs have potential immunogenicity, especially when used for a long time, it is easy to produce anti-drug antibodies (Anti-Drug Antibody, ADA), and then neutralize the drugs in the body, resulting in reduced drug concentration, inactivity, disease recurrence, and severe It can also cause infusion-induced hypersensitivity reactions.
  • Anti-Drug Antibody Antibody
  • ADA anti-drug Antibody
  • induction of immune tolerance is also one of the most effective strategies for preventing and treating diseases or conditions that can be mediated by B-cell immune tolerance, such as hypersensitivity reactions, autoimmune diseases, and transplant rejection.
  • the immune function of the body is mainly regulated by immunomodulators, but long-term medication is required, and it is accompanied by side effects such as easily activating pathogens, tumor development and increasing the probability of infection.
  • B cells are derived from pluripotent stem cells of the bone marrow. Mature B cells migrate out of peripheral blood and enter the spleen and lymph nodes, and are mainly distributed in splenic nodules, splenic cords and lymph nodes, lymphatic cords and submucosal lymph nodes in the digestive tract. After being stimulated by antigen, mature B cells differentiate and proliferate into plasma cells, synthesize antibodies, and exert humoral immune function. B cells are the most numerous antigen-presenting cells in the body, and their characteristic surface markers are membrane surface immunoglobulins, which can act as specific antigen receptors to recognize various antigenic epitopes. Membrane surface immunoglobulin M (mIgM) constitutes the antigen receptor of B cells. Acting on mIgM by specific ligands can cause B cell anergy and induce B cell immune tolerance.
  • mIgM membrane surface immunoglobulin M
  • B cell immune tolerance e.g, hypersensitivity reactions, autoimmune diseases, or transplant rejection
  • targeted diagnosis, treatment, or prevention of mIgM-positive B cell lymphomas e.g, hypersensitivity reactions, autoimmune diseases, or transplant rejection
  • Folic acid is composed of three parts pterin, p-aminobenzoic acid and L-glutamic acid, also known as pteroylglutamic acid. Folic acid plays an important role in the body's metabolism, can participate in the synthesis of purine and ribonucleic acid, etc., help regulate the development of embryonic nerve cells, and prevent newborn babies from suffering from congenital neural tube defects. Folic acid has become a new health vitamin product in the international market after vitamin C and vitamin E. Due to its advantages of safety, stability and low cost, its market prospect is very broad.
  • the inventors have unexpectedly discovered through extensive research that folic acid has specific binding to mIgM expressed on the surface of B cells, and can target B cells with positive mIgM expression, thereby inhibiting the maturation of B cells and their differentiation into germinal center B cells. B-cell anergy induces B-cell immune tolerance.
  • the administration of folic acid or its salts or esters or conjugates can induce the body to develop B-cell immune tolerance, respond to immunogenic substances (such as biological macromolecular drugs, immunogenic drug delivery systems, or lead to abnormal immune responses in the body) Stimulation of pathogenic antigenic substances) exhibits specific immune hyporesponsiveness or non-response, which can be used to reduce the production of antibodies against immunogenic substances and/or for the treatment or prevention of immune tolerance mediated by B cells. disease or condition.
  • immunogenic substances such as biological macromolecular drugs, immunogenic drug delivery systems, or lead to abnormal immune responses in the body
  • Stimulation of pathogenic antigenic substances exhibits specific immune hyporesponsiveness or non-response, which can be used to reduce the production of antibodies against immunogenic substances and/or for the treatment or prevention of immune tolerance mediated by B cells. disease or condition.
  • the inventors have also unexpectedly found that the B cell immune tolerance induced by folic acid or its salts, esters or conjugates is reversible, and the immune function of the body can be recovered after the administration of folic acid or its salts, esters or conjugates is stopped, This is especially advantageous for individuals who, for medical purposes, desire only temporary immune tolerance.
  • modifying an immunogenic substance with folic acid can also significantly reduce the production of antibodies against the immunogenic substance.
  • modification of biomacromolecules with folic acid can significantly reduce the autoimmunity of biomacromolecules and inhibit anti-drug antibodies (ADA) and drug resistance.
  • Modification of pathogenic antigenic substances that cause abnormal immune responses in the body with folic acid can significantly reduce the production of antigen-specific antibodies for the prevention or treatment of hypersensitivity reactions, autoimmune diseases and/or transplant rejection, etc. It can be tolerated through B cell immunity mediated disease or disorder.
  • the present inventors unexpectedly found that the targeted diagnosis, treatment and/or prevention of mIgM-positive B-cell lymphomas can be achieved through the mIgM pathway by modifying drugs (eg, antitumor drugs) or probes with folic acid.
  • drugs eg, antitumor drugs
  • the present invention provides folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof in the manufacture of folic acid for inducing B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and /or use in a medicament for the treatment or prevention of a disease or condition mediated by B cell immune tolerance.
  • the present invention provides folic acid-modified immunogenic substances in the preparation of B-cell immune tolerance, especially for reducing the production of antibodies against immunogenic substances and/or for use in the treatment or prevention of Use in a medicament for a disease or disorder mediated by B cell immune tolerance.
  • the present invention provides the use of a folic acid-modified antitumor drug or a folic acid-modified probe in the preparation of a medicament for targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphoma.
  • the present invention provides for inducing B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for treating or preventing B cell immune tolerance mediated
  • a method of disease or disorder comprising administering to a subject an effective amount of a folic acid, or a pharmaceutically acceptable salt or ester or conjugate thereof, or a folic acid-modified immunogenic substance of the present invention.
  • the present invention provides a method for targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphoma, the method comprising administering to an individual an effective amount of the folic acid-modified antineoplastic drug or folic acid-modified agent of the present invention probe.
  • the present invention provides for inducing B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for treating or preventing B cell immune tolerance mediated A folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof or a folic acid-modified immunogenic substance of the invention for a disease or disorder.
  • the present invention provides a folic acid-modified antineoplastic drug or a folic acid-modified probe of the present invention for targeted diagnosis, treatment or prevention of mIgM-positive B cell lymphoma.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of folic acid of the present invention or a pharmaceutically acceptable salt or ester or conjugate thereof and/or a folic acid-modified immunogenic substance and/or a folic acid-modified Antineoplastic and/or folic acid-modified probes, and optionally one or more pharmaceutically acceptable excipients.
  • the present invention provides a combination product of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof and an immunogenic substance.
  • Embodiment 1 Folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof in the preparation for inducing B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for treatment or prevention Use in a medicament for a disease or disorder mediated by B cell immune tolerance.
  • Embodiment 2 The use according to embodiment 1, wherein said folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof is selected from the group consisting of free folic acid, a pharmaceutically acceptable salt of folic acid, a pharmaceutically acceptable ester of folic acid, and a pharmaceutically acceptable ester of folic acid.
  • Pharmaceutical conjugates eg, folic acid albumin conjugates, folic acid polyethylene glycol conjugates, etc.), or any combination thereof.
  • Embodiment 3 The use according to embodiment 1 or 2, wherein the B cells are mIgM expressing B cells, particularly mIgM highly expressing B cells, more particularly spleen B cells or lymph node B cells.
  • Embodiment 4 The use according to any one of embodiments 1-3, wherein the immunogenic substance is a biomacromolecular active agent, and the medicament is administered in combination with the biomacromolecular active agent, eg, in the presence of a biomacromolecular active agent. before and/or during administration of the agent to reduce the production of anti-drug antibodies against the biomacromolecular active agent;
  • the immunogenic substance is a biomacromolecular active agent
  • the medicament is administered in combination with the biomacromolecular active agent, eg, in the presence of a biomacromolecular active agent. before and/or during administration of the agent to reduce the production of anti-drug antibodies against the biomacromolecular active agent;
  • the biomacromolecular active agent is selected from polypeptide drugs, such as p53 activating peptide, melittin, scorpion venom, antimicrobial peptide or insulin; or protein drugs, such as antibody drugs and especially monoclonal antibody drugs or polyclonal antibodies Drugs, interferons, growth factors, growth factor inhibitors, enzymes, or albumin such as human albumin or chicken ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies or Fully human monoclonal antibodies, such as tumor necrosis factor alpha (TNF ⁇ ) mAbs (such as adalimumab, etanercept, or infliximab), PD1/PD-L1 mAbs (such as nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalizumab, or camrelizumab); HER2 mAbs (such as trast
  • VEGF/VEGFR vascular endothelial growth factor/vascular endothelial growth factor receptor
  • EGFR epidermal growth factor receptor
  • the biological macromolecular active agent is selected from adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab, white protein and insulin.
  • Embodiment 5 The use according to any one of Embodiments 1-3, wherein the immunogenic substance is an immunogenic drug delivery system, such as a microcarrier drug delivery system, preferably selected from liposomes, microspheres , microcapsules, nanoparticles, nanocapsules, lipid nanodiscs or polymeric micelles, and the agent is administered in combination with the delivery system, for example before and/or during administration of the delivery system, To reduce the production of antibodies against an immunogenic delivery system; preferably, the delivery system is loaded with a biomacromolecular active agent as defined in embodiment 4.
  • an immunogenic drug delivery system such as a microcarrier drug delivery system, preferably selected from liposomes, microspheres , microcapsules, nanoparticles, nanocapsules, lipid nanodiscs or polymeric micelles
  • the agent is administered in combination with the delivery system, for example before and/or during administration of the delivery system, to reduce the production of antibodies against an immunogenic delivery system; preferably, the delivery system is loaded with a
  • Embodiment 6 The use according to any one of embodiments 1-3, wherein the medicament is for the treatment or prevention of a disease or disorder mediated by B cell immune tolerance, such as hypersensitivity, autoimmune disease or transplantation Rejection; preferably, the hypersensitivity is selected from anaphylactic shock, respiratory hypersensitivity (such as allergic asthma or allergic rhinitis) and gastrointestinal hypersensitivity, and the autoimmune disease is selected from systemic lupus erythematosus , rheumatoid arthritis, Hashimoto's thyroiditis, toxic diffuse goiter, ankylosing spondylitis, autoimmune encephalomyelitis, neuromyelitis optica spectrum disorders, anticardiolipin syndrome, hemophilia and psoriasis, and the transplant rejection is selected from the group consisting of organ transplant rejection, tissue transplant rejection (eg, bone marrow transplant rejection) and recurrent miscarriage.
  • a disease or disorder mediated by B cell immune tolerance such as hypersensitivity, autoimmune disease or transplantation Rejection
  • Embodiment 7 The preparation of folic acid-modified immunogenic substances for the induction of B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for the treatment or prevention of B cell immune tolerance. Use in a medicament for a mediated disease or disorder.
  • Embodiment 8 The use according to embodiment 7, wherein the B cells are mIgM expressing B cells, particularly mIgM highly expressing B cells, more particularly spleen B cells or lymph node B cells.
  • Embodiment 9 The use according to embodiment 7 or 8, wherein the immunogenic substance is a biomacromolecular active agent;
  • the biomacromolecular active agent is selected from polypeptide drugs, such as p53 activating peptide, melittin, scorpion venom, antimicrobial peptide or insulin; or protein drugs, such as antibody drugs and especially monoclonal antibody drugs or polyclonal antibodies Drugs, interferons, growth factors, growth factor inhibitors, enzymes, or albumin such as human albumin or chicken ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies or Fully human monoclonal antibodies, such as tumor necrosis factor alpha (TNF ⁇ ) mAbs (such as adalimumab, etanercept, or infliximab), PD1/PD-L1 mAbs (such as nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalizumab, or camrelizumab); HER2 mAbs (such as trast
  • VEGF/VEGFR vascular endothelial growth factor/vascular endothelial growth factor receptor
  • EGFR epidermal growth factor receptor
  • the biological macromolecular active agent is selected from adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab, white protein and insulin.
  • Embodiment 10 The use according to embodiment 7 or 8, wherein the immunogenic substance is a pathogenic antigenic substance that causes the body to produce an abnormal immune response, preferably a pathogenic hypersensitivity reaction, autoimmune disease or transplant rejection
  • Antigenic substances such as chicken ovalbumin, proteolipoprotein polypeptide, Dby polypeptide, Uty, myelin basic protein, denatured IgG, thyroglobulin, thyrotropin receptor, histone, acetylcholine receptor, major histocompatibility antigen (including HLA-I and HLA-II), minor histocompatibility antigens, blood group antigens, tissue-specific antigens (eg, vascular endothelial cell antigen, skin SK antigen).
  • the medicament is for the treatment or prevention of hypersensitivity, autoimmune disease or transplant rejection; preferably, the hypersensitivity is selected from anaphylactic shock, respiratory hypersensitivity (eg allergic asthma or allergic rhinitis) and gastrointestinal hypersensitivity, the autoimmune disease selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis, toxic diffuse goiter, compulsive spondylitis, autoimmune encephalomyelitis, neuromyelitis optica spectrum disorders, anticardiolipin syndrome, hemophilia, and psoriasis, and the transplant rejection is selected from the group consisting of organ transplant rejection, tissue transplant rejection (eg, bone marrow transplant rejection) and recurrent miscarriage.
  • the hypersensitivity is selected from anaphylactic shock, respiratory hypersensitivity (eg allergic asthma or allergic rhinitis) and gastrointestinal hypersensitivity
  • the autoimmune disease selected from the group consisting of systemic lupus erythematos
  • Embodiment 12 Use of a folic acid-modified antitumor drug or a folic acid-modified probe in the preparation of a medicament for targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphoma.
  • Embodiment 13 The use according to Embodiment 12, wherein the anti-tumor drug in the folic acid-modified anti-tumor drug is an anti-tumor drug that can be used to treat or prevent mIgM-positive B-cell lymphoma, preferably an anthracycline Drugs, such as doxorubicin or epirubicin; taxane drugs, such as paclitaxel, docetaxel, or cabazitaxel; camptothecin drugs, such as camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or irinotecan; vinblastine drugs such as vincristine or vinorelbine; proteasome inhibitors such as bortezomib or carfilzomib; lactone drugs such as parthenolide; cyclophosphamide, etopo glycosides, gemcitabine, cytarabine, 5-fluorouracil, teniposide, mor
  • Embodiment 14 Method for inducing B-cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for treating or preventing diseases or disorders mediated by B-cell immune tolerance , the method comprises administering to the individual an effective amount of folic acid as defined in any one of Embodiments 1-6 or a pharmaceutically acceptable salt or ester or conjugate thereof and/or as defined in any of Embodiments 7-11 Folic acid-modified immunogenic substances.
  • Embodiment 15 A method for targeted diagnosis, treatment or prevention of mIgM-positively expressing B-cell lymphoma, the method comprising administering to an individual an effective amount of a folic acid-modified antineoplastic drug or folic acid as defined in embodiment 12 or 13 Modified probes.
  • Embodiment 16 For the induction of B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for the treatment or prevention of diseases or conditions that may be mediated by B cell immune tolerance.
  • Embodiment 17 A folic acid-modified antineoplastic drug or a folic acid-modified probe as defined in embodiment 12 or 13, in particular a folic acid-modified carbachol, for use in the targeted diagnosis, treatment or prevention of mIgM-positively expressing B-cell lymphomas Triptolide or Triptolide modified with folic acid.
  • Embodiment 18 A pharmaceutical composition comprising an effective amount of folic acid as defined in any of embodiments 1-6 or a pharmaceutically acceptable salt or ester or conjugate thereof and/or as defined in any of embodiments 7-11 A defined folic acid-modified immunogenic substance and/or a folic acid-modified antineoplastic drug or a folic acid-modified probe as defined in embodiment 12 or 13, and optionally one or more pharmaceutically acceptable excipients.
  • Embodiment 19 A combination product of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof and an immunogenic substance, wherein the immunogenic substance is preferably a biomacromolecular active agent or an immunogenic drug delivery system ;
  • the biomacromolecule active agent is selected from polypeptide drugs, such as p53 activating peptide, melittin, scorpion venom, antimicrobial peptide or insulin; or protein drugs, such as antibody drugs and especially monoclonal antibody drugs or Polyclonal antibodies, interferons, growth factors, growth factor inhibitors, enzymes, or albumin such as human albumin or chicken ovalbumin, including murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies Antibodies or fully human monoclonal antibodies, such as tumor necrosis factor alpha (TNF ⁇ ) mAbs (such as adalimumab, etanercept, or infliximab), PD1/PD-L1 mAbs (such as nivolumab) anti-, pembrolizumab, atezolizumab, sintilimab, toripalizumab, or camrelizumab); HER2 mAbs (e
  • the immunogenic drug delivery system is selected from microcarrier drug delivery systems, such as liposomes, microspheres, microcapsules, nanoparticles, nanocapsules, lipid nanodiscs or polymer gels bundle.
  • the term "about” should be understood to mean adjusting the given numerical value by 20%, such as 10%, such as 5%, above or below the numerical value.
  • folate or a salt or ester or conjugate thereof includes free folic acid, folic acid salt, folic acid ester or folic acid conjugates (eg, folic acid albumin conjugates, folic acid polyethylene glycol conjugates, etc. ), or any combination of them. Accordingly, the term “folate or a pharmaceutically acceptable salt or ester or conjugate thereof” as used herein includes free folate or a pharmaceutically acceptable folate, folate or folate conjugate (eg, folate albumin conjugated). compound, folic acid polyethylene glycol conjugate, etc.), or any combination thereof.
  • fused refers to the chemical binding of folic acid to other substances, such as immunogenic substances (eg, biological macromolecules or pathogenic antigenic substances), antineoplastic drugs, probes, polymeric materials Equijoined to form a complex.
  • immunogenic substances eg, biological macromolecules or pathogenic antigenic substances
  • antineoplastic drugs e.g., antineoplastic drugs, probes, polymeric materials Equijoined to form a complex.
  • the term "substance of the invention” as used herein refers to folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof, folic acid-modified immunogenic substances (eg, folic acid-modified biomacromolecular active agents, folic acid-modified one or more of the pathogenic antigenic substances), folic acid-modified antineoplastic drugs or folic acid-modified probes.
  • folic acid-modified immunogenic substances eg, folic acid-modified biomacromolecular active agents, folic acid-modified one or more of the pathogenic antigenic substances
  • folic acid-modified antineoplastic drugs folic acid-modified probes.
  • the substance of the present invention may also refer to folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof in combination with an immunogenic substance.
  • immunogenic substance refers to a substance capable of activating and inducing an immune response in the body, including but not limited to biological macromolecules such as biological macromolecular active agents, immunogenic drug delivery systems, or causing abnormal immunity in the body responsive to pathogenic antigenic substances.
  • drug delivery system refers to a system that modulates the distribution of a drug in an organism over space, time and/or dose.
  • immunogenic drug delivery system refers to a drug delivery system capable of activating and inducing an immune response in the body, such as a microcarrier drug delivery system.
  • microcarrier delivery system includes a range of micro, submicron, nanoscale, etc. systems, such as liposomes, microspheres, microcapsules, microemulsions, nanoparticles, nanocapsules, nanodiscs, polymeric micelles, etc.
  • biomacromolecule refers to high molecular weight organic substances that are active components in living organisms, such as molecular weights up to thousands of Daltons or higher, including but not limited to polypeptides or proteins such as antibodies and the like.
  • biomacromolecular drug and “biomacromolecular active agent” are used interchangeably and refer to natural or synthetic biomacromolecules with therapeutic or prophylactic activity, such as, but not limited to, polypeptide drugs or protein drugs such as antibodies drugs, etc.
  • causal antigenic material that causes an abnormal immune response in the body or “causative antigenic material” are used interchangeably and refer to causing the body to produce an excessive immune response, thereby causing tissue damage and/or manifesting disease in the body state of immunogenic substances.
  • anti-plastic drug refers to a substance that can be used to prevent or treat tumor diseases or cancer, such as chemotherapeutic drugs, hormone drugs or toxins, and the like.
  • probe refers to functional chemicals such as contrast agents and the like capable of detecting or displaying biomedical problems, such as diagnosing or treating disease.
  • contrast agent refers to a chemical substance administered to a tissue or organ of an individual to enhance image viewing.
  • pharmaceutically acceptable means chemically/toxicologically compatible with other ingredients of the formulation or composition (including excipients and/or active ingredients) and/or with the body.
  • pharmaceutically acceptable salt refers to the salt of the parent compound with a pharmaceutically acceptable acid or base.
  • Pharmaceutically acceptable salts are well known in the art and include, but are not limited to, inorganic acid salts such as hydrochloride, sulfate, phosphate, etc.; organic acid salts such as acetate, propionate, mesylate, ethanesulfonate acid salt, malate salt, citrate salt, tartrate salt, etc.; alkali metal or alkaline earth metal salt, such as sodium, potassium, calcium salt, etc.
  • pharmaceutically acceptable ester refers to a pharmaceutically acceptable ester derivative of the parent carboxylic acid, such as a lower alkyl ester (eg, methyl, ethyl, n- or isopropyl, etc.), Cycloalkyl esters, lower alkenyl esters, benzyl esters, such as ⁇ -(amino, mono- or di-lower alkylamino, carboxyl, lower alkoxycarbonyl)-lower alkyl esters, ⁇ -(lower alkanoyloxy , lower alkoxycarbonyl or di-lower alkylaminocarbonyl)-lower alkyl esters such as pivaloyloxymethyl ester, and the like conventionally used in the art.
  • a lower alkyl ester eg, methyl, ethyl, n- or isopropyl, etc.
  • Cycloalkyl esters such as ⁇ -(amino, mono- or di-lower al
  • conjugate or “folate conjugate” of folic acid as used herein refers to the coupling of folic acid with other substances through chemical bonds to form a complex to improve the in vivo kinetic behavior and action time of folic acid, including but not limited to folic acid white Protein conjugates, folic acid polyethylene glycol conjugates, etc.
  • pharmaceutically acceptable excipient includes any and all pharmaceutically acceptable carriers or diluents, solvents, dispersion media, coatings, surfactants, antioxidants, preservatives, isotonic agents, absorption delaying agents, Salts, preservatives, stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavors, dyes, etc., or any combination thereof, as known to those of ordinary skill in the art. Any adjuvant known in the art can be used in the pharmaceutical composition or pharmaceutical formulation, so long as it is compatible with the active ingredient.
  • B cells as used herein especially refers to mIgM expressing B cells, in particular mIgM highly expressing B cells, eg spleen B cells or lymph node B cells.
  • B-cell immune tolerance refers to a low or no reactivity of B cells to antigenic stimulation, a reduced or no immune response.
  • antibody against an immunogenic substance refers to a specific antibody produced by the body induced by an immunogenic substance.
  • anti-drug antibodies refers to anti-drug antibodies (ADAs) produced by the body induced by biological macromolecules with potential immunogenicity.
  • ADAs anti-drug antibodies
  • the production of ADAs can lead to decreased drug concentration, shortened drug half-life, decreased drug efficacy, or even ineffectiveness, or caused hypersensitivity reactions, even life-threatening.
  • drug resistance refers to a decrease in the efficacy of a drug to treat a disease or improve symptoms.
  • disease or disorder mediated by B cell immune tolerance refers to a disease or disorder that can benefit from, or be treated or prevented by, B cell immune tolerance.
  • hyposensitivity reaction is an abnormal adaptive immune response that occurs when the body is stimulated by certain antigens, such as physiological dysfunction or tissue cell damage.
  • autoimmune disease refers to a disease state caused by the body's abnormal immune response to self-antigens, resulting in the destruction of its own cells, tissue damage, or dysfunction.
  • transplant rejection refers to the recipient's tissue or organ transplantation, especially after allogeneic or xenogeneic tissue or organ transplantation, the transplant of a foreign tissue or organ as a foreign object is recognized by the recipient's immune system, the latter Initiate an immunological response to attack, destruction and clearance of the graft.
  • transplant rejection also includes recurrent miscarriages.
  • the terms “individual” and “patient” are used interchangeably and include any human or non-human animal.
  • the term “non-human animal” includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • the individual is a primate, especially a human.
  • the term "effective amount” refers to the amount of a substance of the invention that, when administered to a cell or tissue or non-cellular biological material or medium of an individual, elicits a biological or medical response, such as an improvement in symptoms, amelioration of a disorder, disease Delay or delay of progress or prevention of disease, etc. It is understood by those of skill in the art that the specific effective amount may vary depending on factors such as the intended biological endpoint, the drug to be delivered, the target tissue, etc., and that an "effective amount” may be administered in a single dose or may be obtained by administering multiple doses accomplish.
  • treating refers to ameliorating, alleviating, alleviating or preventing a disease or disorder or at least one clinical symptom thereof.
  • “treating” refers to alleviating or improving at least one physical parameter, including those that a patient can and/or cannot feel.
  • “treating” refers to physically modulating a disease or disorder (eg, stabilizing a perceived symptom) or physiologically modulating a disease or disorder (eg, stabilizing a physical parameter), or both.
  • treating refers to preventing or delaying the onset or development or progression of a disease or disorder.
  • the terms "prevent,” “prevent,” and the like refer to preventing the onset, progression or progression of one or more symptoms of a disease or disorder in an individual by administering a therapy (eg, a therapeutic agent) or a combination of therapies (eg, a combination of therapeutic agents). relapse.
  • a therapy eg, a therapeutic agent
  • a combination of therapies eg, a combination of therapeutic agents
  • diagnosis refers to making a judgment about an individual's disease state after collecting information through methods such as examination or investigation.
  • combination refers to the simultaneous, separate or sequential administration of two or more active agents by the same or different routes, the active agents may be included in the same pharmaceutical composition or in separate pharmaceutical combinations in (eg in the form of a kit).
  • the term “combination product” includes both two or more active agents in the same pharmaceutical composition and two or more active agents in separate pharmaceutical compositions (eg, in a set box).
  • Folic acid is composed of three parts pterin, p-aminobenzoic acid and L-glutamic acid, so it is also called pteroylglutamic acid.
  • the folic acid of the present invention may be of natural or synthetic origin.
  • folic acid albumin conjugates and folic acid polyethylene glycol conjugates are commercially available from Ruixi Bioavailability.
  • the body can induce B cell immune tolerance.
  • the folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof of the present invention may be administered alone, or may be administered in combination with an immunogenic substance, preferably before and/or during the administration of the immunogenic substance, to Inducing B cell immune tolerance, for reducing the production of antibodies against immunogenic substances and/or for treating or preventing diseases or conditions that may be mediated by B cell immune tolerance.
  • the folic acid of the present invention is administered to an individual who will be administered, is being administered, or has been administered an immunogenic substance (eg, a biomacromolecular active agent, an immunogenic delivery system). Salts or esters or conjugates to reduce the production of antibodies against immunogenic substances.
  • an immunogenic substance eg, a biomacromolecular active agent, an immunogenic delivery system. Salts or esters or conjugates to reduce the production of antibodies against immunogenic substances.
  • folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof is administered before and/or during administration of the immunogenic substance. More preferably, administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof precedes administration of the immunogenic substance (eg 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 day) a period of time that begins and lasts for years, months, weeks, or days.
  • the administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof continues until such as 3 months, 2 months, 1 month, 3 weeks, 2 weeks or 1 week after the end of administration of the immunogenic substance, This depends on properties such as metabolism or half-life of the immunogenic substance and can be routinely judged by the participating physician.
  • an individual at risk for a disease or disorder mediated by B-cell immune tolerance eg, hypersensitivity, autoimmune disease, or transplant rejection
  • a disease or disorder mediated by B-cell immune tolerance is administered to an individual Administration of a folic acid of the present invention, or a pharmaceutically acceptable salt or ester or conjugate thereof, to a subject of a mediated disease or condition to prevent the occurrence of the disease or condition or to treat the disease or condition when the disease or condition occurs or A disorder, eg, alleviation of one or more symptoms thereof.
  • Immunogenic substances that can be used in the present invention include, but are not limited to, biomacromolecules such as biomacromolecular active agents, immunogenic drug delivery systems, and pathogenic antigenic substances that cause abnormal immune responses in the body.
  • immunogenic substances useful in the present invention are biomacromolecular active agents, including but not limited to polypeptide drugs, such as p53 activating peptides, melittin, scorpion venom, antimicrobial peptides, or insulin; or proteins Drugs, such as antibody drugs and especially monoclonal or polyclonal antibodies, interferons, growth factors, growth factor inhibitors, enzymes, or albumins such as human albumin or chicken ovalbumin, including murine monoclonal antibodies, Chimeric monoclonal antibodies, humanized monoclonal antibodies, or fully human monoclonal antibodies, such as tumor necrosis factor alpha (TNF ⁇ ) mAbs (eg, adalimumab, etanercept, or infliximab); PD1/PD-L1 mAbs (eg, nivolumab, pembrolizumab, atezolizumab, sintilimab, toripalizumab, or camrelizuma
  • trastuzumab trastuzumab, pertuzumab, or lapatinib
  • CD20 mAbs e.g., rituximab, tiimumab, or tositumumab
  • VEGF/VEGFR vascular endothelial growth factor/vascular endothelial Growth factor receptor
  • EGFR epidermal growth factor receptor
  • the biomacromolecular active agent that can be used in the present invention is selected from adalimumab, infliximab, atezolizumab, sintilimab, toripalimab, trastuzumab , albumin and insulin.
  • the immunogenic substance useful in the present invention is an immunogenic drug delivery system, including but not limited to a microcarrier drug delivery system, preferably selected from the group consisting of liposomes, microspheres, microcapsules, Nanoparticles, nanocapsules, lipid nanodiscs or polymeric micelles.
  • the drug delivery system can be loaded with any suitable drug, such as a biomacromolecular active agent as described herein, an antineoplastic drug, an antibacterial drug, a hormone, and the like.
  • the folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof of the present invention can reduce antibodies against the drug delivery system and the biomacromolecular active agent production of anti-drug antibodies.
  • the preparation of drug delivery systems is known in the art, eg, by membrane-forming hydration using polymeric materials such as phospholipids or polymers.
  • the immunogenic substance that can be used in the present invention is a pathogenic antigenic substance that causes an abnormal immune response in the body, preferably a hypersensitivity reaction, an autoimmune disease or a pathogenic antigenic substance of transplant rejection, such as Chicken ovalbumin, protein lipoprotein (PLP) polypeptide, Dby polypeptide, Uty, myelin basic protein, denatured IgG, thyroglobulin, thyrotropin receptor, histone, acetylcholine receptor, major histocompatibility antigen ( Including HLA-I and HLA-II), minor histocompatibility antigens, blood group antigens, tissue-specific antigens (such as vascular endothelial cell antigen, skin SK antigen).
  • PLP protein lipoprotein
  • Dby polypeptide Uty
  • myelin basic protein denatured IgG
  • thyroglobulin thyrotropin receptor
  • histone acetylcholine receptor
  • the folic acid-modified immunogenic substance of the present invention can be prepared by forming a complex between folic acid and the immunogenic substance via a chemical bond or a linker.
  • the folate-modified immunogenic substance may be a folate-modified biomacromolecular active agent.
  • Administration of biomacromolecule active agents, especially long-term administration, is prone to the development of anti-drug antibodies and/or drug resistance.
  • the biomacromolecular drugs include, but are not limited to, those defined above.
  • the folic acid-modified biomacromolecule active agent of the present invention is folic acid-modified human albumin, folic acid-modified insulin, folic acid-modified adalimumab, folic acid-modified infliximab, folic acid-modified Ate folic acid-modified sintilimab, folic acid-modified toripalimab, or folic acid-modified trastuzumab.
  • Folic acid modification can reduce the production of anti-drug antibodies of biomacromolecular active agents.
  • the folic acid-modified immunogenic substances of the present invention are folic acid-modified pathogenic antigenic substances that cause an abnormal immune response in the body, such as those as defined above.
  • the method of modifying an immunogenic substance with folic acid can be carried out according to methods known in the art or as exemplified in the Examples below.
  • it can be prepared by condensing folic acid with biological macromolecules such as polypeptides or proteins by direct chemical modification (eg, amide condensation), bifunctional bridging groups, or solid phase synthesis.
  • the folate-modified immunogenic substances of the present invention can be used to reduce the production of antibodies against the immunogenic substances and/or can be used to treat or prevent diseases or disorders that can be mediated by B cell immune tolerance.
  • the folic acid-modified antitumor drug of the present invention can be prepared by forming a complex between folic acid and the antitumor drug via a chemical bond or a linker.
  • the antineoplastic agents that can be used in the present invention are preferably those that can be used for the treatment or prevention of mIgM-positive B cell lymphoma.
  • the antitumor drugs that can be used in the present invention include, but are not limited to, anthracycline drugs, such as doxorubicin or epirubicin; taxane drugs, such as paclitaxel, docetaxel or cabazitaxel; camptothecin drugs, Examples include camptothecin, hydroxycamptothecin, 9-nitrocamptothecin or irinotecan; vinblastine drugs such as vincristine or vinorelbine; proteasome inhibitors such as bortezomib or carfilzomib ; Lactone drugs such as parthenolide; cyclophosphamide, etoposide, gemcitabine, cytarabine, 5-fluorouracil, teniposide, moritinib, epot
  • the preparation method of the folic acid-modified antitumor drug of the present invention is known in the art or exemplified in the Examples.
  • the folic acid-modified antitumor drug of the present invention can be prepared as follows: (a) For a drug containing a boronic acid group, such as bortezomib, folic acid can be modified with dopamine and then reacted with the boronic acid group of the drug to form a drug containing a boronic acid group.
  • Folic acid complexes of pH-sensitive boronate esters (b) For drugs containing ketone or aldehyde groups, such as anthracycline drugs such as doxorubicin, epirubicin, etc., sulfhydryl groups can be introduced into folic acid, and then combined with the drugs.
  • drugs containing ketone or aldehyde groups such as anthracycline drugs such as doxorubicin, epirubicin, etc.
  • sulfhydryl groups can be introduced into folic acid, and then combined with the drugs.
  • the folic acid-modified antitumor drug of the present invention can be used for targeted therapy or prevention of mIgM-positive B-cell lymphoma.
  • the folic acid-modified probe of the present invention can be prepared by forming a complex between folic acid and the probe via a chemical bond or a linker.
  • Probes useful in the present invention may be contrast agents, including but not limited to fluorescent substances such as fluorescein, carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), hexachlorofluorescein (HEX), coumarin 6, near-infrared dyes Cy5, Cy5.5, Cy7, ICG, IR820 , DiR, DiD, etc.; and radioactive substances, such as magnetic resonance contrast agents such as Gd-DTPA or radio contrast agents such as 99mTc-DTPA, etc.
  • the probes that can be used in the present invention are carboxyfluorescein (FAM), near-infrared dyes Cy5, Cy5.5, ICG, IR820, DiR, DiD or Gd-DTPA and the like.
  • the folate-modified probes of the present invention can be prepared by amide condensation, functionalized derivatization, chelation, and the like.
  • the folic acid-modified probe of the present invention can be used for targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphoma.
  • folic acid free or in the form of a pharmaceutically acceptable salt or ester or conjugate
  • Administration of folic acid in combination with an immunogenic substance can reduce the production of antibodies against the immunogenic substance.
  • the immunogenic substance is preferably a biomacromolecular active agent, such as those described above.
  • the immunogenic substance is an immunogenic delivery system, such as a microcarrier delivery system, preferably liposomes, microspheres, microcapsules, nanoparticles, nanocapsules, lipids Nanodiscs or polymeric micelles.
  • the drug delivery system can be loaded with any suitable drug, such as a biomacromolecular active agent as described herein, an antineoplastic drug, an antibacterial drug, a hormone, and the like.
  • the immunogenic substance is a drug delivery system loaded with a biomacromolecule active agent.
  • the administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof and the administration of the immunogenic substance can be performed simultaneously, sequentially or alternately.
  • folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof is administered prior to and/or during administration of the immunogenic substance.
  • administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof precedes administration of the immunogenic substance (eg 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 prior to days) and lasts for years, months, weeks, or days.
  • the administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof continues until such as 3 months, 2 months, 1 month, 3 weeks, 2 weeks or 1 week after the end of administration of the immunogenic substance, This depends on properties such as metabolism or half-life of the immunogenic substance and can be routinely judged by the participating physician. More preferably, the administration of folic acid, or a pharmaceutically acceptable salt or ester or conjugate thereof, and the administration of the immunogenic substance are performed simultaneously.
  • the routes of administration of folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof and the immunogenic substance may be the same or different.
  • folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof can be administered orally, by inhalation, injection, etc.
  • the immunogenic substance can be the same as folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof or different routes of administration.
  • folic acid, or a pharmaceutically acceptable salt or ester or conjugate thereof, and the immunogenic substance may be included in the same pharmaceutical composition, or in separate pharmaceutical compositions.
  • folic acid, or a pharmaceutically acceptable salt or ester or conjugate thereof, and the immunogenic substance may be presented as a combination product, such as a kit.
  • the agents of the present invention may be administered by any suitable route in the art, including but not limited to oral, parenteral such as subcutaneous, intramuscular, intravenous, intraperitoneal, inhalation, intranasal, rectal, transdermal, implant, topical Wait.
  • the agents of the present invention are administered by injection, eg, continuous infusion, subcutaneous injection, intramuscular injection, or intravenous injection.
  • the substances of the present invention may be administered orally.
  • the substances of the present invention may be administered by inhalation.
  • the substances of the present invention can also be administered via an implanted device.
  • the substances of the present invention can be prepared into pharmaceutical preparations or pharmaceutical compositions by any suitable operation in the art.
  • Pharmaceutical formulations or compositions can be in solid form (including but not limited to tablets, lozenges, coated tablets, capsules, pills, granules, cachets, lyophilisates, powders or suppositories, etc.), liquid forms (including but not limited to solutions, suspensions or emulsions, etc.) or semi-solid forms (including but not limited to creams, ointments, pastes, gels, etc.), for any suitable route in the art, such as oral administration , parenteral such as subcutaneous, intramuscular, intravenous, intraperitoneal, inhalation or nasal, rectal, transdermal, implant, topical administration and the like.
  • compositions contain an effective amount of a substance of the present invention and optionally one or more pharmaceutically acceptable excipients, such as diluents, isotonic agents, buffers, preservatives, stabilizers, wetting agents, emulsifiers, lubricants etc, as long as they are compatible.
  • pharmaceutical formulation or pharmaceutical composition may also be subjected to any suitable conventional pharmaceutical procedures, such as granulation, tableting, lyophilization, sterilization, and the like.
  • the pharmaceutical composition or formulation is in a form suitable for oral administration, eg, tablet, coated tablet, lozenge, capsule (soft or hard), powder, granule, cachet, emulsion , aqueous or oily suspensions, syrups or elixirs, etc.
  • the pharmaceutical composition or formulation is in a form suitable for injectable administration, including but not limited to sterile aqueous solutions (where soluble in water) or dispersions and for the extemporaneous preparation of sterile injectable solutions or Sterile powders for dispersion, preferably aqueous isotonic solutions or suspensions.
  • the pharmaceutical composition or formulation is for continuous infusion, subcutaneous injection, intramuscular injection or intravenous injection.
  • Formulation components and methods suitable for administration by injection are known in the art and include, for example, physiological saline, stabilizers, buffers, isotonic agents, and the like.
  • the composition should be sterile and fluid to the extent that it is easy to inject immediately before use.
  • the pH of the formulation is generally about 3 to 11, more preferably about 5 to 9 or 6 to 8, most preferably about 7 to 8, eg about 7 to 7.5.
  • the pharmaceutical composition or formulation is in a form suitable for inhalation or nasal administration. They are conveniently delivered as an aerosol or spray from a pressurized container, pump, nebulizer, nebulizer or diffuser, with or without a suitable propellant, or as a dry powder (alone or as a mixture) , such as a dry blend with lactose) is delivered by a dry powder inhaler.
  • a dry powder alone or as a mixture
  • lactose a dry blend with lactose
  • an aerosol in the case of an aerosol, it preferably comprises, for example, a hydrofluoroalkane (HFA) propellant, such as dichlorodifluoromethane, HFA134a or HFA227, or any mixture thereof, and may comprise one or more known in the art known co-solvents (eg ethanol) and/or one or more surfactants (eg oleic acid or sorbitan trioleate) and/or one or more fillers (eg lactose).
  • HFA hydrofluoroalkane
  • the pharmaceutical composition or formulation is a spray, it preferably contains the active agent, eg dissolved or suspended in a vehicle containing water, a cosolvent such as ethanol or propylene glycol, and a stabilizer (which may be a surfactant).
  • the pharmaceutical composition or formulation when it is a dry powder formulation, it preferably contains, for example, the active agent having a particle size of up to 10 microns, optionally also containing a diluent or carrier (eg lactose) having the desired particle size distribution and to help prevent moisture induced Compounds that reduce product performance (such as magnesium stearate).
  • a diluent or carrier eg lactose
  • the pharmaceutical composition or formulation is in a form suitable for transdermal or topical administration, including powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhaler.
  • the materials of the present application can be prepared by mixing under sterile conditions a pharmaceutically acceptable carrier and any adjuvants that may be required such as penetration enhancers, preservatives or buffers and the like.
  • the agents of the invention may be administered intermittently or continuously, eg, via an implanted device or catheter, for a day or days or longer, eg, weeks, months, or years, eg, at least about 3 days, at least About 1 week, at least about 2 weeks, or at least 1 month.
  • the substances of the present invention are generally effective over a wide dosage range.
  • the daily dose may be about 0.01-2000 mg, preferably about 1-1000 mg, more preferably about 2-100 mg, and most preferably about 5-80 mg.
  • dosages below the lower limit of the above-mentioned dosage ranges may be employed, while in other cases dosages above the upper limit of the above-mentioned dosage ranges may be employed. Accordingly, the above dosage ranges are not intended to limit the scope of the invention in any way.
  • the actual dosage to be administered will be determined by the physician in consideration of the relevant circumstances, including the condition to be treated, the route of administration chosen, the active agent(s) actually administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms. sex to determine.
  • the substances of the present application may be administered as a single dose or as two or more doses (each dose may be the same or different).
  • the pharmaceutical formulations or compositions of the present invention may also contain other active agents, so long as they are compatible.
  • the other active agents may have the same or different pharmaceutical activity as the substances of the present application.
  • the substances of the present invention can be used for diagnosis, treatment and/or prevention.
  • the substance of the present invention can specifically bind to mIgM on the surface of B cells and target B cells expressing mIgM, especially B cells with high mIgM expression, causing B cell anergy and inducing B cell immune tolerance.
  • the substances of the invention are useful for inducing B cell immune tolerance, in particular for reducing the production of antibodies against immunogenic substances and/or for treatment or prophylaxis that may be mediated through B cell immune tolerance diseases or conditions, including but not limited to hypersensitivity reactions, autoimmune diseases or transplant rejection.
  • the B cells are mIgM expressing B cells, particularly mIgM highly expressing B cells, more particularly spleen B cells or lymph node B cells.
  • the substances of the present invention can be used to reduce biomacromolecular active agents such as polypeptides or proteins such as antibody drugs anti-drug antibodies and/or drug resistance.
  • biomacromolecular active agents such as polypeptides or proteins such as antibody drugs anti-drug antibodies and/or drug resistance.
  • Biomacromolecule drugs have potential immunogenicity, especially when they are used for a long time, they are prone to produce anti-drug antibodies (ADA).
  • B cell immune tolerance can be induced to make the body tolerant to the biomacromolecule , effectively reducing the production of anti-drug antibodies and improving the efficacy and safety.
  • chronic administration of adalimumab causes the body to produce antibodies against adalimumab.
  • the anti-drug antibody rapidly neutralizes adalimumab, resulting in a decrease in drug concentration, inactivity, and induction of immune side effects.
  • the folic acid-modified adalimumab is administered, the body can tolerate the adalimumab, which effectively reduces the production of anti-drug antibodies and improves the efficacy and safety.
  • the agents of the invention can be used to reduce the production of antibodies against an immunogenic drug delivery system.
  • liposomes are commonly used drug delivery systems, however, repeated injections of liposomes can cause the body to produce antibodies against liposomes, resulting in rapid clearance of liposomes from the body.
  • folic acid or its pharmaceutically acceptable salts or esters or conjugates can be effectively reduced and the exposure of liposomes to the body can be increased.
  • the agents of the invention may be used to treat or prevent hypersensitivity reactions, for example, by administering folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof, or folic acid-modified hypersensitivity causative antigenic agents such as polypeptides or protein.
  • the hypersensitivity reactions include, but are not limited to, anaphylactic shock, respiratory hypersensitivity reactions (eg, allergic asthma or allergic rhinitis), gastrointestinal hypersensitivity reactions, and the like.
  • the substances of the present invention may be used to treat or prevent autoimmune diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, Hashimoto's thyroiditis, toxic diffuse goiter, ankylosing spine inflammation, autoimmune encephalomyelitis, neuromyelitis optica spectrum disorders, anticardiolipin syndrome, hemophilia, psoriasis, etc., for example, by administering folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof or folic acid-modified
  • autoimmune diseases such as polypeptides or proteins.
  • the causative antigenic substances of autoimmune diseases include, but are not limited to, PLP polypeptides, myelin basic protein, denatured IgG, thyroglobulin, thyrotropin receptors, histones, and acetylcholine receptors.
  • the agents of the invention may be used to treat or prevent transplant rejection, eg, by administering folic acid, or a pharmaceutically acceptable salt or ester or conjugate thereof, or a folic acid-modified antigenic agent of transplant rejection.
  • folic acid or a pharmaceutically acceptable salt or ester or conjugate thereof
  • WMHHNMDLI folic acid-modified antigenic agent of transplant rejection.
  • Dby NAGFNSNRANSSRSS
  • Uty WMHHNMDLI
  • Pre-stimulation of recipient mice with folic acid-modified Dby or Uty to induce immune tolerance can increase the success rate of bone marrow transplantation in donor mice.
  • the transplant rejection includes organ transplant rejection (eg, but not limited to, suppressed rejection of the heart, liver, spleen, lung, kidney), tissue transplant rejection (eg, bone marrow transplant rejection), and recurrent miscarriage.
  • organ transplant rejection eg, but not limited to, suppressed rejection of the heart, liver, spleen, lung, kidney
  • tissue transplant rejection eg, bone marrow transplant rejection
  • recurrent miscarriage e.g, organ transplant rejection (eg, but not limited to, suppressed rejection of the heart, liver, spleen, lung, kidney), tissue transplant rejection (eg, bone marrow transplant rejection), and recurrent miscarriage.
  • the causative antigenic substances of transplant rejection include but are not limited to major histocompatibility antigens (including HLA-I, HLA-II), minor histocompatibility antigens, blood group antigens, tissue-specific antigens (such as vascular endothelial cells) antigen, skin SK anti
  • the substances of the present invention can also be used for targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphomas.
  • the substance of the present invention can target mIgM-positive B-cell lymphoma to achieve targeted diagnosis, treatment or prevention of mIgM-positive B-cell lymphoma .
  • FIG. 1 provides the NMR spectrum of the folic acid-OVA complex of Example 1.
  • Figure 2 provides a quantitative standard curve for folic acid molecules.
  • FIG. 3 provides the NMR spectrum of the folic acid-PEG 2000 -Cy5 of Example 3.
  • Figure 4 provides the binding mode of folic acid and mouse mIgM in Example 4 (a: binding site of folic acid and mIgM; b: analysis of the binding force of folic acid and mIgM).
  • Figure 5 provides the flow analysis of the binding of folic acid to mouse mIgM in Example 4 (a: the effect of anti-IgM antibody to eliminate mIgM on the surface of B cells; b: the results of the uptake of folic acid and B cells under different anti-IgM antibody concentrations; c: Linear fitting curve of B cell uptake of folic acid and mIgM expression level).
  • Figure 6 provides confocal images of folic acid binding to mouse mIgM in Example 4.
  • Figure 7 provides the distribution in the spleen of FA-PEG2000-Cy5 or PEG2000 -Cy5 in Example 5 at 1 h (a) and 4 h (b) after tail vein injection. (*p ⁇ 0.05, **p ⁇ 0.01, T-test)
  • Figure 8 provides flow sorting plots (a: 1 h; c: 4 h) and statistical analysis (b) of spleen lymphocytes at 1 h and 4 h after tail vein injection of FA-PEG 2000 -Cy5 or PEG 2000 -Cy5 in Example 5 :1h; d:4h). (ns: no statistical difference, ***p ⁇ 0.001, T-test)
  • Figure 9 provides immunofluorescence photographs of the distribution in the spleen of FA-PEG 2000 -Cy5 or PEG 2000 -Cy5 in Example 5 at 1 h after tail vein injection.
  • FIG. 10 provides immunofluorescence photographs of binding to IgM-positive marginal B cells in the spleen at 1 h after tail vein injection of FA-PEG 2000 -Cy5 in Example 5.
  • FIG. 10 provides immunofluorescence photographs of binding to IgM-positive marginal B cells in the spleen at 1 h after tail vein injection of FA-PEG 2000 -Cy5 in Example 5.
  • Figure 11 provides the results of B cell proliferation (a, b) and differentiation (c, d) analyzed by flow cytometry after the mice in Example 6 were stimulated three times with FA-OVA, OVA and saline, respectively. (*p ⁇ 0.05, ***p ⁇ 0.001, one-way ANOVA)
  • Figure 12 provides the serum OVA-specific antibody expression (a: IgE; b: IgG; c: IgG1; d: IgG2a) of the mice in Example 6 after three stimulations with FA-OVA, OVA and saline, respectively.
  • Figure 13 provides the serum FVIII-specific antibody expression of the mice in Example 6 after three stimulations with FVIII, folic acid-FVIII and normal saline, respectively.
  • Figure 14 provides the expression of OVA-specific IgG antibody in mouse serum after oral administration of folic acid in Example 7 (a: day 14; b: day 21; c: day 28).
  • Figure 15 provides the expression of KLH-specific IgG antibody in mouse serum after oral administration of folic acid in Example 7 (a: day 21; b: day 28; c: day 35).
  • Figure 16 provides the serum-specific antibody expression of the mice after stimulation in Example 8 (a: adalimumab as a model mAb; b: infliximab as a model mAb; c: trastuzumab as a model mAb).
  • Figure 17 provides serum sLip-specific antibody expression after stimulation with sLip, sLip+FA (10 ⁇ g), sLip+FA (50 ⁇ g) and normal saline in mice in Example 9, respectively.
  • FIG. 18 provides the temperature change curves of mice treated with FA-OVA, OVA and saline in Example 10 after re-stimulation with OVA.
  • Figure 19 provides serum OVA-specific antibody levels (a: IgE; b: IgGi; c : IgG2a ) of mice treated with FA-OVA, OVA and saline in Example 10 after re-stimulation with OVA.
  • Figure 20 provides the body temperature change curve of the sensitized mice in Example 11 after being treated with FA-OVA, OVA and normal saline, respectively, after being stimulated with OVA again. (ns: no statistical difference, **p ⁇ 0.01, ***p ⁇ 0.001, one-way ANOVA)
  • Figure 21 provides serum OVA-specific antibody levels (a: IgE; b: IgG 2a ; c: IgG 1 ) after re-stimulation with OVA in sensitized mice treated with FA-OVA, OVA, and saline, respectively, in Example 11 ). (ns: no statistical difference, ***p ⁇ 0.001, one-way ANOVA)
  • EDC carbodiimide
  • FIG. 1 provides the obtained H NMR spectrum of the folic acid-OVA complex.
  • the signals ⁇ 7.45 and ⁇ 6.45 are the signal peaks of a group of symmetrical "AA'BB'" spin systems, so it can be determined that the signal is
  • the two sets of proton signal peaks on the para-disubstituted benzene ring in the folic acid molecule indicate that the molecule contains folic acid structure, and the product structure is further determined to be folic acid-OVA.
  • the OD 365nm of the obtained folic acid complex was measured by UV spectrophotometer.
  • Figure 2 provides a standard curve used to quantify the degree of folate modification. As shown in Table 1, the degree of modification of folic acid in folic acid-OVA was calculated to be about 8.66. The degree of folic acid modification indicates the ratio of folic acid to folic acid complex in the folic acid complex.
  • folic acid-modified protein complex was synthesized by taking folic acid-coagulation factor VIII (FA-FVIII) as an example.
  • Folic acid-monoclonal antibody complexes were prepared by taking folic acid-adalimumab complex (FA-Adalimumab) and folic acid-trastuzumab complex (FA-Trastuzumab) as examples.
  • a 1 mg/mL solution of each mAb in PBS was prepared, and tris(2-carboxyethyl)phosphine and EDTA were added to the concentrations of 10 mM and 5 mM, respectively. The reaction was shaken at room temperature for 90 minutes.
  • the reaction solution was mixed with 15 equivalents of folic acid-PEG 400 -Mal (folic acid-PEG 400 -maleimide, purchased from Shanghai Yisheng Biotechnology Co., Ltd.), and the reaction was shaken at room temperature for 20 minutes. To block unreacted sulfhydryl groups, the solution was further reacted with 15 equivalents of maleimide with shaking at room temperature for 20 minutes. Subsequently, the reaction solution was dialyzed against PBS for 72 hours to remove small molecules in the system to obtain each folic acid-monoclonal antibody complex solution. Table 1 provides the degree of modification of folate in the folate-mAb complex.
  • Table 1 Results of the degree of folic acid modification in each folic acid-modified biomacromolecule prepared in Example 1-2
  • Folic acid-contrast agent complexes were prepared by taking folic acid-PEG 2000 -Cy5 (ie FA-PEG 2000 -Cy5) as an example. Weigh 0.57 g (1.28 mmol) of folic acid, 0.37 g (2.8 mmol) of NHS, and 0.6 g (2.8 mmol) of DCC into a 250 mL round-bottom flask, and add 75 mL of DMSO. The reaction was carried out under nitrogen protection at 25°C.
  • FIG. 3 is the hydrogen NMR spectrum of the obtained FA-PEG 2000 -Cy5.
  • the signals ⁇ 7.65 and ⁇ 6.65 are a group of signal peaks of a symmetrical "AA'BB ⁇ " spin system, which can be determined to be two sets of proton signal peaks on the para-disubstituted benzene ring in the folic acid molecule, indicating that The molecule contains folic acid structure.
  • the signal ⁇ 3.51 was a typical PEG signal peak, and the product structure was further determined to be FA-PEG 2000 -Cy5.
  • Example 4 Specific binding of folic acid to mIgM on the surface of B cells
  • FIG. 4a shows the binding mode of folic acid to mouse mIgM. It can be seen that the binding pocket of folic acid to mIgM is located between C ⁇ 3 and C ⁇ 4 of the Fc fragment.
  • Figure 4b further provides a force analysis of the binding of folic acid to mouse IgM monomer, where the binding pocket of folic acid is located at the junction of the two heavy chains (chain A and chain B) of the IgM monomer, and the pteridine fragment in the structure of folic acid can Forms hydrogen bonds with Phe287, Tyr288 and Pro285 in chain A and Glu52 in chain B; Phe55 in chain B forms ⁇ - ⁇ interactions with the benzene fragment of folic acid; Lys25 in chain B forms ionic interactions with carboxyl groups; folic acid
  • the predicted binding energy to the Fc fragment of IgM is -10.929 kcal/mol, and the affinity reaches the level of 10 -8 mol.
  • the binding of folic acid to mIgM was examined using mIgM-positive spleen B cells.
  • the spleen lymphocytes of BALB/c mice were extracted with mouse spleen lymphocyte separation solution (purchased from Solebao Technology Co., Ltd.), counted to 2 ⁇ 10 6 /mL, and plated in 24-well culture plates (1 mL/well).
  • Anti-IgM (ab97230, 1 mg/mL) was added to each well to make the working concentrations 0, 2.5, 5, and 12.5 ⁇ g/mL.
  • FIG. 5 provides the results of a flow cytometry analysis of folic acid binding to mIgM.
  • Figure 5a shows that with the increase of anti-IgM antibody concentration, the mIgM of mouse spleen B cells can be effectively depleted.
  • Figure 5b showed that the uptake of FA-PEG-Cy5 gradually decreased with increasing anti-IgM concentration, while the uptake of PEG-Cy5 had little effect.
  • bovine serum albumin (5%) was incubated with cells at room temperature for 1 hour and then with PEG 2000 -Cy5 (purchased from Xi'an Ruixi Biological Co., Ltd.) for 1 hour at room temperature. B cells were screened with CD19 marker, and the binding of folic acid to mIgM was observed by laser confocal microscopy.
  • Figure 6 provides a confocal laser photomicrograph of folic acid binding to mIgM (Carl Zeiss LSM710, Germany), which shows that B cells bind folic acid (FA-PEG 2000 -Cy5) much higher than PEG 2000 -Cy5, and the binding can be Inhibited by anti-IgM antibodies and free folic acid molecules, not affected by serum complement proteins, ie, independent of complement receptors.
  • mIgM Carl Zeiss LSM710, Germany
  • Example 5 Evaluation of B-cell targeting of splenic marginal region with folic acid
  • mice Male, 6 weeks old, Slack
  • folic acid-contrast agent complex FA-PEG 2000 -Cy5 prepared according to Example 3
  • PEG 2000 -Cy5 as a control
  • 100nmol/a three in each group.
  • Mice were sacrificed after 1 hour and 4 hours, respectively, and the spleen was isolated.
  • tissue homogenization according to the fluorescence standard curve of FA-PEG 2000 -Cy5 or PEG 2000 -Cy5, the concentration of folic acid in the spleen homogenate was determined by a fluorescence microplate reader.
  • Figure 7 shows that the spleen accumulation of FA-PEG 2000 -Cy5 and PEG 2000 -Cy5 was 9.35 ⁇ 2.10 nmol/g tissue and 4.57 ⁇ 1.19 nmol/g tissue, respectively, 1 hour after injection; 4 hours after injection, FA-PEG The spleen accumulation of 2000 -Cy5 and PEG 2000 -Cy5 was 10.20 ⁇ 2.44 nmol/g tissue and 7.07 ⁇ 1.44 nmol/g tissue, respectively. The accumulation of FA-PEG 2000 -Cy5 in the spleen was significantly higher than that of the control PEG 2000 -Cy5, suggesting the spleen targeting ability of FA-PEG 2000 -Cy5.
  • FIG. 8 shows flow sorting plots (a: 1 h; c: 4 h) and statistical analysis (b: 4 h) of mouse spleen lymphocytes 1 hour or 4 hours after administration of FA-PEG 2000 -Cy5 or PEG 2000 -Cy5, respectively 1h; d: 4h).
  • the results showed that folic acid has a significant B cell targeting ability.
  • FIG. 9 shows immunofluorescence photographs of the distribution of FA-PEG 2000 -Cy5(a) and PEG 2000 -Cy5(b) in the spleen, with DAPI-labeled nuclei in blue and CD19-PE antibody (553786, BD Pharmingen) in green ) labeled B cells, red is Cy5, and yellow is the overlapping color of green and red.
  • folic acid was mainly distributed in the marginal region of the spleen, with obvious overlap with marginal B cells, that is, folic acid targeted to marginal B cells of the spleen.
  • FIG. 10 shows the immunofluorescence photographs of the distribution of FA-PEG 2000 -Cy5 in marginal B cells of the spleen, in which blue is DAPI-labeled nuclei, green is anti-IgM-FITC-labeled marginal B cells, red is Cy5, and yellow is green Overlapping color with red. The results showed that folic acid has significant overlap with marginal B cells, that is, folic acid targets marginal B cells of the spleen.
  • Example 6 The role of folic acid modification in mediating humoral immunosuppression and reducing antidrug antibodies of protein biomacromolecule drugs
  • OVA ovalbumin
  • ADA anti-drug antibodies
  • FIG. 11a and 11b provide CD86 flow sorting plots and statistical analysis of mouse spleen lymphocytes, respectively.
  • Figures 11c and 11d provide GL7 flow-sorting plots and statistical analysis of mouse spleen lymphocytes, respectively.
  • the B cells of the OVA group of mice proliferated significantly and differentiated into germinal center B cells, while in the FA-OVA group of mice, B cell proliferation and differentiation into germinal center B cells were significantly inhibited, indicating that The efficacy of folic acid modification in inhibiting B cell proliferation and differentiation, resulting in B cell anergy.
  • mice whole blood of mice was collected one week after the last administration for routine blood analysis.
  • the results showed that there was no obvious abnormality in the blood routine indexes of the mice in each group, indicating that the folic acid modification did not produce obvious side effects.
  • the data are shown in Table 2 below.
  • folic acid modification in reducing anti-drug antibodies (ADA) of protein-based biomacromolecular drugs was studied using coagulation factor VIII (FVIII) as a model protein.
  • FVIII coagulation factor VIII
  • BALB/c female mice (5 weeks old) were randomly divided into FVIII group, FA-FVIII group and normal saline control group, 5 mice in each group.
  • FVIII, FA-FVIII (prepared according to Example 1) and normal saline were administered by tail vein injection at a dose of 0.5 IU/mouse, once a week, three times in total.
  • mouse serum was collected and the expression of FVIII-specific antibodies in the serum was determined by ELISA.
  • the results in Figure 13 show that the antibody level of the FA-FVIII group was significantly lower than that of the FVIII group, indicating that folic acid modification effectively reduced the production of anti-drug antibodies of protein drugs.
  • Example 7 Effects of folic acid on humoral immunosuppression
  • BALB/c female mice (5-week-old) were randomly divided into 4 groups, 6 mice in each group, and were treated as follows: (1) FA(ig) group: each mouse was given 3 mg of free folic acid daily for 2 days. Weeks; (2) FA(ig)+OVA group: each mouse was intragastrically administered 3 mg of free folic acid every day for two weeks, and OVA (10 ⁇ g/mice) was injected into the tail vein on days 0 and 7; (3) OVA group: each mouse was injected with OVA (10 ⁇ g/mice) through the tail vein on the 0th and 7th day, respectively; (4) Normal saline group: each mouse was injected with physiological saline on the 0th and 7th day, respectively. brine.
  • mice were injected with 100ul of mixed solution of OVA and KLH (hemocyanin) in water (containing 100ug/mL OVA and 100ug/mL KLH) through tail vein, respectively.
  • Serum was collected on days 14, 21 and 28 before administration of the mixed solution and on day 35, and specific antibodies in the sera were detected by ELISA.
  • Figure 14 shows OVA-specific antibody levels in mouse serum at day 14 (a), day 21 (b), and day 28 (c), respectively.
  • the results showed that on the 14th and 21st days, the serum OVA-specific IgG level of mice in the FA(ig)+OVA group was significantly lower than that in the OVA group, indicating that folic acid produced a humoral immunosuppressive effect, and the combined administration of folic acid and OVA could reduce the OVA-specific antibody production.
  • the serum OVA-specific IgG level of the mice in the FA(ig)+OVA group was comparable to that in the OVA group, indicating that the immunosuppressive effect of folic acid is reversible and non-persistent. After stopping folic acid administration, the body can restore its immune function.
  • Figure 15 shows KLH-specific antibody levels in mouse serum at day 21 (a), day 28 (b), and day 35 (c), respectively.
  • the results showed that the levels of KLH-specific antibodies in mice in the FA(ig) group and the normal saline group were similar, and both increased with the number of injections.
  • the results also proved that after stopping the administration of folic acid, the immune function of the body was restored.
  • Example 8 Efficacy of folic acid and folic acid modification in reducing antidrug antibody production of antibody-based biomacromolecules
  • Adalimumab and Infliximab are humanized monoclonal antibodies against human tumor necrosis factor (TNF), and Trastuzumab is an anti-human epidermal growth factor receptor- 2 (HER2) humanized monoclonal antibody. Their repeated injections are easy to induce the body to produce antibodies against the monoclonal antibody, which affects the efficacy of the monoclonal antibody.
  • Adalimumab, infliximab and trastuzumab were used as model mAbs to investigate the effect of folic acid and folic acid modification on reducing the anti-drug antibodies of antibody drugs.
  • mice BALB/c female mice (6 weeks old) were randomly divided into adalimumab group, FA-adalimumab group (prepared according to Example 2) and normal saline group, with 5 mice in each group.
  • Adalimumab group and FA-adalimumab group were administered subcutaneously at a dose of 30 ⁇ g antibody/mouse on days 0, 7 and 14.
  • mice were subcutaneously injected with saline on days 0, 7 and 14.
  • One week after the last administration (day 21) mouse sera were collected, and antibodies against adalimumab were detected in the sera by ELISA.
  • the results are shown in Figure 16a. The results showed that the antibody level of the FA-adalimumab group was significantly lower than that of the adalimumab group, indicating that folic acid modification effectively reduced the production of anti-drug antibodies of the antibody drug.
  • mice BALB/c female mice (6 weeks old) were randomly divided into infliximab group, FA gavage + infliximab group and normal saline group, with 5 mice in each group.
  • doses of 60 ⁇ g antibody/mouse were administered via tail vein injection on days 0, 7 and 14.
  • FA gavage + infliximab group each mouse was gavaged with 3 mg of free folic acid per day for two weeks and injected via tail vein at a dose of 60 ⁇ g antibody/mouse on days 0, 7 and 14 Infliximab.
  • mice were injected with saline via the tail vein on days 0, 7 and 14.
  • BALB/c female mice (6 weeks old) were randomly divided into trastuzumab group, folic acid-trastuzumab group (prepared according to Example 2), folic acid gavage + trastuzumab group and Normal saline group, 5 animals in each group.
  • trastuzumab group and the folic acid-trastuzumab group doses of 60 ⁇ g antibody/mouse were administered via tail vein injection on days 0, 7 and 14.
  • folic acid gavage + intrastuzumab group each mouse was gavaged with 3 mg of free folic acid per day for two weeks and via the tail vein at a dose of 60 ⁇ g antibody/mouse on days 0, 7 and 14 Inject trastuzumab.
  • mice were injected with saline via the tail vein on days 0, 7 and 14. One week after the last administration (day 21), mouse sera were collected, and antibodies against trastuzumab were detected in the sera by ELISA. The results are shown in Figure 16c. The results showed that the antibody levels in the folic acid gavage + trastuzumab group and the folic acid-trastuzumab group were significantly lower than those in the trastuzumab group, indicating that folic acid and folic acid modification effectively reduced the production of anti-drug antibodies of antibody drugs.
  • Example 9 Efficacy of folic acid in reducing antibody production in anti-drug delivery systems
  • Liposomes are first prepared. HSPC, Chol, mPEG 2000 -DSPE (all purchased from Aiwei Tuo (Shanghai) Pharmaceutical Technology Co., Ltd.) were weighed, and the molar ratio was 52:43:5. The membrane material was dissolved in 5 mL of chloroform, and the chloroform was removed by rotary evaporation under reduced pressure to obtain a uniform lipid membrane. Residual organic solvent was removed by vacuum drying overnight. Shake in a water bath and shaker at 60°C until the liposome membrane is completely hydrated to obtain a white liposome suspension. The liposomal suspension was sequentially extruded through 200 and 100 nuclear pore membranes using a micro-extruder, resulting in a light blue opalescent liquid, ie, a liposome (sLip) solution.
  • mice BALB/c female mice (6 weeks old) were randomly divided into sLip group, sLip+FA (10 ⁇ g) group, sLip+FA (50 ⁇ g) group and normal saline group, with 4 mice in each group.
  • sLip group a liposome solution of 5 mg liposome/kg mouse body weight was administered through the tail vein; for the mice in the sLip+FA (10 ⁇ g) group, 5 mg of lipid was administered through the tail vein at one time.
  • Example 10 Efficacy of folic acid modification in the prevention of anaphylactic shock
  • mice Five-week-old BALB/c female mice were randomly divided into FA-OVA group, OVA group and normal saline group, 10 mice in each group. Mice were administered FA-OVA or OVA or an equal volume of normal saline by tail vein injection at a dose of 10 ⁇ g OVA/mouse, once a week, three times in total. One week after the last administration, 10 ⁇ g of OVA (allergen) was injected into the tail vein of each mouse. Changes in mouse body temperature within 2 hours after OVA stimulation were recorded ( Figure 18). At the same time, the mouse serum was collected, and the expression of OVA-specific antibody in the serum was determined by ELISA method (Fig. 19).
  • OVA allergen
  • Figure 18 shows that the body temperature of the mice in the FA-OVA group did not change significantly, which was basically the same as the normal saline group; while the mice in the OVA group developed severe anaphylactic shock, the body temperature dropped significantly, and two mice died.
  • Figure 19 shows that the FA-OVA group is basically the same as the normal saline group, while the OVA group mice produced a large amount of OVA-specific antibodies, indicating that folic acid modification can significantly reduce the antibody production caused by OVA. Therefore, folic acid modification can effectively prevent anaphylactic shock caused by allergens.
  • Example 11 Efficacy of folic acid modification in the treatment of anaphylactic shock
  • mice 5-week-old BALB/c female mice were sensitized by subcutaneous injection of 10 ⁇ g OVA, once a week, three times in total.
  • OVA-sensitized mice were injected with FA-OVA, OVA and normal saline by tail vein at a dose of 4 ⁇ g/mouse, once every two days, three times in total.
  • mice were injected with 25 ⁇ g of OVA (allergen) in the tail vein. Changes in mouse body temperature 2 hours after injection were recorded ( Figure 25).
  • OVA allergen
  • Figure 20 shows the temperature change curve of sensitized mice treated with FA-OVA, OVA and saline, and then stimulated with OVA again.
  • the FA-OVA treatment showed desensitization, and the drop in the body temperature of the mice was controlled and returned to normal within 2 hours.
  • Figure 21 shows serum OVA-specific antibody levels in sensitized mice treated with FA-OVA, OVA and saline, respectively, after re-stimulation with OVA. It can be seen that in the FA-OVA group, the expression of OVA-specific antibodies IgE (a), IgG 1 (b) and IgG 2a (c) was comparable to the saline group and significantly decreased compared to the OVA group, indicating that folic acid Efficacy of modifications in the treatment of antigen allergic diseases.
  • Example 12 Efficacy of folic acid modification in autoimmune encephalomyelitis
  • EAE experimental autoimmune encephalomyelitis
  • mice Female C57/BL6 mice were injected intravenously with PLP polypeptide (amino acid sequence: HSLGKWLGHPDKF) or folic acid-PLP complex (FA-PLP) at a dose of 10 ⁇ g/mouse, once a week, three times in total.
  • PLP polypeptide amino acid sequence: HSLGKWLGHPDKF
  • FA-PLP folic acid-PLP complex
  • emulsions of PLP polypeptides in Freund's complete adjuvant were formulated at a PLP concentration of 1 ⁇ g/ ⁇ L.
  • Four points were taken on both sides of the back of the mice, and 0.05 mL of PLP emulsifier was injected subcutaneously at each point to induce inflammation.
  • 150ng pertussis toxin was injected intraperitoneally 2h and 24h after PLP injection.
  • mice The incidence of mice was observed every day, and the mice were scored according to the 5-point scoring standard (Kono method), and the incidence scoring curve was drawn. The results showed that the modification of PLP with folic acid resulted in the immune tolerance of the body to PLP and helped to reduce the inflammatory response.
  • Example 13 Efficacy of folic acid modification in bone marrow transplant rejection
  • mice were injected intravenously with 5 x 106 cells. After bone marrow transplantation, the mice were bled once a week, and the lymphocyte chimerism was measured by flow cytometry. Pure CD45.1 mice were used as the control group. The measurement indicators were CD45.1/CD45.2 and CD45.1/CD90.2.
  • Example 14 Efficacy of folic acid-human albumin conjugates for reducing the immunogenicity of biological macromolecules and drug delivery systems
  • Folic acid-human serum albumin (FA-HSA, purchased from Xi'an Ruixi Bio-effective Co., Ltd.) was used as the representative of folic acid conjugates, and its effects on reducing biological macromolecular drugs (OVA, trastuzumab) and drug delivery systems (slip) were investigated. ) efficacy in immunogenicity.
  • mice BALB/c female mice (5 weeks old) were randomly divided into OVA group, FA-HSA+OVA group and normal saline control group, with 6 mice in each group.
  • the following treatments were performed respectively: (1) OVA group: each mouse was injected with 10 ⁇ g OVA in the tail vein on the 0th, 7th and 14th days; (2) FA-HSA+OVA group: each mouse was treated with On the 0th, 7th and 14th days, 10 ⁇ g OVA and 10 ⁇ g FA-HSA mixed solution were injected into the tail vein; (3) Normal saline group: each mouse was injected into the tail vein on the 0th day, the 7th day and the 14th day, respectively An equal volume of normal saline was injected. Serum was collected from all mice on day 21, and the titer of OVA-specific antibody (IgG) in serum was detected by ELISA.
  • IgG OVA-specific antibody
  • mice BALB/c female mice (6 weeks old) were randomly divided into sLip group (sLip prepared according to Example 9), sLip+FA-HSA group and normal saline group, with 6 mice in each group.
  • sLip group a liposome solution of 5 mg liposome/kg of mouse body weight was administered through the tail vein at one time; for the mice in the sLip+FA-HSA group, 5 mg of lipid was administered through the tail vein at one time.
  • serum was collected from all mice, and the antibody (IgM) titer against sLip in the serum was detected by ELISA method.
  • Example 15 Efficacy of folic acid conjugates for reducing the immunogenicity of biological macromolecules and drug delivery systems
  • FA-HSA was replaced with folic acid-polyethylene glycol 40kDa (FA-PEG 40kDa , purchased from Xi'an Ruixi Bio-effective Co., Ltd.), to investigate the effect of FA-PEG 40kDa on reducing biological macromolecular drugs (OVA, trastuz Efficacy in the immunogenicity of benzumab) and the delivery system (slip).
  • OVA biological macromolecular drugs
  • the dose of FA-PEG 40KDa was 100 nM per mouse.
  • Injection solutions containing the substances of the present invention can be produced in a conventional manner:
  • the material of the present invention was dissolved in a mixture of polyethylene glycol 400 and water for injection (parts).
  • the pH was adjusted to 5.0 by adding acetic acid. Adjust the volume to 1.0 mL by adding the remaining amount of water.
  • the solution is filtered, appropriately overfilled into vials, and sterilized.
  • Oral tablets containing the following ingredients can be produced in a conventional manner:
  • Oral capsules containing the following ingredients can be produced in a conventional manner:
  • the ingredients are sieved, mixed, and filled into capsule shells.
  • Aerosols containing the following ingredients can be produced in a conventional manner:
  • the substance of the present invention and oleic acid are mixed, filled into an aerosol bottle in divided doses, and then injected with dichlorodifluoromethane under pressure.

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Abstract

叶酸或其可药用盐或酯或偶联物或叶酸修饰的免疫原性物质在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途。诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤的药物组合物和组合产品，含有叶酸或其可药用盐或酯或偶联物或叶酸修饰的免疫原性物质。

Description

叶酸和叶酸修饰在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途 技术领域
概括而言，本发明涉及叶酸和叶酸修饰在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途，特别是用于减少抗免疫原性物质的抗体的产生，用于预防或治疗可通过B细胞免疫耐受介导的疾病或病症(例如超敏反应、自身免疫性疾病或移植排斥)，和/或用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤。本发明还涉及诱导B细胞免疫耐受和靶向治疗、诊断或预防mIgM阳性表达的B细胞淋巴瘤的方法、药物组合物和组合产品。
背景技术
多肽和蛋白质等生物大分子药物在免疫相关疾病的治疗中有着显著效果。但是，这类药物具有潜在的免疫原性，尤其在长期使用时易产生抗药抗体(Anti-Drug Antibody,ADA)，进而中和体内药物，造成药物浓度降低、无活性、疾病复发，严重的还会引起输液性超敏反应等。例如，在阿达木单抗(adalimumab)的I期单剂量研究中，健康受试者的ADA发生率高达70-100％。诱导机体免疫耐受是降低生物大分子药物的ADA和耐药性的有效策略之一。免疫耐受是机体免疫系统接触某种抗原刺激后所表现出的特异性免疫低应答或无应答状态。
而且，诱导机体免疫耐受也是预防和治疗超敏反应、自身免疫性疾病和移植排斥等可通过B细胞免疫耐受介导的疾病或病症最有效的策略之一。
目前主要通过免疫调节剂来调控机体免疫功能，但需长期用药，且伴有易激活病原体、肿瘤发展和增大感染几率等副作用。
在免疫耐受的诱导中，B细胞所起的作用越来越受到人们的重视。B细胞来源于骨髓的多能干细胞。成熟的B细胞经外周血迁出，进入脾脏、淋巴结，主要分布于脾小结、脾索及淋巴小结、淋巴索及消化道粘膜下的淋巴小结中。成熟B细胞受抗原刺激后，分化增殖为浆细胞，合成抗体，发挥体液免疫功能。B细胞是体内数目最多的抗原提呈细胞，其特征性表面标志是膜表面免疫球蛋白，后者可作为特异性抗原受体识别各种不同的抗原表位。膜表面免疫球蛋白M(mIgM)构成了B细胞的抗原受体。通过特异性配体作用于mIgM可造成B细胞无反应性，诱导B细胞免疫耐受。
因此，在本领域需要新的医药选择，其能够特异性地结合B细胞表面表达的mIgM受体，诱导B细胞免疫耐受，用于减少抗免疫原性物质的抗体的产生和/或用于预防或治疗可通过B细胞免疫耐受介导的疾病或病症(例如超敏反应、自身免疫性疾病或移植排斥)，和/或靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤。
本申请通过下文以及所附权利要求书中记载的技术方案满足了上述需求。
发明内容
发明简述
叶酸(FA)由喋呤、对氨基苯甲酸和L-谷氨酸三部分构成，也称为蝶酰谷氨酸。叶酸在机 体代谢中发挥重要作用，可参与嘌呤及核糖核酸的合成等，帮助调节胚胎神经细胞发育，防止新生婴儿患先天性神经管缺陷症等。叶酸成为继维生素C、维生素E之后国际市场上新崛起的一种保健维生素产品，由于其安全、稳定、廉价等优点，其市场前景十分广阔。
本发明人通过大量研究出人意料地发现，叶酸与B细胞表面表达的mIgM存在特异性结合，可靶向于mIgM阳性表达的B细胞，通过抑制B细胞的成熟及其向生发中心B细胞的分化造成B细胞无能，诱导B细胞免疫耐受。因此，施用叶酸或其盐或酯或偶联物可诱导机体产生B细胞免疫耐受，对免疫原性物质(例如生物大分子药物、具有免疫原性的递药系统或导致机体产生异常免疫反应的致病抗原物质)的刺激表现出特异性免疫低应答或无应答，从而可用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症。而且，发明人还出人意料地发现，叶酸或其盐或酯或偶联物引起的B细胞免疫耐受是可逆的，停止施用叶酸或其盐或酯或偶联物后机体的免疫功能可恢复，这对于出于医学目的而希望仅暂时性地免疫耐受的个体是尤其有利的。
发明人还出人意料地发现，用叶酸修饰免疫原性物质也能够显著减少抗免疫原性物质的抗体的产生。例如，用叶酸修饰生物大分子活性剂可以显著降低生物大分子活性剂的自身免疫原性，抑制抗药抗体(ADA)和耐药性。用叶酸修饰导致机体产生异常免疫应答的致病抗原物质可显著减少抗原特异性抗体的产生，用于预防或治疗超敏反应、自身免疫性疾病和/或移植排斥等可通过B细胞免疫耐受介导的疾病或病症。
而且，本发明人还出人意料地发现，用叶酸修饰药物(例如抗肿瘤药)或探针还能够通过mIgM途径实现mIgM阳性表达的B细胞淋巴瘤的靶向诊断、治疗和/或预防。
因此，在第一方面，本发明提供了叶酸或其可药用盐或酯或偶联物在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药剂中的用途。
在第二方面，本发明提供了叶酸修饰的免疫原性物质在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药剂中的用途。
在第三方面，本发明提供了叶酸修饰的抗肿瘤药或叶酸修饰的探针在制备用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的药剂中的用途。
在第四方面，本发明提供了用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的方法，该方法包括给个体施用有效量的本发明的叶酸或其可药用盐或酯或偶联物或者叶酸修饰的免疫原性物质。
在第五方面，本发明提供了用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的方法，该方法包括给个体施用有效量的本发明的叶酸修饰的抗肿瘤药或叶酸修饰的探针。
在第六方面，本发明提供了用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的本发明的叶酸或其可药用盐或酯或偶联物或者叶酸修饰的免疫原性物质。
在第七方面，本发明提供了用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的本发明的叶酸修饰的抗肿瘤药或叶酸修饰的探针。
在第八方面，本发明提供了药物组合物，其包含有效量的本发明的叶酸或其可药用盐或酯或偶联物和/或叶酸修饰的免疫原性物质和/或叶酸修饰的抗肿瘤药和/或叶酸修饰的探针，以及任选的一种或多种可药用辅料。
在第九方面，本发明提供了叶酸或其可药用盐或酯或偶联物与免疫原性物质的组合产品。
发明详述
本发明提供了如下的实施方案：
实施方案1.叶酸或其可药用盐或酯或偶联物在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药剂中的用途。
实施方案2.根据实施方案1的用途，其中所述的叶酸或其可药用盐或酯或偶联物选自游离叶酸、叶酸的可药用盐、叶酸的可药用酯和叶酸的可药用偶联物(例如叶酸白蛋白偶联物、叶酸聚乙二醇偶联物等)，或它们的任意组合。
实施方案3.根据实施方案1或2所述的用途，其中所述B细胞是表达mIgM的B细胞，特别是高表达mIgM的B细胞，更特别是脾脏B细胞或淋巴结B细胞。
实施方案4.根据实施方案1-3任一项的用途，其中所述的免疫原性物质是生物大分子活性剂，并且所述药剂与生物大分子活性剂组合施用，例如在生物大分子活性剂的施用之前和/或期间施用，以减少抗生物大分子活性剂的抗药抗体的产生；
优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；
更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素。
实施方案5.根据实施方案1-3任一项的用途，其中所述的免疫原性物质是具有免疫原性的递药系统，例如微载体递药系统，优选选自脂质体、微球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束，并且所述药剂与所述递药系统组合施用，例如在所述递药系统的施用之前和/或期间施用，以减少抗具有免疫原性的递药系统的抗体的产生；优选地，所述递药系统载有如实施方案4中所定义的生物大分子活性剂。
实施方案6.根据实施方案1-3任一项的用途，其中所述药剂用于治疗或预防可通过B 细胞免疫耐受介导的疾病或病症，例如超敏反应、自身免疫性疾病或移植排斥；优选地，所述超敏反应选自过敏性休克、呼吸道超敏反应(如过敏性哮喘或过敏性鼻炎)和胃肠道超敏反应，所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、桥本甲状腺炎、毒性弥漫性甲状腺肿、强制性脊柱炎、自身免疫性脑脊髓炎、视神经脊髓炎谱系疾病、抗心磷脂综合征、血友病和银屑病，和所述移植排斥选自器官移植排斥、组织移植排斥(例如骨髓移植排斥)和复发性流产。
实施方案7.叶酸修饰的免疫原性物质在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药剂中的用途。
实施方案8.根据实施方案7所述的用途，其中所述B细胞是表达mIgM的B细胞，特别是高表达mIgM的B细胞，更特别是脾脏B细胞或淋巴结B细胞。
实施方案9.根据实施方案7或8的用途，其中所述的免疫原性物质是生物大分子活性剂；
优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；
更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素。
实施方案10.根据实施方案7或8的用途，其中所述的免疫原性物质是导致机体产生异常免疫应答的致病抗原物质，优选是超敏反应、自身免疫性疾病或移植排斥的致病抗原物质，例如鸡卵白蛋白、蛋白脂蛋白多肽、Dby多肽、Uty、髓鞘碱性蛋白、变性IgG、甲状腺球蛋白、促甲状腺素受体、组蛋白、乙酰胆碱受体、主要组织相容性抗原(包括HLA-I类和HLA-II类)、次要组织相容性抗原、血型抗原、组织特异性抗原(如血管内皮细胞抗原、皮肤SK抗原)。
实施方案11.根据实施方案10的用途，其中所述药剂用于治疗或预防超敏反应、自身免疫性疾病或移植排斥；优选地，所述超敏反应选自过敏性休克、呼吸道超敏反应(如过敏性哮喘或过敏性鼻炎)和胃肠道超敏反应，所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、桥本甲状腺炎、毒性弥漫性甲状腺肿、强制性脊柱炎、自身免疫性脑脊髓炎、视神经脊髓炎谱系疾病、抗心磷脂综合征、血友病和银屑病，和所述移植排斥选自器官移植排斥、组织移植排斥(例如骨髓移植排斥)和复发性流产。
实施方案12.叶酸修饰的抗肿瘤药或叶酸修饰的探针在制备用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的药剂中的用途。
实施方案13.根据实施方案12所述的用途，其中所述叶酸修饰的抗肿瘤药中的抗肿瘤药是可用于治疗或预防mIgM阳性表达的B细胞淋巴瘤的抗肿瘤药，优选是蒽环药物，例如阿霉素或表阿霉素；紫杉烷药物，例如紫杉醇、多烯紫杉醇或卡巴他赛；喜树碱药物，例如喜树碱、羟基喜树碱、9-硝基喜树碱或伊立替康；长春花碱药物，例如长春新碱或长春瑞滨；蛋白酶体抑制剂，例如硼替佐米或卡非佐米；内酯药物，例如小白菊内酯；环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、雷公藤甲素、贝伐单抗或曲妥单抗；和其中所述叶酸修饰的探针中的探针是造影剂，优选是荧光物质，例如荧光素、羧基荧光素(FAM)、异硫氰酸荧光素(FITC)、六氯荧光素(HEX)、香豆素6、近红外染料Cy5、Cy5.5、Cy7、ICG、IR820、DiR或DiD；或放射性物质，例如磁共振造影剂如Gd-DTPA或放射造影剂如 99mTc-DTPA。
实施方案14.用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的方法，该方法包括给个体施用有效量的如实施方案1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如实施方案7-11任一项所定义的叶酸修饰的免疫原性物质。
实施方案15.用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的方法，该方法包括给个体施用有效量的如实施方案12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针。
实施方案16.用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的如实施方案1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如实施方案7-11任一项所定义的叶酸修饰的免疫原性物质，特别是叶酸白蛋白偶联物、叶酸聚乙二醇偶联物、叶酸修饰的人血白蛋白、叶酸修饰的胰岛素、叶酸修饰的阿达木单抗、叶酸修饰的英夫利昔单抗、叶酸修饰的阿特珠单抗或叶酸修饰的曲妥珠单抗。
实施方案17.用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的如实施方案12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针，特别是叶酸修饰的卡巴他赛或叶酸修饰的雷公藤甲素。
实施方案18.药物组合物，包含有效量的如实施方案1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如实施方案7-11任一项所定义的叶酸修饰的免疫原性物质和/或如实施方案12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针，以及任选的一种或多种可药用辅料。
实施方案19.叶酸或其可药用盐或酯或偶联物与免疫原性物质的组合产品，其中所述的免疫原性物质优选是生物大分子活性剂或具有免疫原性的递药系统；
其中更优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎 毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素；
或者
其中更优选地，所述的具有免疫原性的递药系统选自微载体递药系统，例如脂质体、微球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束。
可以理解，在每个实施方案中指定的特征可以与任意其它指定特征组合以提供其它实施方案，这些组合的实施方案也落入本申请的范围内。
本申请所用的术语和符号具有本领域技术人员通常理解的含义，另有说明除外。特别地，本申请的下列术语具有下文指示的含义，另有说明除外。
本文所用的表述“一种”、“一个”、“该”、“所述”或实体本身包括其单数形式和复数形式，另有指示除外。
本文所用的术语“包含”、“含有”、“包括”和类似术语应理解为除所指明物质外还可以包含未具体指明的物质，只要它们是相容的。
本文所用的术语“和/或”当连接两个或多个可选项时，应理解为指可选项中的任意一项或多项。
本文所用的术语“约”应理解为表示将所给数值调整至高于或低于该数值20％、例如10％、例如5％。
本文所用的术语“叶酸或其盐或酯或偶联物”包括游离叶酸、叶酸盐、叶酸酯或叶酸偶联物(例如叶酸白蛋白偶联物、叶酸聚乙二醇偶联物等)，或它们的任意组合。相应地，本文所用的术语“叶酸或其可药用盐或酯或偶联物”包括游离叶酸或者药学上可接受的叶酸盐、叶酸酯或叶酸偶联物(例如叶酸白蛋白偶联物、叶酸聚乙二醇偶联物等)，或它们的任意组合。
本文所用的术语“叶酸修饰”或“叶酸修饰的”指叶酸通过化学结合与其它物质、例如免疫原性物质(例如生物大分子或致病抗原物质)、抗肿瘤药、探针、高分子材料等连接形成复合物。
本文所用的术语“本发明的物质”指如本文所定义的叶酸或其可药用盐或酯或偶联物、叶酸修饰的免疫原性物质(例如叶酸修饰的生物大分子活性剂、叶酸修饰的致病抗原物质)、叶酸修饰的抗肿瘤药或叶酸修饰的探针中的一种或多种。而且，本发明的物质还可以指叶酸或其可药用盐或酯或偶联物与免疫原性物质的组合。
本文所用的术语“免疫原性物质”指能够激活和诱导机体产生免疫应答的物质，包括但不 限于生物大分子如生物大分子活性剂、具有免疫原性的递药系统或导致机体产生异常免疫应答的致病抗原物质。
本文所用的术语“递药系统”指在空间、时间和/或剂量上调控药物在生物体内分布的体系。
本文所用的术语“具有免疫原性的递药系统”指能够激活和诱导机体产生免疫应答的递药系统，例如微载体递药系统。
本文所用的术语“微载体递药系统”包括一系列的微米、亚微米、纳米级等体系，例如脂质体、微球、微囊、微乳、纳米粒、纳米囊、纳米圆盘、聚合物胶束等。
本文所用的术语“生物大分子”指作为生物体内活性成分的高分子量有机物质，例如分子量达到上千道尔顿或更高，包括但不限于多肽或蛋白质如抗体等。
本文所用的术语“生物大分子药物”和“生物大分子活性剂”可互换使用，指具有治疗或预防活性的天然或合成的生物大分子，例如包括但不限于多肽药物或蛋白质药物如抗体药物等。
本文所用的术语“导致机体产生异常免疫应答的致病抗原物质”或“致病抗原物质”可互换使用，指导致机体产生过高的免疫应答、从而引起机体组织损伤和/或表现出疾病状态的具有免疫原性的物质。
本文所用的术语“抗肿瘤药”指可用于预防或治疗肿瘤疾病或癌症的物质，例如化疗药、激素类药物或毒素等。
本文所用的术语“探针”指能检测或显示生物医学问题、例如诊断或治疗疾病的功能性化学物质，例如造影剂等。
本文所用的术语“造影剂”指为增强影像观察效果而施用至个体组织或器官的化学物质。
本文所用的术语“可药用”指在化学上/毒理学上与制剂或组合物的其它成分(包括辅料和/活性成分)和/或与机体相容。
本文所用的术语“可药用盐”指母体化合物与可药用酸或碱形成的盐。可药用盐是本领域熟知的，包括但不限于无机酸盐，如盐酸盐、硫酸盐、磷酸盐等；有机酸盐，如乙酸盐、丙酸盐、甲磺酸盐、乙磺酸盐、苹果酸盐、柠檬酸盐、酒石酸盐等；碱金属或碱土金属盐，如钠、钾、钙盐等。
本文所用的术语“可药用酯”指母体羧酸的可药用的酯衍生物，例如低级烷基酯(例如甲基酯、乙基酯、正丙基酯或异丙基酯等)、环烷基酯、低级烯基酯、苄基酯、如ω-(氨基、单或二低级烷基氨基、羧基、低级烷氧基羰基)-低级烷基酯、α-(低级烷酰基氧基、低级烷氧基羰基或二低级烷基氨基羰基)-低级烷基酯如新戊酰氧基甲基酯，以及本领域中常规使用的类似物质。
本文所用的术语叶酸的“偶联物”或“叶酸偶联物”指叶酸通过化学键与其它物质偶联形成复合物以改善叶酸的体内动力学行为和作用时间等性质，包括但不限于叶酸白蛋白偶联物、叶酸聚乙二醇偶联物等。
本文所用的术语“可药用辅料”包括任意和所有药学上可接受的载体或稀释剂、溶剂、分散介质、包衣料、表面活性剂、抗氧化剂、防腐剂、等张剂、吸收延迟剂、盐、防腐剂、稳定 剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等或其任意组合，如本领域普通技术人员已知的那样。在药物组合物或药物制剂中可以使用任何本领域已知的辅料，只要它与活性成分相容。
本文所用的术语“B细胞”尤其指表达mIgM的B细胞，特别是高表达mIgM的B细胞，例如脾脏B细胞或淋巴结B细胞。
本文所用的术语“B细胞免疫耐受”指B细胞对抗原刺激低反应或无反应性，免疫应答降低或不产生免疫应答。
本文所用的术语“抗免疫原性物质的抗体”指具有免疫原性的物质诱导机体产生的特异性抗体。
本文所用的术语“抗药抗体”指具有潜在免疫原性的生物大分子诱导机体产生的抗药抗体(Anti-Drug Antibodies，ADAs)。ADAs的产生可导致药物浓度降低、药物半衰期缩短、药物疗效下降、甚至无效，或者引起超敏反应，甚至危及生命。
本文所用的术语“耐药性”指药物治疗疾病或改善症状的效力降低。
本文所用的术语“可通过B细胞免疫耐受介导的疾病或病症”指可获益于B细胞免疫耐受或者可通过B细胞免疫耐受治疗或预防的疾病或病症。
本文所用的术语“超敏反应”是机体受到某些抗原刺激时出现生理功能紊乱或组织细胞损伤等异常的适应性免疫应答。
本文所用的术语“自身免疫性疾病”指机体对自身抗原产生异常免疫应答、导致自身细胞破坏、组织损伤或功能异常所引起的疾病状态。
本文所用的术语“移植排斥”指受者进行组织或器官移植、特别是同种异体或异种组织或器官移植后，外来组织或器官等移植物作为外来物被受者的免疫系统识别，后者发起针对移植物的攻击、破坏和清除的免疫学反应。术语“移植排斥”还包括反复性流产。
本文所用的术语“个体”和“患者”可互换使用，包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。优选地，个体是灵长类，特别是人。
本文所用的术语“有效量”指本发明的物质当施用于个体的细胞或组织或非细胞生物材料或介质时引起生物学或医学响应的量，所述响应例如是症状改善、病症缓解、疾病进程延缓或延迟或者预防疾病等。本领域技术人员理解，具体的有效量可根据诸如预期生物学终点、待递送的药物、靶组织等的因素而变化，而且“有效量”可以以单一剂量施用或者可通过多个剂量的施用来实现。
本文所用的术语“治疗”指改善、缓解、减轻或阻止疾病或病症或其至少一种临床症状。在另一项实施方案中，“治疗”指缓解或改善至少一个身体参数，包括患者能够感受到和/或不能感受到的那些。在另一项实施方案中，“治疗”指在身体方面调控疾病或障碍(例如稳定可感受到的症状)或在生理学方面调控疾病或障碍(例如稳定身体参数)或二者。在另一个实施方案中，治疗指阻止或延迟疾病或障碍的发作或发展或进程。
本文所用的术语“预防”、“阻止”等指由施用疗法(例如治疗剂)或疗法组合(例如治疗剂的 组合)来阻止个体的疾病或病症的一种或多种症状的发作、发展或复发。
本文所用的术语“诊断”指通过检查或调查等方法采集信息后对个体疾病状态做出判断。
本文所用的术语“组合”指两种或更多种活性剂以相同或不同的途径同时、分别或依次施用，所述活性剂可以包括在同一药物组合物中，或者被包括在分开的药物组合物中(例如以套盒的形式)。
本文所用的术语“组合产品”既包括两种或更多种活性剂在同一药物组合物中的情况，也包括两种或更多种活性剂在分开的药物组合物中的情况(例如以套盒的形式)。
叶酸或其可药用盐或酯或偶联物
叶酸由蝶呤、对氨基苯甲酸和L-谷氨酸三部分组成，因此也称为蝶酰谷氨酸。本发明的叶酸可以是天然或合成来源的。
叶酸的可药用盐或酯或偶联物可以购买获得，或者可以按照本领域的常规方法来制备。例如，叶酸白蛋白偶联物和叶酸聚乙二醇偶联物可购自瑞禧生物有效公司。
通过施用本发明的叶酸或其可药用盐或酯或偶联物，诱导机体产生了B细胞免疫耐受。因此，本发明的叶酸或其可药用盐或酯或偶联物可以单独施用，或者可以与免疫原性物质组合施用，优选在所述免疫原性物质的施用之前和/或期间施用，以诱导B细胞免疫耐受，用于减少抗免疫原性物质的抗体的产生和/或可用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症。
在一项实施方案中，给将要施用、正在施用或已经施用了免疫原性物质(例如生物大分子活性剂、具有免疫原性的递药系统)的个体施用本发明的叶酸或其可药用盐或酯或偶联物，以减少抗免疫原性物质的抗体的产生。优选地，叶酸或其可药用盐或酯或偶联物在施用免疫原性物质之前和/或期间进行施用。更优选地，叶酸或其可药用盐或酯或偶联物的施用在免疫原性物质的施用之前(例如提前15、10、9、8、7、6、5、4、3、2、1天)开始并持续数年、数月、数周或数天的时间段。特别地，叶酸或其可药用盐或酯或偶联物的施用持续至免疫原性物质的施用结束后诸如3个月、2个月、1个月、3周、2周或1周，这取决于免疫原性物质的诸如代谢或半衰期的性质并且可以由参与医师常规判断。
在另一项实施方案中，给处于罹患可通过B细胞免疫耐受介导的疾病或病症(例如超敏反应、自身免疫性疾病或移植排斥)的风险的个体或罹患可通过B细胞免疫耐受介导的疾病或病症的个体施用本发明的叶酸或其可药用盐或酯或偶联物，以预防所述疾病或病症的发生或者当发生所述疾病或病症时治疗所述疾病或病症、例如减轻其一种或多种症状。
免疫原性物质
可用于本发明的免疫原性物质包括但不限于生物大分子如生物大分子活性剂、具有免疫原性的递药系统和导致机体产生异常免疫应答的致病抗原物质。
在一项实施方案中，可用于本发明的免疫原性物质是生物大分子活性剂，包括但不限于多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如 人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)；PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；或表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)。
更优选地，可用于本发明的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素。
在另一项实施方案中，可用于本发明的免疫原性物质是具有免疫原性的递药系统，包括但不限于微载体递药系统，优选选自脂质体、微球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束。所述递药系统可以载有任意适宜的药物，例如如本文所述的生物大分子活性剂、抗肿瘤药、抗菌药、激素等。当递药系统载有如本文所述的生物大分子活性剂时，本发明的叶酸或其药用盐或酯或偶联物可以减少抗所述递药系统的抗体和所述生物大分子活性剂的抗药抗体两者的产生。递药系统的制备是本领域已知的，例如采用高分子材料如磷脂或聚合物通过成膜水化来制备。
在另一项实施方案中，可用于本发明的免疫原性物质是导致机体产生异常免疫应答的致病抗原物质，优选是超敏反应、自身免疫性疾病或移植排斥的致病抗原物质，例如鸡卵白蛋白、蛋白脂蛋白(PLP)多肽、Dby多肽、Uty、髓鞘碱性蛋白、变性IgG、甲状腺球蛋白、促甲状腺素受体、组蛋白、乙酰胆碱受体、主要组织相容性抗原(包括HLA-I类和HLA-II类)、次要组织相容性抗原、血型抗原、组织特异性抗原(如血管内皮细胞抗原、皮肤SK抗原)。
叶酸修饰的免疫原性物质
本发明的叶酸修饰的免疫原性物质可以通过将叶酸和免疫原性物质经由化学键或连接基形成复合物来制备。
在一项实施方案中，叶酸修饰的免疫原性物质可以是叶酸修饰的生物大分子活性剂。生物大分子活性剂的施用、尤其是长期施用易产生抗药抗体和/或耐药性。优选地，所述生物大分子药物包括但不限于上文所定义的那些。特别地，本发明的叶酸修饰的生物大分子活性剂是叶酸修饰的人血白蛋白、叶酸修饰的胰岛素、叶酸修饰的阿达木单抗、叶酸修饰的英夫利昔单抗、叶酸修饰的阿特珠单抗、叶酸修饰的信迪利单抗、叶酸修饰的特瑞普利单抗或叶酸修饰的曲妥珠单抗。叶酸修饰能够减少生物大分子活性剂的抗药抗体的产生。
在另一项实施方案中，本发明的叶酸修饰的免疫原性物质是叶酸修饰的导致机体产生异常免疫应答的致病抗原物质，例如如上文所定义的那些。
用叶酸修饰免疫原性物质的方法可以按照本领域已知的方法或下文实施例中所示例的方法来进行。例如，可以通过直接化学修饰(例如酰胺缩合)、双功能桥连基团或固相合成使叶酸与生物大分子如多肽或蛋白质缩合来制备。
通过本发明的叶酸修饰，诱导了B细胞免疫耐受。因此，本发明的叶酸修饰的免疫原性 物质可用于减少抗免疫原性物质的抗体的产生和/或可用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症。
叶酸修饰的抗肿瘤药
本发明的叶酸修饰的抗肿瘤药可以通过将叶酸和抗肿瘤药经由化学键或连接基形成复合物来制备。
可用于本发明的抗肿瘤药优选是可用于治疗或预防mIgM阳性表达的B细胞淋巴癌的那些。优选地，可用于本发明的抗肿瘤药包括但不限于蒽环药物，例如阿霉素或表阿霉素；紫杉烷药物，例如紫杉醇、多烯紫杉醇或卡巴他赛；喜树碱药物，例如喜树碱、羟基喜树碱、9-硝基喜树碱或伊立替康；长春花碱药物，例如长春新碱或长春瑞滨；蛋白酶体抑制剂，例如硼替佐米或卡非佐米；内酯药物，例如小白菊内酯；环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、雷公藤甲素、贝伐单抗、曲妥单抗；等。在一些实施方案中，本发明的叶酸修饰的抗肿瘤药是叶酸修饰的卡巴他赛或叶酸修饰的雷公藤甲素。
本发明的叶酸修饰的抗肿瘤药的制备方法是本领域已知的或者如实施例中所示例。例如，本发明的叶酸修饰的抗肿瘤药可以如下制备：(a)对于含硼酸基团的药物，例如硼替佐米，可以通过将叶酸用多巴胺修饰、然后与药物的硼酸基团反应，形成含pH敏感性硼酸酯的叶酸复合物；(b)对于含酮或醛基的药物，例如蒽环药物如阿霉素、表阿霉素等，可以通过在叶酸中引入巯基、然后与药物的马来酰亚胺己肼衍生物反应，形成含pH敏感性腙键的叶酸复合物；(c)对于含羟基或氨基的药物，例如紫杉醇、多烯紫杉醇、卡巴他赛、喜树碱、羟基喜树碱、9-硝基喜树碱、伊立替康、长春新碱和长春瑞滨等，可以通过在叶酸中引入巯基、然后与药物的3-(2-吡啶二巯基)丙酸衍生物反应，形成含二硫键的叶酸复合物；(d)对于含活性氨基的药物，例如氨基磷酸酯、氨基糖苷、阿霉素等，其可以直接通过酰胺缩合来形成；和(e)对于含活性羧基的药物，例如苯丁酸氮芥，其可以通过在叶酸中引入氨基、然后通过酰胺缩合来形成。
本发明的叶酸修饰的抗肿瘤药可用于mIgM阳性表达的B细胞淋巴瘤的靶向治疗或预防。
叶酸修饰的探针
本发明的叶酸修饰的探针可以通过将叶酸和探针经由化学键或连接基形成复合物来制备。
可用于本发明的探针可以是造影剂，包括但不限于荧光物质，例如荧光素、羧基荧光素(FAM)、异硫氰酸荧光素(FITC)、六氯荧光素(HEX)、香豆素6、近红外染料Cy5、Cy5.5、Cy7、ICG、IR820、DiR、DiD等；和放射性物质，例如磁共振造影剂如Gd-DTPA或放射造影剂如 99mTc-DTPA等。优选地，可用于本发明的探针是羧基荧光素(FAM)、近红外染料Cy5、Cy5.5、ICG、IR820、DiR、DiD或Gd-DTPA等。
叶酸修饰的探针的制备方法是本领域已知的或者如下文实施例中所示例。例如，本发明的叶酸修饰的探针可以通过酰胺缩合、功能化衍生、螯合等来制备。
本发明的叶酸修饰的探针可用于mIgM阳性表达的B细胞淋巴瘤的靶向诊断、治疗或预防。
叶酸或其可药用盐或酯或偶联物与免疫原性物质的组合
将叶酸(游离或其可药用盐或酯或偶联物的形式)和免疫原性物质组合施用，能够减少抗免疫原性物质的抗体的产生。
在一项实施方案中，所述免疫原性物质优选是生物大分子活性剂，例如上文所述的那些。
在另一项实施方案中，所述免疫原性物质是具有免疫原性的递药系统，例如微载体递药系统，优选脂质体、微球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束。所述递药系统可以载有任意适宜的药物，例如如本文所述的生物大分子活性剂、抗肿瘤药、抗菌药、激素等。进一步地，所述免疫原性物质是装载生物大分子活性剂的递药系统。
叶酸或其可药用盐或酯或偶联物的施用和免疫原性物质的施用可以同时、依次或交替进行。例如，叶酸或其可药用盐或酯或偶联物在施用免疫原性物质之前和/或期间进行施用。优选地，叶酸或其可药用盐或酯或偶联物的施用在免疫原性物质的施用之前(例如提前15、10、9、8、7、6、5、4、3、2、1天)开始并持续数年、数月、数周或数天的时间段。特别地，叶酸或其可药用盐或酯或偶联物的施用持续至免疫原性物质的施用结束后诸如3个月、2个月、1个月、3周、2周或1周，这取决于免疫原性物质的诸如代谢或半衰期的性质并且可以由参与医师常规判断。更优选地，叶酸或其可药用盐或酯或偶联物的施用和免疫原性物质的施用同时进行。
叶酸或其可药用盐或酯或偶联物和免疫原性物质的施用途径可以相同或不同。优选地，叶酸或其可药用盐或酯或偶联物可以以口服、吸入、注射等途径进行施用，而免疫原性物质可以以与叶酸或其可药用盐或酯或偶联物相同或不同的途径进行施用。而且，叶酸或其可药用盐或酯或偶联物和免疫原性物质可以包括在同一药物组合物中，或者被包括在分开的药物组合物中。例如，叶酸或其可药用盐或酯或偶联物和免疫原性物质可以以组合产品如套盒的形式呈现。
药物组合物和施用
本发明的物质可以通过本领域任何适宜的途径来施用，包括但不限于口服、胃肠外如皮下、肌内、静脉内、腹腔内、吸入、鼻内、直肠、透皮、植入、局部等。在一些实施方案中，本发明的物质通过注射施用，例如连续输注、皮下注射、肌内注射或静脉内注射。在另一些实施方案中，本发明的物质可以口服施用。在另一些实施方案中，本发明的物质可以吸入施用。进一步地，本发明的物质还可以通过植入装置进行施用。
本发明的物质可以通过本领域的任意适宜操作制备成药物制剂或药物组合物。药物制剂或药物组合物可以是固体形式(包括但不限于片剂、锭剂、包衣片剂、胶囊剂、丸剂、颗粒剂、扁囊剂、冻干物、粉剂或栓剂等)、液体形式(包括但不限于溶液剂、混悬剂或乳剂等)或半固 体形式(包括但不限于霜剂、软膏剂、糊剂、凝胶剂等)，用于本领域任何适宜的途径，如口服、胃肠外如皮下、肌内、静脉内、腹腔内、吸入或经鼻、直肠、透皮、植入、局部施用等。药物制剂含有有效量的本发明的物质和任选的一种或多种可药用辅料，例如稀释剂、等张剂、缓冲剂、防腐剂、稳定剂、润湿剂、乳化剂、润滑剂等，只要它们是相容的。药物制剂或药物组合物还可以接受任何适宜的常规医药操作，例如制粒、压片、冻干、灭菌等。
在一些实施方案中，药物组合物或药物制剂是适于口服施用的形式，例如片剂、包衣片剂、锭剂、胶囊剂(软或硬)、散剂、颗粒剂、扁囊剂、乳剂、水性或油性混悬剂、糖浆剂或酏剂等。
在另一些实施方案中，药物组合物或药物制剂是适于注射施用的形式，包括但不限于无菌水溶液(当可溶于水时)或分散液和用于临时制备无菌可注射溶液或分散液的无菌粉末，优选是水性等张溶液或混悬液。优选地，药物组合物或制剂用于连续输注、皮下注射、肌内注射或静脉内注射。适于注射施用的制剂组分和方法是本领域已知的，例如包括生理盐水、稳定剂、缓冲剂和等张剂等。在所有情形下，组合物均应当是无菌的，并且在临用前应当是以易于注射的程度流动的。制剂的pH通常为约3至11，更优选为约5至9或6至8，最优选为约7至8，例如约7至7.5。
在另一些实施方案中，药物组合物或药物制剂是适于吸入或经鼻施用的形式。它们可以方便地以气雾剂或喷雾剂形式由加压容器、泵、喷雾器、雾化器或喷散器在使用或不使用适宜抛射剂的情况下递送，或者以干粉形式(单独或作为混合物、例如与乳糖的干混物)由干粉吸入器递送。适于制备吸入或经鼻施用药物组合物或制剂的方法和组分是本领域已知的。特别地，就气雾剂而言，其优选包含例如氢氟烷(HFA)抛射剂，如二氯二氟甲烷、HFA134a或HFA227或它们的任意混合物，并且可以包含一种或多种本领域已知的助溶剂(如乙醇)和/或一种或多种表面活性剂(如油酸或三油酸脱水山梨醇)和/或一种或多种填充剂(如乳糖)。当药物组合物或制剂为喷雾剂时，其优选含有例如溶于或混悬于含有水、助溶剂(如乙醇或丙二醇)和稳定剂(其可以是表面活性剂)的溶媒中的活性剂。当药物组合物或制剂是干粉制剂时，其优选含有例如粒径至多10微米的活性剂，任选地还含有具有预期粒度分布的稀释剂或载体(如乳糖)和有助于防止由潮湿引起的产品性能降低的化合物(如硬脂酸镁)。
在另一些实施方案中，药物组合物或药物制剂是适于透皮或局部施用的形式，包括粉末、喷雾剂、软膏剂、糊剂、霜剂、洗剂、凝胶、溶液、贴剂和吸入剂。可以将本申请的物质在无菌条件下与可药用载体和与可能需要的任意辅料如渗透促进剂、防腐剂或缓冲剂等混合来制备。
在一些实施方案中，本发明的物质可以例如通过植入装置或导管间歇或连续施用，持续一天或数天或更长时间，例如数周、数月或数年，例如至少约3天、至少约1周、至少约2周或至少1个月。
本发明的物质在宽剂量范围通常是有效的。例如，日剂量可以为约0.01-2000mg，优选约1-1000mg，更优选约2-100mg，最优选约5-80mg。在一些情况中可以采用低于上述剂量范围下限的剂量，而在另一些情况中可以采用高于上述剂量范围上限的剂量。因此，上述剂量范 围不意欲以任何方式限制本发明的范围。可以理解，实际施用的剂量将由医师考虑相关的情况、包括待治疗的病症、所选的施用途径、实际施用的一种或多种活性剂、个体患者的年龄、体重和响应以及患者症状的严重性来确定。而且，本申请的物质可以作为单一剂量或作为两个或更多个剂量(各剂量可以相同或不同)施用。
本发明的药物制剂或药物组合物还可以含有其它活性剂，只要它们是相容的。所述其它活性剂可以具有与本申请的物质相同或不同的医药活性。
药理学功效和用途
本发明的物质可用于诊断、治疗和/或预防。特别地，本发明的物质能够特异性地结合B细胞表面的mIgM和靶向于表达mIgM的B细胞、特别是高表达mIgM的B细胞，造成B细胞无能，诱导B细胞免疫耐受。
因此，在一个方面，本发明的物质可用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症，包括但不限于超敏反应、自身免疫性疾病或移植排斥。优选地，B细胞是表达mIgM的B细胞，特别是高表达mIgM的B细胞，更特别是脾脏B细胞或淋巴结B细胞。
在一些实施方案中，本发明的物质、特别是叶酸或其可药用盐或酯或偶联物或者叶酸修饰的生物大分子活性剂可用于减少生物大分子活性剂如多肽或蛋白质如抗体药物的抗药抗体和/或耐药性。生物大分子药物具有潜在的免疫原性，尤其在长期使用时易产生抗药抗体(ADA)。通过给个体施用叶酸或其可药用盐或酯或偶联物和生物大分子的组合或者施用经叶酸修饰的生物大分子，能够诱导B细胞免疫耐受，使机体对该生物大分子耐受，有效降低了抗药抗体的产生，提高了疗效和安全性。例如，长期施用阿达木单抗使机体产生了针对阿达木单抗的抗体。当再次给予阿达木单抗时，抗药抗体快速中和阿达木单抗，造成药物浓度降低、无活性，诱发免疫副反应。当施用经叶酸修饰的阿达木单抗后，机体对阿达木单抗耐受，有效降低了抗药抗体的产生，提高了疗效和安全性。
在另一些实施方案中，本发明的物质、特别是叶酸或其可药用盐或酯或偶联物可用于减少抗具有免疫原性的递药系统的抗体的产生。例如，脂质体是常用的药物递送系统，但是，反复注射脂质体会使机体产生抗脂质体的抗体，导致脂质体在体内快速清除。通过将叶酸或其可药用盐或酯或偶联物与脂质体联用，可有效减少抗脂质体抗体的产生，增加脂质体对机体的暴露。
在另一些实施方案中，本发明的物质可用于治疗或预防超敏反应，例如通过施用叶酸或其可药用盐或酯或偶联物或者叶酸修饰的超敏反应的致病抗原物质如多肽或蛋白质。所述超敏反应包括但不限于过敏性休克、呼吸道超敏反应(如过敏性哮喘或过敏性鼻炎)、胃肠道超敏反应等。
在另一些实施方案中，本发明的物质可用于治疗或预防自身免疫性疾病，包括但不限于系统性红斑狼疮、类风湿性关节炎、桥本甲状腺炎、毒性弥漫性甲状腺肿、强制性脊柱炎、自身免疫性脑脊髓炎、视神经脊髓炎谱系疾病、抗心磷脂综合征、血友病、银屑病等，例如通过施用叶酸或其可药用盐或酯或偶联物或者叶酸修饰的自身免疫性疾病的致病抗原物质如 多肽或蛋白质。自身免疫性疾病的致病抗原物质包括但不限于PLP多肽、髓鞘碱性蛋白、变性IgG、甲状腺球蛋白、促甲状腺素受体、组蛋白和乙酰胆碱受体。
在另一些实施方案中，本发明的物质可用于治疗或预防移植排斥，例如通过施用叶酸或其可药用盐或酯或偶联物或者叶酸修饰的移植排斥的致病抗原物质。例如，Dby(NAGFNSNRANSSRSS)和Uty(WMHHNMDLI)是介导雌性小鼠骨髓移植排斥的重要抗原。用叶酸修饰的Dby或Uty提前刺激受体小鼠造成免疫耐受，可增加供体小鼠骨髓移植的成功率。所述移植排斥包括器官移植排斥(例如但不限于心脏、肝脏、脾脏、肺、肾脏的抑制排斥)、组织移植排斥(例如骨髓移植排斥)和复发性流产。移植排斥的致病抗原物质包括但不限于主要组织相容性抗原(包括HLA-I类、HLA-II类)、次要组织相容性抗原、血型抗原、组织特异性抗原(如血管内皮细胞抗原、皮肤SK抗原)等。
在另一方面，本发明的物质还可用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤。通过特异性地识别B细胞淋巴瘤表面表达的mIgM受体，本发明的物质能够靶向于mIgM阳性表达的B细胞淋巴瘤，实现mIgM阳性表达的B细胞淋巴瘤的靶向诊断、治疗或预防。
附图说明
图1提供了实施例1的叶酸-OVA复合物的核磁共振图谱。
图2提供了叶酸分子的定量标准曲线。
图3提供了实施例3的叶酸-PEG 2000-Cy5的核磁共振图谱。
图4提供了实施例4中叶酸与小鼠mIgM的结合模式(a：叶酸与mIgM的结合位点；b：叶酸与mIgM结合作用力分析)。
图5提供了实施例4中叶酸与小鼠mIgM结合的流式分析(a：抗-IgM抗体消除B细胞表面mIgM的效果；b：不同抗-IgM抗体浓度下叶酸与B细胞摄取的结果；c：叶酸的B细胞摄取量与mIgM表达水平的线性拟合曲线)。
图6提供了实施例4中叶酸与小鼠mIgM结合的共聚焦图片。
图7提供了实施例5中FA-PEG 2000-Cy5或PEG 2000-Cy5尾静脉注射后1h(a)和4h(b)时在脾脏中的分布。(*p<0.05，**p<0.01，T检验)
图8提供了实施例5中FA-PEG 2000-Cy5或PEG 2000-Cy5尾静脉注射后1h和4h时脾脏淋巴细胞的流式分选图(a:1h；c:4h)和统计分析(b:1h；d:4h)。(ns:无统计学差异，***p<0.001，T检验)
图9提供了实施例5中FA-PEG 2000-Cy5或PEG 2000-Cy5尾静脉注射后1h时在脾脏中分布的免疫荧光照片。
图10提供了实施例5中FA-PEG 2000-Cy5尾静脉注射后1h时在脾脏中与IgM阳性表达的边缘B细胞结合的免疫荧光照片。
图11提供了实施例6中小鼠分别用FA-OVA、OVA和生理盐水刺激三次后通过流式细胞仪分析的B细胞增殖(a,b)与分化(c,d)结果。(*p<0.05，***p<0.001，单因素方差分析)
图12提供了实施例6中小鼠分别用FA-OVA、OVA和生理盐水刺激三次后的血清OVA 特异性抗体表达(a:IgE；b:IgG；c:IgG1；d:IgG2a)。
图13提供了实施例6中小鼠分别用FVIII、叶酸-FVIII和生理盐水刺激三次后的血清FVIII特异性抗体表达。
图14提供了实施例7中口服叶酸后小鼠血清中OVA特异性IgG抗体表达(a:第14天；b:第21天；c:第28天)。
图15提供了实施例7中口服叶酸后小鼠血清中KLH特异性IgG抗体表达(a:第21天；b:第28天；c:第35天)。
图16提供了实施例8中小鼠分别进行刺激后的血清特异性抗体表达(a:阿达木单抗作为模型单抗；b:英夫利昔单抗作为模型单抗；c:曲妥珠单抗作为模型单抗)。
图17提供了实施例9中小鼠分别用sLip、sLip+FA(10μg)、sLip+FA(50μg)和生理盐水刺激后的血清sLip特异性抗体表达。
图18提供了实施例10中分别用FA-OVA、OVA和生理盐水处置后小鼠再次用OVA刺激后的体温变化曲线。
图19提供了实施例10中分别用FA-OVA、OVA和生理盐水处置后小鼠再次用OVA刺激后的血清OVA特异性抗体水平(a:IgE；b:IgG 1；c:IgG 2a)。
图20提供了实施例11中致敏小鼠分别用FA-OVA、OVA和生理盐水处置后再次用OVA刺激后的体温变化曲线。(ns:无统计学差异，**p<0.01，***p<0.001，单因素方差分析)
图21提供了实施例11中致敏小鼠分别用FA-OVA、OVA和生理盐水处置后再次用OVA刺激后的血清OVA特异性抗体水平(a:IgE；b:IgG 2a；c:IgG 1)。(ns:无统计学差异，***p<0.001，单因素方差分析)
实施例
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。所用的实验材料和试剂如无特别说明均可从市售渠道获得或者按照本领域已知的方法制备。本文中所用的缩写具有本领域通常理解的含义，另有指示除外。
特别地，本文中的缩写具有以下含义：
缩写 含义
BSA 牛血清白蛋白
Chol 胆固醇
DAPI 荧光染料4',6-二脒基-2-苯基吲哚
Dby NAGFNSNRANSSRSS
DCC 二环己基碳二亚胺
DMSO 二甲亚砜
DSPE 二硬脂酰基磷脂酰乙醇胺
EDC 碳二亚胺
EDTA 乙二胺四乙酸
FA 叶酸
FVIII 凝血因子VIII
h 小时
HPLC 高效液相色谱法
HSPC 氢化大豆磷脂
mPEG 聚乙二醇单甲醚
NHS N-羟基琥珀酰亚胺
OVA 鸡卵白蛋白
PBS 磷酸盐缓冲溶液，pH＝7.4
PEG 聚乙二醇
PLGA 聚乳酸羟基乙酸共聚物
PLP 蛋白脂蛋白
POPC 1-棕榈酰基-2-油酰基卵磷脂
TEA 三乙醇胺
TLC 薄层色谱法
实施例1：叶酸-蛋白质复合物的制备
以叶酸-OVA为例合成了叶酸修饰的蛋白质复合物。称取4.4mg叶酸，分散于3mL PBS(pH＝7.4)中。加入15mg碳二亚胺(EDC)，避光搅拌3小时使叶酸活化。称取4.3mg鸡卵白蛋白(OVA)，充分溶于0.2mL PBS中。将活化叶酸溶液缓慢滴入OVA溶液中，避光搅拌3小时，于4℃摇晃过夜。次日用10mM PBS透析72小时，每12小时更换透析液。冻干获取产物。
叶酸自身不溶于水。与OVA连接后，由于蛋白水溶性较好，叶酸-OVA复合物易溶于水，表明叶酸成功连接到OVA上。图1提供了所获得的叶酸-OVA复合物的核磁氢谱，信号δ7.45、δ6.45为一组对称的“AA′BB′”自旋体系的信号峰，由此可确定该信号为叶酸分子中对位二取代苯环上的两组质子信号峰，说明分子中含叶酸结构，进一步判定产物结构为叶酸-OVA。采用紫外分光光度计测定所得叶酸复合物的OD 365nm。图2提供了用于定量叶酸修饰度的标准曲线。如表1所示，计算得叶酸-OVA中叶酸修饰度约为8.66。叶酸修饰度表示叶酸复合物中的叶酸与叶酸复合物的比例。
另用以叶酸-凝血因子VIII(FA-FVIII)为例合成了叶酸修饰的蛋白质复合物。FVIII药品原液(20IU/mL,购自上海莱士)采用0.1M浓度的碳酸盐缓冲液(pH＝9.5)对半稀释，加入50倍当量的叶酸和60倍当量的EDC(均溶解在0.1M碳酸盐缓冲液中)，加入后的15分钟内滴加2M盐酸溶液，调节反应液的pH为6.6，室温震荡反应过夜，透析72小时，收集产物。如表1所示，计算得FA-FVIII中叶酸修饰度约为8.03。
实施例2：叶酸-单克隆抗体复合物的制备
以叶酸-阿达木单抗复合物(FA-Adalimumab)和叶酸-曲妥珠单抗复合物(FA-Trastuzumab)为例制备了叶酸-单克隆抗体复合物。分别配制1mg/mL各单抗的PBS溶液，加入三(2-羧乙基)膦和EDTA至浓度分别为10mM和5mM。室温震荡反应90分钟。随后将反应溶液与15当量的叶酸-PEG 400-Mal(叶酸-PEG 400-马来酰亚胺,购自上海翊圣生物科技有限公司)混合，室温震荡反应20分钟。为了封闭未反应的巯基，将溶液进一步与15当量的马来酰亚胺于室温震荡反应20分钟。随后，将反应溶液在PBS中透析72小时，除去体系中的小分子，获得各叶酸-单克隆抗体复合物溶液。表1提供了叶酸-单抗复合物中叶酸的修饰度。
表1：实施例1-2中制备的各叶酸修饰的生物大分子中叶酸修饰度结果
Figure PCTCN2021125346-appb-000001
实施例3：叶酸-造影剂复合物的制备
以叶酸-PEG 2000-Cy5(即FA-PEG 2000-Cy5)为例制备了叶酸-造影剂复合物。分别称取叶酸0.57g(1.28mmol)、NHS 0.37g(2.8mmol)、DCC 0.6g(2.8mmol)于250mL圆底烧瓶中，加DMSO75mL。25℃氮气保护下反应。TLC监测叶酸基本活化后，过0.22μm有机滤膜，加入3g NH 2-PEG 2000-Cy5(1.15mmol，购自西安瑞禧生物有效公司)和TEA 350μl。2小时后，HPLC监测反应完全。反应液经G50硅胶柱纯化(洗脱剂为0.01M的PBS，pH8.0)，获得FA-PEG 2000-Cy5。
叶酸自身不溶于水，引入PEG 2000提高了叶酸的水溶性。图3为所获得的FA-PEG 2000-Cy5的核磁氢谱。信号δ7.65、δ6.65为一组对称的“AA′BBˊ”自旋体系的信号峰，由此可确定该信号为叶酸分子中对位二取代苯环上的两组质子信号峰，说明分子中含叶酸结构。信号δ3.51为典型的PEG信号峰，进一步判定产物结构为FA-PEG 2000-Cy5。
实施例4：叶酸与B细胞表面的mIgM的特异性结合
利用同源建模和分子对接研究了叶酸在鼠IgM Fc域中的结合模式。使用Schrodinger2015软件进行了分子对接研究，以研究叶酸在IgM Fc中的结合模式。图4a显示了叶酸与小鼠mIgM的结合模式。可以看出，叶酸与mIgM的结合口袋位于Fc片段的Cμ3和Cμ4之间。图4b进一步提供了叶酸与小鼠IgM单体结合的作用力分析，其中叶酸的结合口袋位于IgM单体两条重链(链A和链B)的交界处，叶酸结构中的蝶啶片段可以与链A中的Phe287、Tyr288和Pro285和链B中的Glu52形成氢键；链B中的Phe55与叶酸的苯片段形成π-π相互作用；链B中的Lys25与羧基形成离子相互作用；叶酸与IgM的Fc片段的预测结合能为-10.929kcal/mol，亲和力达到10 -8mol水平。
使用mIgM阳性表达的脾脏B细胞考察了叶酸与mIgM的结合。用小鼠脾脏淋巴细胞分离液(购自索莱宝科技有限公司)提取BALB/c小鼠脾脏淋巴细胞，计数至2×10 6/mL，铺于24孔培养板(1mL/孔)。每孔分别加入抗-IgM(ab97230,1mg/mL)，使工作浓度为0、2.5、5、12.5 μg/mL。于37℃细胞培养24h后，将细胞分为FA-PEG-Cy5组和PEG-Cy5组(n＝3-4)，分别加入10μL的FA-PEG-Cy5(0.1mM,按照实施例3制得)或PEG-Cy5(0.1mM,购自西安瑞禧生物有限公司)，并于37℃摄取0.5h。离心洗涤细胞后，加入FITC-IgM和CD19-PE，于4℃孵育1h，标记细胞表面mIgM和CD19，用流式细胞仪检测小鼠脾脏B细胞mIgM表达水平以及细胞对FA-PEG-Cy5的摄取差异。图5提供了叶酸与mIgM结合的流式分析结果。图5a结果显示，随着抗-IgM抗体浓度的提高，小鼠脾脏B细胞mIgM可被有效消减。图5b结果显示，FA-PEG-Cy5的摄取随着抗-IgM浓度提高而逐渐降低，而PEG-Cy5的摄取影响不大。将mIgM表达水平与FA-PEG-Cy5的摄取能力进行线性拟合，如图5c所示，相关系数R 2＝0.82，表明FA-PEG-Cy5的摄取能力与mIgM表达水平正相关。
利用共聚焦显微镜进一步观察叶酸与mIgM的结合，将提取的BALB/c小鼠脾脏淋巴细胞计数为2×10 6/mL。分别加入抗IgM抗体(抗-鼠IgM,ab97230,10μg/mL)、游离叶酸分子(0.5M)、补体灭活血清(血清56℃热处理30min)或牛血清白蛋白(5％)，与细胞室温孵育1小时后再与FA-PEG 2000-Cy5(按照实施例3制得)于室温共孵育1小时。相似的，将牛血清白蛋白(5％)与细胞室温孵育1小时后再与PEG 2000-Cy5(购自西安瑞禧生物有限公司)于室温共孵育1小时。用CD19标记筛选B细胞，通过激光共聚焦显微镜观察叶酸与mIgM的结合。图6提供了叶酸与mIgM结合的激光共聚焦显微照片(Carl Zeiss LSM710,德国)，结果显示B细胞与叶酸(FA-PEG 2000-Cy5)的结合远高于PEG 2000-Cy5，且结合可被抗-IgM抗体和游离叶酸分子抑制，不受血清补体蛋白的影响，即与补体受体无关。以上结果说明叶酸可特异性结合靶向B细胞的mIgM。
实施例5：叶酸的脾脏边缘区域B细胞靶向性评价
给BALB/c小鼠(雄性，6周龄，斯莱克)经尾静脉注射叶酸-造影剂复合物FA-PEG 2000-Cy5(按照实施例3制得)或PEG 2000-Cy5(作为对照)，100nmol/只，每组三只。分别在1小时和4小时后处死小鼠，分离脾脏。组织匀浆后，根据FA-PEG 2000-Cy5或PEG 2000-Cy5的荧光标准曲线，利用荧光酶标仪测定脾脏匀浆中的叶酸浓度，数据显示在图7中(a:1小时；b：4小时，*p<0.05，**p<0.01，***p<0.001，单因素方差分析)。图7显示，注射1小时后，FA-PEG 2000-Cy5和PEG 2000-Cy5的脾脏蓄积量分别为9.35±2.10nmol/g组织和4.57±1.19nmol/g组织；注射4小时后，FA-PEG 2000-Cy5和PEG 2000-Cy5的脾脏蓄积量分别为10.20±2.44nmol/g组织和7.07±1.44nmol/g组织。FA-PEG 2000-Cy5在脾脏中的蓄积显著高于对照PEG 2000-Cy5，提示了FA-PEG 2000-Cy5的脾靶向能力。
另外取1或4小时处死小鼠的脾脏，分离脾脏淋巴细胞，进行流式分析(CytoFlex S,Beckman)。图8显示了分别施用FA-PEG 2000-Cy5或PEG 2000-Cy5后1小时或4小时的小鼠脾脏淋巴细胞的流式分选图(a:1h；c:4h)和统计分析(b:1h；d:4h)。结果表明，叶酸具有显著的B细胞靶向能力。
取1小时处死小鼠的脾脏，进行冷冻切片分析。图9显示了FA-PEG 2000-Cy5(a)和PEG 2000-Cy5(b)在脾脏中分布的免疫荧光照片，其中蓝色为DAPI标记的细胞核，绿色为CD19-PE抗体(553786，BD Pharmingen)标记的B细胞，红色为Cy5，黄色为绿色与红色的重叠色。结果 表明，叶酸主要分布在脾脏的边缘区域，与边缘B细胞具有明显重叠，即叶酸靶向于脾脏边缘B细胞。
由于脾脏边缘B细胞高表达mIgM，利用抗-IgM-FITC抗体(148445,Jackson)标记边缘B细胞。图10显示了FA-PEG 2000-Cy5在脾脏边缘B细胞分布的免疫荧光照片，其中蓝色为DAPI标记的细胞核，绿色为抗-IgM-FITC标记的边缘B细胞，红色为Cy5，黄色为绿色与红色的重叠色。结果表明，叶酸与边缘B细胞具有明显重叠，即叶酸靶向于脾脏边缘B细胞。
实施例6：叶酸修饰在介导体液免疫抑制和降低蛋白质生物大分子药物的抗药抗体中的作用
利用鸡卵白蛋白(OVA)作为模型蛋白研究了叶酸修饰在介导体液免疫抑制和在降低生物大分子药物的抗药抗体(ADA)中的作用。OVA具有强免疫原性，可诱导机体产生免疫反应，用其反复刺激小鼠可使小鼠体内产生大量特异性抗体。将BALB/c雌性小鼠(5周龄)随机分为FA-OVA(叶酸-OVA复合物，实施例1)组、OVA组和生理盐水对照组，每组6只。以10μg/只小鼠的剂量经尾静脉注射分别施用FA-OVA、OVA和生理盐水，每周一次，共三次。
在最后一次施用后一周时，收集脾脏淋巴细胞混悬液，通过流式细胞仪分析了脾脏B淋巴增殖及分化。分别用CD86作为细胞增殖标志，GL-7作为生发中心B细胞表面标志。图11a和11b分别提供了小鼠脾脏淋巴细胞的CD86流式分选图和统计分析结果。图11c和11d分别提供了小鼠脾脏淋巴细胞的GL7流式分选图和统计分析结果。由图11可以看出，OVA组小鼠的B细胞显著增殖且向生发中心B细胞分化，而在FA-OVA组小鼠中B细胞增殖和向生发中心B细胞的分化均被显著抑制，表明了叶酸修饰在抑制B细胞增殖和分化中的功效，造成B细胞无反应性。
在最后一次施用后一周时，收集小鼠血清，通过ELISA法测定血清中OVA特异性抗体的表达。结果显示在图12中，其中图12a、12b、12c和12d分别显示了OVA特异性IgE、IgG、IgG 1和IgG 2a的测定结果。数据显示，重复注射OVA诱导机体产生大量OVA特异性抗体，而FA-OVA组小鼠的抗体表达显著被抑制，与生理盐水对照组的水平相当。这表明用叶酸修饰可以显著降低抗原类物质如OVA的免疫原性，有效抑制体液免疫，减低生物大分子蛋白质药物的抗药抗体的产生。
另外，最后一次施用后一周时收集小鼠全血，进行血常规分析。结果显示，各组小鼠的血常规指标均未出现明显异常，说明叶酸修饰未产生明显副作用。数据显示在下表2中。
表2：
Figure PCTCN2021125346-appb-000002
Figure PCTCN2021125346-appb-000003
利用凝血因子VIII(FVIII)作为模型蛋白研究了叶酸修饰在降低蛋白质类生物大分子药物的抗药抗体(ADA)中的作用。将BALB/c雌性小鼠(5周龄)随机分为FVIII组、FA-FVIII组和生理盐水对照组，每组5只。以0.5IU/只小鼠的剂量经尾静脉注射分别施用FVIII、FA-FVIII(按照实施例1制得)和生理盐水，每周一次，共三次。在最后一次施用后一周时，收集小鼠血清，通过ELISA法测定血清中FVIII特异性抗体的表达。图13结果显示，FA-FVIII组的抗体水平显著低于FVIII组，表明叶酸修饰有效降低了蛋白质药物的抗药抗体产生。
实施例7：叶酸介导体液免疫抑制的作用
将BALB/c雌性小鼠(5周龄)随机分为4组，每组6只，分别做如下处理：(1)FA(ig)组：每只小鼠每天灌胃3mg游离叶酸，持续两周；(2)FA(ig)+OVA组：每只小鼠每天灌胃3mg游离叶酸，持续两周，并在第0天和第7天尾静脉注射OVA(10μg/只)；(3)OVA组：每只小鼠分别在第0天和第7天尾静脉注射OVA(10μg/只)；(4)生理盐水组：每只小鼠分别在第0天和第7天尾静脉注射生理盐水。然后，在第14、21和28天，所有小鼠分别尾静脉注射OVA和KLH(血蓝蛋白)在水中的混合溶液100ul(含100ug/mL OVA和100ug/mL KLH)。于第14、21和28天在施用所述混合溶液之前和在第35天收集血清，ELISA法检测血清中的特异性抗体。
图14分别显示了第14天(a)、第21天(b)和第28天(c)小鼠血清中OVA特异性抗体水平。结果显示，在第14天和第21天，FA(ig)+OVA组小鼠血清OVA特异性IgG水平明显低于OVA组，说明叶酸产生了体液免疫抑制效果，叶酸与OVA的组合施用可降低OVA特异性抗体产生。第28天时，FA(ig)+OVA组小鼠血清OVA特异性IgG水平与OVA组相当，说明叶酸的免疫抑制效果是可逆以及非持久性的，停止施用叶酸后，机体可恢复其免疫功能。
图15分别显示了第21天(a)、第28天(b)和第35天(c)小鼠血清中KLH特异性抗体水平。结果显示，FA(ig)组和生理盐水组小鼠KLH特异性抗体水平类似，都随注射次数增多而提高。结果同样证明，停止施用叶酸后，机体免疫功能恢复。
实施例8：叶酸和叶酸修饰在降低抗体类生物大分子药物的抗药抗体产生中的功效
阿达木单抗(Adalimumab)和英夫利昔单抗(Infliximab)为抗人肿瘤坏死因子(TNF)的人源化单克隆抗体，曲妥珠单抗(Trastuzumab)为抗人表皮生长因子受体-2(HER2)的人源化单克隆抗体。它们的反复注射易诱导机体产生针对所述单抗的抗体，影响所述单抗的药效。分别以阿达木单抗、英夫利昔单抗和曲妥珠单抗为模型单抗，考察了叶酸和叶酸修饰用于降低抗体药物的抗药抗体的效果。
将BALB/c雌性小鼠(6周龄)随机分为阿达木单抗组、FA-阿达木单抗组(按照实施例2制得)和生理盐水组，每组5只。对阿达木单抗组、FA-阿达木单抗组，在第0、7和14天以30μg抗体/只小鼠的剂量经皮下注射给药。对于生理盐水组，在第0、7和14天给小鼠经皮下注射生理盐水。在最后一次施用后一周(第21天)，收集小鼠血清，利用ELISA法检测血清中针对阿达木单抗的抗体。结果显示在图16a中。结果显示，FA-阿达木单抗组的抗体水平显著低于阿达木单抗组，表明叶酸修饰有效降低了抗体药物的抗药抗体产生。
将BALB/c雌性小鼠(6周龄)随机分为英夫利昔单抗组、FA灌胃+英夫利昔单抗组和生理盐水组，每组5只。对英夫利昔单抗组而言，在第0、7和14天以60μg抗体/只小鼠的剂量经尾静脉注射给药。对于FA灌胃+英夫利昔单抗组，给每只小鼠每天灌胃3mg游离叶酸，持续两周，并且在第0、7和14天以60μg抗体/只小鼠的剂量经尾静脉注射英夫利昔单抗。对于生理盐水组，在第0、7和14天给小鼠经尾静脉注射生理盐水。在最后一次施用后一周(第21天)，收集小鼠血清，利用ELISA法检测血清中针对英夫利昔单抗的抗体。结果显示在图16b中。结果显示，FA灌胃+英夫利昔单抗组的抗体水平显著低于英夫利昔单抗组，表明叶酸有效降低了抗体药物的抗药抗体产生。
将BALB/c雌性小鼠(6周龄)随机分为曲妥珠单抗组、叶酸-曲妥珠单抗组(按照实施例2制得)、叶酸灌胃+曲妥珠单抗组和生理盐水组，每组5只。对曲妥珠单抗组和叶酸-曲妥珠单抗组而言，在第0、7和14天以60μg抗体/只小鼠的剂量经尾静脉注射给药。对于叶酸灌胃+英曲妥珠单抗组，给每只小鼠每天灌胃3mg游离叶酸，持续两周，并且在第0、7和14天以60μg抗体/只小鼠的剂量经尾静脉注射曲妥珠单抗。对于生理盐水组，在第0、7和14天给小鼠经尾静脉注射生理盐水。在最后一次施用后一周(第21天)，收集小鼠血清，利用ELISA法检测血清中针对曲妥珠单抗的抗体。结果显示在图16c中。结果显示，叶酸灌胃+曲妥珠单抗组和叶酸-曲妥珠单抗组抗体水平显著低于曲妥珠单抗组，表明叶酸以及叶酸修饰有效降低了抗体药物的抗药抗体产生。
实施例9：叶酸在降低抗递药系统抗体产生中的功效
以脂质体为例，考察了叶酸在降低抗递药系统抗体的效果。
首先制备脂质体(sLip)。称取HSPC、Chol、mPEG 2000-DSPE(均购自艾伟拓(上海)医药科技有限公司)，摩尔比为52:43:5。将膜材溶于5mL氯仿中，减压旋转蒸发以除去氯仿，得到均匀脂质膜。真空干燥过夜除去残留有机溶剂。60℃水浴摇床震荡至脂质体膜水化完全，得到白色脂质体混悬液。使用微型挤出器将脂质体混悬液依次挤压通过200和100核孔膜，得到有淡蓝色乳光的液体，即脂质体(sLip)溶液。
将BALB/c雌性小鼠(6周龄)随机分为sLip组、sLip+FA(10μg)组、sLip+FA(50μg)组和生 理盐水组，每组4只。对sLip组小鼠而言，经尾静脉一次性给予5mg脂质体/kg小鼠体重的脂质体溶液；对sLip+FA(10μg)组小鼠而言，经尾静脉一次性给予5mg脂质体/kg小鼠体重的脂质体和10μg FA的混合溶液；对sLip+FA(50μg)组小鼠而言，经尾静脉一次性给予5mg脂质体/kg小鼠体重的脂质体和50μg FA的混合溶液；对生理盐水组小鼠而言，每只小鼠尾静脉一次性注射相同体积的生理盐水。所有小鼠在给药5天后收集血清，利用ELISA法检测血清中针对sLip的抗体浓度。结果显示在图17中，结果显示将叶酸与脂质体组合可有效降低抗脂质体抗体的产生，且随着叶酸浓度增加效果越好。
实施例10：叶酸修饰在预防过敏性休克中的功效
将5周龄BALB/c雌性小鼠随机分为FA-OVA组、OVA组和生理盐水组，每组10只。以10μg OVA/只小鼠的剂量经尾静脉注射给小鼠分别施用FA-OVA或OVA或等体积的生理盐水，每周一次，共三次。在最后一次施用后一周，给每只小鼠尾静脉注射10μg OVA(过敏原)。记录OVA刺激后2小时内的小鼠体温变化(图18)。同时收集小鼠血清，通过ELISA法测定血清中OVA特异性抗体的表达(图19)。图18显示，FA-OVA组小鼠的体温无明显变动，与生理盐水组基本一致；而OVA组小鼠出现了严重的过敏性休克，体温显著下降，并有两只小鼠死亡。图19显示，FA-OVA组与生理盐水组基本一致，而OVA组小鼠产生了大量的OVA特异性抗体，表明叶酸修饰可显著降低OVA所致的抗体产生。因此，叶酸修饰可有效地预防过敏原所致的过敏性休克。
实施例11：叶酸修饰在治疗过敏性休克中的功效
给5周龄BALB/c雌性小鼠皮下注射10μg OVA致敏，每周一次，共三次。在最后一次致敏后一周，以4μg/只小鼠的剂量给OVA致敏的小鼠分别尾静脉注射FA-OVA、OVA和生理盐水，每两天一次，共三次。在最后一次施用后，给小鼠尾静脉注射25μg OVA(过敏原)。记录注射后2小时的小鼠体温变化(图25)。同时收集小鼠血清，通过ELISA法测定血清中OVA特异性抗体水平(图26)。无OVA致敏但尾静脉注射OVA刺激的小鼠作为
Figure PCTCN2021125346-appb-000004
对照组。
图20显示了致敏小鼠分别用FA-OVA、OVA和生理盐水处置后再次用OVA刺激后的体温变化曲线。可以看到，FA-OVA治疗显示出脱敏作用，小鼠体温下降得到控制，并在2小时恢复正常。
图21显示了致敏小鼠分别用FA-OVA、OVA和生理盐水处置后再次用OVA刺激后的血清OVA特异性抗体水平。可以看到，在FA-OVA组中，OVA特异性抗体IgE(a)、IgG 1(b)和IgG 2a(c)的表达与生理盐水组相当，与OVA组相比显著下降，表明了叶酸修饰在治疗抗原过敏性疾病中的功效。
实施例12：叶酸修饰在自身免疫性脑脊髓炎中的功效
采用实验性自身免疫性脑脊髓炎(EAE)模型考察了叶酸修饰在自身免疫性脑脊髓炎中的功效。该模型如下建立：给动物注射诱导神经细胞髓鞘和周边蛋白的全蛋白或蛋白片段(如 PLP多肽)，引起动物的炎症和脱髓鞘症状，诱发动物的自身免疫反应，从而将所注射的自身蛋白视为异源而导致免疫系统攻击自身的髓鞘。
给雌性C57/BL6小鼠以10μg/只小鼠的剂量静脉注射PLP多肽(氨基酸序列为HSLGKWLGHPDKF)或叶酸-PLP复合物(FA-PLP)，每周一次，共三次。一周后，配制PLP多肽在弗氏完全佐剂中的乳液，PLP浓度为1μg/μL。在小鼠背部两侧取4点，每点皮下注射0.05mL PLP乳化剂致炎。注射PLP后2h和24h分别腹腔注射150ng百日咳毒素。每天观察小鼠发病情况，给小鼠按照5分法评分标准(Kono法)进行评分，绘制发病评分曲线。结果显示，PLP经叶酸修饰后，造成机体对PLP免疫耐受，有助于降低炎症反应。
实施例13：叶酸修饰在骨髓移植排斥中的功效
CD45.1小鼠捐献骨髓，CD45.2小鼠接收骨髓移植。骨髓移植前7天和后1天，以10μg/只小鼠的剂量给CD45.2小鼠尾静脉注射FA-Dby(叶酸修饰的Dby多肽，Dby的氨基酸序列为NAGFNSNRANSSRSS)或FA+Dby(叶酸与Dby在水中的物理混合液)。在接受骨髓移植前1天，每只CD45.2小鼠接收200cGy低剂量辐射。供体小鼠处死，在超净台中剪断股骨和胫骨，反复吹洗空腔，提取骨髓细胞，cPBS洗涤。给受体小鼠静脉注射5×10 6个细胞。骨髓移植后，小鼠每周取血一次，流式测量淋巴细胞嵌合情况，纯种CD45.1小鼠作为对照组。测量指标为CD45.1/CD45.2以及CD45.1/CD90.2。
实施例14：叶酸-人血白蛋白偶联物用于降低生物大分子及递药系统免疫原性的功效
以叶酸-人血白蛋白(FA-HSA，购自西安瑞禧生物有效公司)代表叶酸偶联物，考察其在降低生物大分子药物(OVA、曲妥珠单抗)及递药系统(slip)免疫原性中的功效。
将BALB/c雌性小鼠(5周龄)随机分为OVA组、FA-HSA+OVA组和生理盐水对照组，每组6只。分别做如下处理：(1)OVA组：每只小鼠分别在第0天、第7天和第14天尾静脉注射10μg OVA；(2)FA-HSA+OVA组：每只小鼠分别在第0天、第7天和第14天尾静脉注射10μg OVA和10μg FA-HSA混合溶液；(3)生理盐水组：每只小鼠分别在第0天、第7天和第14天尾静脉注射同等体积生理盐水。所有小鼠在第21天收集血清，ELISA法检测血清中的OVA特异性抗体(IgG)滴度。
用曲妥珠单抗替代OVA，调整剂量为60μg抗体/只小鼠，重复上述实验。
将BALB/c雌性小鼠(6周龄)随机分为sLip组(sLip按照实施例9制备)、sLip+FA-HSA组和生理盐水组，每组6只。对sLip组小鼠而言，经尾静脉一次性给予5mg脂质体/kg小鼠体重的脂质体溶液；对sLip+FA-HSA组小鼠而言，经尾静脉一次性给予5mg脂质体/kg小鼠体重的脂质体和10μg的FA-HSA的混合溶液；对生理盐水组小鼠而言，每只小鼠尾静脉一次性注射相同体积的生理盐水。所有小鼠在给药5天后，收集血清，利用ELISA法检测血清中针对sLip的抗体(IgM)滴度。
实施例15：叶酸偶联物用于降低生物大分子及递药系统免疫原性的功效
类似于实施例14，用叶酸-聚乙二醇 40kDa(FA-PEG 40kDa，购自西安瑞禧生物有效公司)代 替FA-HSA，考察FA-PEG 40KDa在降低生物大分子药物(OVA、曲妥珠单抗)和递药系统(slip)免疫原性中的功效。FA-PEG 40KDa的剂量为100nM每只小鼠。
制剂实施例
实施例A
可以以常规方式生产含有本发明的物质的注射溶液：
成分 量(每mL)
本发明的物质 5.0mg
聚乙二醇400 150.0mg
乙酸 适量至pH 5.0
注射用水 加至1.0mL
将本发明的物质溶于聚乙二醇400和注射用水(部分)的混合物中。通过添加乙酸调节pH至5.0。通过添加剩余量的水调节体积至1.0mL。过滤溶液，适当超量地装填入小瓶中，灭菌。
实施例B
可以以常规方式生产含有以下成分的口服片剂：
成分 每片片剂
本发明的物质 10mg
玉米淀粉 80mg
乳糖 95mg
硬脂酸镁 5mg
将以上成分混合均匀，压制成片剂。
实施例C
可以以常规方式生产含有以下成分的口服胶囊：
成分 每粒胶囊
本发明的物质 10.0mg
乳糖 95.0mg
玉米淀粉 20.0mg
滑石粉 5.0mg
将各组分过筛，混合，填充入胶囊壳中。
实施例D
可以以常规方式生产含有以下成分的气雾剂：
成分 含量(每100mL)
本发明的物质 80.0mg
油酸 适量
二氯二氟甲烷 适量
将本发明的物质和油酸混合，分剂量灌装入气雾瓶中，再加压注入二氯二氟甲烷。
出于清楚和理解的目的，已经通过说明和实施例详细解释了本发明。可以理解，上述说明和实施例仅仅是说明性的，而非限制性的，在本发明的范围内可以进行各种变通和改变。因此，本发明的范围不应当限于上述说明和实施例，而是应当取决于随后所附的权利要求以及满足这些权利要求的等价方式的全部范围。
本文引用的专利、专利申请和科学文献以如同它们各自被具体和单独提到的程度整体并入本文。在本文引用的任何参考文献和本说明书的具体教导之间的任何分歧应当在有利于后者的情况下来解决。同样，在本领域所理解的词语或短语的定义和本说明书中所具体教导的词语或短语的定义之间的任何分歧也应当在有利于后者的情况下来解决。

Claims (19)

  1. 叶酸或其可药用盐或酯或偶联物在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药剂中的用途。
  2. 根据权利要求1的用途，其中所述的叶酸或其可药用盐或酯或偶联物选自游离叶酸、叶酸的可药用盐、叶酸的可药用酯和叶酸的可药用偶联物(例如叶酸白蛋白偶联物、叶酸聚乙二醇偶联物等)，或它们的任意组合。
  3. 根据权利要求1或2所述的用途，其中所述B细胞是表达mIgM的B细胞，特别是高表达mIgM的B细胞，更特别是脾脏B细胞或淋巴结B细胞。
  4. 根据权利要求1-3任一项的用途，其中所述的免疫原性物质是生物大分子活性剂，并且所述药剂与生物大分子活性剂组合施用，例如在生物大分子活性剂的施用之前和/或期间施用，以减少抗生物大分子活性剂的抗药抗体的产生；
    优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；
    更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素。
  5. 根据权利要求1-3任一项的用途，其中所述的免疫原性物质是具有免疫原性的递药系统，例如微载体递药系统，优选选自脂质体、微球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束，并且所述药剂与所述递药系统组合施用，例如在所述递药系统的施用之前和/或期间施用，以减少抗具有免疫原性的递药系统的抗体的产生；优选地，所述递药系统载有如权利要求4中所定义的生物大分子活性剂。
  6. 根据权利要求1-3任一项的用途，其中所述药剂用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症，例如超敏反应、自身免疫性疾病或移植排斥；优选地，所述超敏反应选自过敏性休克、呼吸道超敏反应(如过敏性哮喘或过敏性鼻炎)和胃肠道超敏反应，所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、桥本甲状腺炎、毒性弥漫性甲状腺肿、强制性脊柱炎、自身免疫性脑脊髓炎、视神经脊髓炎谱系疾病、抗心磷脂综合征、血友病和银屑病，和所述移植排斥选自器官移植排斥、组织移植排斥(例如骨髓移植排斥)和复发性流产。
  7. 叶酸修饰的免疫原性物质在制备用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的药 剂中的用途。
  8. 根据权利要求7所述的用途，其中所述B细胞是表达mIgM的B细胞，特别是高表达mIgM的B细胞，更特别是脾脏B细胞或淋巴结B细胞。
  9. 根据权利要求7或8的用途，其中所述的免疫原性物质是生物大分子活性剂；
    优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；
    更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素。
  10. 根据权利要求7或8的用途，其中所述的免疫原性物质是导致机体产生异常免疫应答的致病抗原物质，优选是超敏反应、自身免疫性疾病或移植排斥的致病抗原物质，例如鸡卵白蛋白、蛋白脂蛋白多肽、Dby多肽、Uty、髓鞘碱性蛋白、变性IgG、甲状腺球蛋白、促甲状腺素受体、组蛋白、乙酰胆碱受体、主要组织相容性抗原(包括HLA-I类和HLA-II类)、次要组织相容性抗原、血型抗原、组织特异性抗原(如血管内皮细胞抗原、皮肤SK抗原)。
  11. 根据权利要求10的用途，其中所述药剂用于治疗或预防超敏反应、自身免疫性疾病或移植排斥；优选地，所述超敏反应选自过敏性休克、呼吸道超敏反应(如过敏性哮喘或过敏性鼻炎)和胃肠道超敏反应，所述自身免疫性疾病选自系统性红斑狼疮、类风湿性关节炎、桥本甲状腺炎、毒性弥漫性甲状腺肿、强制性脊柱炎、自身免疫性脑脊髓炎、视神经脊髓炎谱系疾病、抗心磷脂综合征、血友病和银屑病，和所述移植排斥选自器官移植排斥、组织移植排斥(例如骨髓移植排斥)和复发性流产。
  12. 叶酸修饰的抗肿瘤药或叶酸修饰的探针在制备用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的药剂中的用途。
  13. 根据权利要求12所述的用途，其中所述叶酸修饰的抗肿瘤药中的抗肿瘤药是可用于治疗或预防mIgM阳性表达的B细胞淋巴瘤的抗肿瘤药，优选是蒽环药物，例如阿霉素或表阿霉素；紫杉烷药物，例如紫杉醇、多烯紫杉醇或卡巴他赛；喜树碱药物，例如喜树碱、羟基喜树碱、9-硝基喜树碱或伊立替康；长春花碱药物，例如长春新碱或长春瑞滨；蛋白酶体抑制剂，例如硼替佐米或卡非佐米；内酯药物，例如小白菊内酯；环磷酰胺、依托泊苷、吉西他滨、阿糖胞苷、5-氟尿嘧啶、替尼泊苷、莫立替尼、埃博霉素、放线菌素D、米托蒽醌、丝裂霉素、博来霉素、顺铂、奥沙利铂、p53激活肽、蜂毒肽、蝎毒肽、雷公藤甲素、贝伐单抗或曲妥单抗；和其中所述叶酸修饰的探针中的探针是造影剂，优选是荧光物质，例如荧光素、 羧基荧光素(FAM)、异硫氰酸荧光素(FITC)、六氯荧光素(HEX)、香豆素6、近红外染料Cy5、Cy5.5、Cy7、ICG、IR820、DiR或DiD；或放射性物质，例如磁共振造影剂如Gd-DTPA或放射造影剂如 99mTc-DTPA。
  14. 用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的方法，该方法包括给个体施用有效量的如权利要求1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如权利要求7-11任一项所定义的叶酸修饰的免疫原性物质。
  15. 用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的方法，该方法包括给个体施用有效量的如权利要求12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针。
  16. 用于诱导B细胞免疫耐受、特别是用于减少抗免疫原性物质的抗体的产生和/或用于治疗或预防可通过B细胞免疫耐受介导的疾病或病症的如权利要求1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如权利要求7-11任一项所定义的叶酸修饰的免疫原性物质，特别是叶酸白蛋白偶联物、叶酸聚乙二醇偶联物、叶酸修饰的人血白蛋白、叶酸修饰的胰岛素、叶酸修饰的阿达木单抗、叶酸修饰的英夫利昔单抗、叶酸修饰的阿特珠单抗或叶酸修饰的曲妥珠单抗。
  17. 用于靶向诊断、治疗或预防mIgM阳性表达的B细胞淋巴瘤的如权利要求12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针，特别是叶酸修饰的卡巴他赛或叶酸修饰的雷公藤甲素。
  18. 药物组合物，包含有效量的如权利要求1-6任一项所定义的叶酸或其可药用盐或酯或偶联物和/或如权利要求7-11任一项所定义的叶酸修饰的免疫原性物质和/或如权利要求12或13所定义的叶酸修饰的抗肿瘤药或叶酸修饰的探针，以及任选的一种或多种可药用辅料。
  19. 叶酸或其可药用盐或酯或偶联物与免疫原性物质的组合产品，其中所述的免疫原性物质优选是生物大分子活性剂或具有免疫原性的递药系统；
    其中更优选地，所述的生物大分子活性剂选自多肽药物，例如p53激活肽、蜂毒肽、蝎毒肽、抗菌肽或胰岛素；或蛋白质药物，例如抗体药物且特别是单抗药物或多抗药物、干扰素、生长因子、生长因子抑制剂、酶、或白蛋白如人血白蛋白或鸡卵白蛋白，包括鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体或全人源单克隆抗体，例如肿瘤坏死因子α(TNFα)单抗(例如阿达木单抗、依那西普或英夫利昔单抗)、PD1/PD-L1单抗(例如纳武单抗、派姆单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗或卡瑞利珠单抗)；HER2单抗(例如曲妥珠单抗、帕妥珠单抗或拉帕替尼)；CD20单抗(例如利妥昔单抗、替伊莫单抗或托西莫单抗)；血管内皮生长因子/血管内皮生长因子受体(VEGF/VEGFR)单抗(例如贝伐单抗、雷珠单抗、阿柏西普或雷莫芦单抗)；表皮生长因子受体(EGFR)单抗(例如西妥昔单抗、帕尼单抗或耐昔妥珠单抗)；更优选地，所述的生物大分子活性剂选自阿达木抗体、英夫利昔单抗、阿特珠单抗、信迪利单抗、特瑞普利单抗、曲妥珠单抗、白蛋白和胰岛素；
    或者
    其中更优选地，所述的具有免疫原性的递药系统选自微载体递药系统，例如脂质体、微 球、微囊、纳米粒、纳米囊、脂质纳米圆盘或聚合物胶束。
PCT/CN2021/125346 2020-10-21 2021-10-21 叶酸和叶酸修饰在诱导B细胞免疫耐受和靶向mIgM阳性表达的B细胞淋巴瘤中的用途 WO2022083694A1 (zh)

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