WO2019201161A1 - 一种ifn与抗pd-l1抗体的融合蛋白及其应用 - Google Patents

一种ifn与抗pd-l1抗体的融合蛋白及其应用 Download PDF

Info

Publication number
WO2019201161A1
WO2019201161A1 PCT/CN2019/082360 CN2019082360W WO2019201161A1 WO 2019201161 A1 WO2019201161 A1 WO 2019201161A1 CN 2019082360 W CN2019082360 W CN 2019082360W WO 2019201161 A1 WO2019201161 A1 WO 2019201161A1
Authority
WO
WIPO (PCT)
Prior art keywords
ifn
tumor
seq
fusion protein
cells
Prior art date
Application number
PCT/CN2019/082360
Other languages
English (en)
French (fr)
Inventor
傅阳心
梁永
彭华
Original Assignee
中国科学院生物物理研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院生物物理研究所 filed Critical 中国科学院生物物理研究所
Priority to JP2020557311A priority Critical patent/JP7133241B2/ja
Priority to US17/048,106 priority patent/US20210147548A1/en
Priority to EP19789504.8A priority patent/EP3783035A4/en
Publication of WO2019201161A1 publication Critical patent/WO2019201161A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of genetic engineering and biomedical medical technology, and particularly relates to a fusion protein of IFN and anti-PD-L1 antibody, a pharmaceutical composition and a kit comprising the fusion protein, and a use of the fusion protein in treating tumor diseases.
  • PD-1 Programmed cell death protein 1
  • PD-1/PD-L1 blocking therapy can induce a long-lasting immune response to tumor cells in various cancer patients.
  • objective and effective immune response was only observed in a small number of patients who underwent PD therapy.
  • drug resistance of acquired PD therapy has received increasing attention, and the mechanism is still unclear. Therefore, the study analyzes why some tumors are unable to respond to PD therapy or develop drug resistance, which has become a top priority in PD-related research.
  • TILs tumor infiltrating lymphocytes
  • Type I interferons contain IFN ⁇ and IFN ⁇ , which activate T cells by promoting the maturation of dendritic cells (DCs), treating and presenting antigens, acting as a bridge between natural and adaptive immunity.
  • DCs dendritic cells
  • Type I interferons inhibit tumor cell proliferation and promote apoptosis, and are therefore approved for the treatment of clinically specific tumors, including lymphoma, melanoma, and renal cell tumors.
  • type I interferon signaling pathway-mediated APC and T cell activation play a crucial role in tumor radiotherapy and chemotherapy.
  • the expression of type I interferon in tumor tissues is very low.
  • Administration of exogenous type I interferon can inhibit tumor proliferation and survival, and activate anti-tumor immune responses, thus becoming an important research target.
  • type I interferon therapy will result in better tumor control.
  • high doses of type I interferon can induce severe side effects, including flu-like symptoms (fever, headache, etc.), vomiting, leukopenia, anemia, thrombocytopenia and other symptoms.
  • type I interferon up-regulates the expression of the immunosuppressive molecule PD-L1, which in turn inhibits the anti-tumor immune response and reduces the therapeutic effect. How to overcome the systemic toxicity of type I interferon and the immunosuppression brought about is an important problem to be solved.
  • IFN type I interferon
  • the present invention provides an IFN-anti-PD-L1 antibody (IFN-anti-PD-L1) fusion protein which can simultaneously target PD-L1 and IFN receptors, It was observed in the experiment that the IFN-anti-PD-L1 fusion protein was able to aggregate in tumor tissues, significantly increasing antigen cross-presentation and overcoming PD-L1-mediated immunosuppression.
  • the IFN-anti-PD-L1 fusion protein can simultaneously (re)activate T cells by releasing immunosuppressive signals and providing costimulatory signals, and can be used as a new generation of anti-PD-L1 antibodies for the treatment of tumor diseases.
  • the present invention provides a fusion protein which is fused to a PD-L1 binding protein by interferon (IFN) to form IFN-anti-PD-L1, which is a homodimeric protein or a heterologous source.
  • IFN interferon
  • the homodimeric protein of the present invention comprises a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide being identical; the first polypeptide and the second polypeptide are from N-terminal to C-
  • the terminal sequence comprises IFN, a PD-L1 binding protein, and an immunoglobulin Fc region.
  • the heterodimeric protein of the present invention comprises a first polypeptide and a second polypeptide, the first polypeptide being different from the second polypeptide; the first polypeptide comprising a PD-L1 binding protein, the The dipeptide comprises IFN and an immunoglobulin Fc region, and the IFN is located at the N-terminus of the Fc region; the first polypeptide comprises an Fc region and the second polypeptide comprises an Fc region derived from immunoglobulins having the same or different subtypes protein.
  • the interferon (IFN) of the present invention may be selected from a type I interferon, a type I interferon mutant, a type II interferon and/or a type III interferon, such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN. - ⁇ 1 (IL-29), IFN- ⁇ 2 (IL-28a), IFN- ⁇ (IL-28b), IFN- ⁇ , etc., preferably type I interferon, preferably IFN- ⁇ 4, more preferably IFN- ⁇ 4 mutant,
  • the IFN may be derived from a human or murine source; preferably IFN- ⁇ 4 (SEQ ID NO.
  • IFN- ⁇ 4 mutant more preferably an IFN- ⁇ 4 mutant; more preferably the mutant mIFN- ⁇ 4 (L30A) (SEQ ID NO. 25), mIFN - ⁇ 4 (R144A) (SEQ ID NO. 27), mIFN- ⁇ 4 (A145G) (SEQ ID NO. 29), mIFN- ⁇ 4 (R149A) (SEQ ID NO. 31), mIFN- ⁇ 4 (S152A) (SEQ ID NO. 33), hIFN- ⁇ 2 (Q124R) (SEQ ID NO. 35).
  • the immunoglobulin Fc region of the invention may be selected from the constant region amino acid sequence of IgG1, IgG2, IgG3 and/or IgG4, preferably IgG1.
  • IgG1 has strong ability to induce ADCC and CDC effects and long serum half-life, and is the most common antibody subtype of antibody drugs;
  • IgG2 and IgG4 have weak ability to induce ADCC and CDC effects, but serum half-life is longer. .
  • the PD-L1 binding protein of the present invention may be selected from the group consisting of an anti-PD-L1 antibody (intact antibody), a single-chain antibody (ScFv), a Fab fragment, and an F(ab') 2 fragment;
  • the anti-PD-L1 antibody is preferably selected from: Tecentriq, Bavencio, Imfinzi, KN035, CS1001, KL-A167, SHR-1316 and/or YW243.55.S70; more preferably a single-chain antibody (ScFv) that binds PD-L1; more preferably YW243.55.S70.
  • the first polypeptide and the second polypeptide of the homodimeric protein of the present invention preferably comprise the amino acid sequence shown in SEQ ID NO. 3; the heterodimeric first polypeptide preferably comprises SEQ ID NO.
  • the amino acid sequence shown, the second polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 2, 37, 39, 41, 43, 45, 47.
  • the invention also provides for the use of a fusion protein that administers IFN-anti-PD-L1 to tumor cells that are capable of inhibiting tumor cell growth and/or migration.
  • the invention provides an application of an IFN-anti-PD-L1 fusion protein in the treatment of tumor diseases; and an application of the IFN-anti-PD-L1 fusion protein in preparing a medicament or a kit.
  • the tumor is a tumor or a late tumor which is ineffective in the treatment of PD-1/PD-L1 alone, and more preferably a tumor which is resistant or ineffective against the treatment of the PD-1/PD-L1 antibody alone; Cell lymphoma, colon cancer, and melanoma.
  • the present invention provides a pharmaceutical preparation or a pharmaceutical composition comprising the active protein IFN-anti-PD-L1 of the present invention.
  • the present invention provides a kit comprising the fusion protein IFN-anti-PD-L1 of the present invention.
  • the invention provides a nucleic acid molecule encoding the fusion protein IFN-anti-PD-L1 of the invention.
  • the nucleic acid molecule 1) the nucleic acid molecule encoding the homodimer is preferably the nucleotide sequence shown in SEQ ID NO.
  • the nucleic acid molecule encoding the heterodimer may be selected from the nucleotide sequences shown in SEQ ID NO. 4, 5, 7, 8, 9, 10, 38, 40, 42, 44, 46, 48; Preferred are SEQ ID NO. 4 and SEQ ID NO. 5, SEQ ID NO. 4 and SEQ ID NO. 40, or SEQ ID NO. 4 and SEQ ID NO.
  • the present invention provides a vector comprising the above nucleic acid molecule.
  • the present invention provides a cell comprising the fusion protein IFN-anti-PD-L1 of the present invention or a nucleic acid molecule encoding a fusion protein for use in the production of a fusion protein.
  • the cells are selected from non-human mammalian cells, preferably CHO and HEK293 cells.
  • the invention provides a method of treating a tumor comprising administering to a cancer patient an effective amount of the fusion protein IFN-anti-PD-L1.
  • the tumor, PD-1/PD-L1 blocks tumors that are ineffective in treatment alone.
  • the present invention provides a method of treating PD-1/PD-L1 blocking treatment of a null or advanced tumor alone, comprising administering to a patient an effective amount of an IFN- ⁇ and an anti-PD-L1 antibody; and at the same time, the present invention provides a The use of IFN- ⁇ and an anti-PD-L1 antibody for the preparation of a pharmaceutical composition, a pharmaceutical preparation or a kit; the present invention also provides a pharmaceutical composition, a pharmaceutical preparation or the like comprising IFN- ⁇ and an anti-PD-L1 antibody Kit; the IFN- ⁇ described above needs to be administered intratumorally.
  • the present invention provides a combination therapy of anti-PD-1/PD-L1 antibody and IFN-anti-PD-L1, and an anti-PD-1/PD-L1 antibody and IFN-anti-PD-
  • a pharmaceutical composition or kit for an L1 fusion protein comprises administering to the patient an effective amount of an anti-PD-1/PD-L1 antibody and IFN-anti-PD-L1, either sequentially or simultaneously.
  • the tumor is a tumor that is resistant or ineffective against PD-1/PD-L1 blockade alone, or an advanced tumor; preferably a tumor that is resistant or ineffective against the PD-1/PD-L1 antibody alone.
  • the patient of the tumor has a peripheral lymphocyte transport defect/disorder-related disease in which the peripheral lymphocytes of the patient cannot migrate to the tumor tissue.
  • the present invention provides the use of a fusion protein IFN-anti-PD-L1 in combination with an anti-PD-1/PD-L1 antibody for the preparation of a pharmaceutical composition or kit for treating tumors.
  • the tumor is a tumor that is ineffective against PD-1/PD-L1 blockade alone, or an advanced tumor; preferably a tumor that is resistant or ineffective against the PD-1/PD-L1 antibody alone.
  • the patient of the tumor has a peripheral lymphocyte transport defect/disorder-related disease in which the peripheral lymphocytes of the patient cannot migrate to the tumor tissue.
  • the present invention provides a use of a IFN- ⁇ and an anti-PD-L1 antibody for the preparation of a pharmaceutical composition, a pharmaceutical preparation or a kit for treating a tumor which is ineffective for the treatment of PD-1/PD-L1 alone or for advanced tumors.
  • the IFN- ⁇ needs to be administered intratumorally.
  • the present invention provides a pharmaceutical composition, pharmaceutical preparation or kit comprising an IFN- ⁇ and an anti-PD-L1 antibody, which is administered to a tumor.
  • the present invention provides the use of a fusion protein IFN-anti-PD-L1 for upregulating IFN receptor expression in leukocytes; the leukocytes are preferably CD45+ cells; and the IFN receptor is preferably IFNAR.
  • a use of the fusion protein IFN-anti-PD-L1 for the preparation of a composition for upregulating IFN receptor expression in leukocytes is provided.
  • the present invention provides the use of a fusion protein IFN-anti-PD-L1 for activating DC cells or TIL cells.
  • a use of the fusion protein IFN-anti-PD-L1 for the preparation of a composition for activating DC cells or TIL cells is provided.
  • the invention provides the use of a fusion protein IFN-anti-PD-L1 for activating tumor-resident T cells.
  • a use of the fusion protein IFN-anti-PD-L1 for the preparation of a composition for activating tumor-resident T cells is provided.
  • the present invention provides an IFN- ⁇ 4 mutant which is a weak affinity mutation to a receptor, including mIFN- ⁇ 4 (L30A) (SEQ ID NO. 25), mIFN- ⁇ 4 (R144A) (SEQ ID NO. 27) ), mIFN- ⁇ 4 (A145G) (SEQ ID NO. 29), mIFN- ⁇ 4 (R149A) (SEQ ID NO. 31), mIFN- ⁇ 4 (S152A) (SEQ ID NO. 33), and/or hIFN- ⁇ 2 (Q124R) (SEQ ID NO. 35).
  • the present invention provides an isolated nucleic acid molecule encoding the above IFN- ⁇ 4 mutant, the nucleotide sequence of which is shown in SEQ ID NO. 26, 28, 30, 32, 34, 36.
  • the present invention provides the use of an IFN- ⁇ 4 mutant for the preparation of a fusion protein or drug for treating a tumor.
  • tumor site refers to an in vivo or ex vivo location that contains or is suspected of containing tumor cells.
  • the tumor site includes a solid tumor and a location near or adjacent to where the tumor is growing.
  • administering refers to systemic and/or topical administration.
  • systemic administration refers to administration non-locally such that the substance being administered may affect several organs or tissues throughout the body; or thus the substance being administered may cross several organs or tissues throughout the body to reach the target site point.
  • administration to a subject's circulatory system can cause the therapeutic product to be expressed from the administered vector in more than one tissue or organ, or can cause the therapeutic product to be expressed at the specific site by the administered vector, for example This is due to natural tropism or due to operably linked to tissue-specific promoter elements.
  • systemic administration encompasses various forms of administration including, but not limited to, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intratumoral administration, oral administration, and the like. .
  • topical administration refers to administration at or around a specific site.
  • topical administration encompasses various forms of administration, such as direct injection to a particular site or injection into it (e.g., intratumoral administration).
  • the term "therapeutically effective amount” refers to an interferon of the invention required to achieve a disease or condition for treatment (eg, a tumor/cancer, eg, to regress a tumor or reduce the size of a tumor), or The amount of the components in the kit of the invention.
  • the effective amount can be determined for a particular purpose by practice, in a conventional manner.
  • the therapeutically effective amount can be an amount required to achieve: reducing the number of cancer cells; reducing tumor size; inhibiting (ie, slowing or stopping) infiltration of cancer cells into peripheral organs; inhibiting (ie, slowing or stopping) Tumor metastasis; inhibiting tumor growth; and/or alleviating one or more symptoms associated with cancer.
  • antibody encompasses, for example, monoclonal antibodies, polyclonal antibodies, single chain antibodies, antibody fragments (which exhibit the desired biological or immunological activity).
  • immunoglobulin Ig
  • the antibody can specifically target tumor antigens, such as surface tumor antigens, such as EGFR, CD4, CD8, Neu, and the like.
  • the "tumor” of the present invention may be selected from B cell lymphoma, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nerve Systemic cancer, esophageal cancer, cervical cancer, melanoma, uterus or endometrial cancer, oral or laryngeal cancer, liver cancer, kidney cancer, cholangiocarcinoma, small bowel cancer or appendical cancer, salivary gland cancer, thymic cancer, adrenal cancer, flesh Tumor, chondrosarcoma, lipoma, testicular cancer, and malignant fibrous histiocytoma.
  • the "tumor cells” of the present invention may be selected from B cell lymphoma, lung cancer, bronchial carcinoma, colorectal cancer, prostate cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral Nervous system cancer, esophageal cancer, cervical cancer, melanoma, uterus or endometrial cancer, oral or laryngeal cancer, liver cancer, kidney cancer, cholangiocarcinoma, small bowel cancer or appendical cancer, salivary gland cancer, thymic cancer, adrenal cancer, Cells produced by cancers of osteosarcoma, chondrosarcoma, lipoma, testicular cancer, and malignant fibrous histiocytoma.
  • the "application” or “use” as used in the present invention may mean both an application for the purpose of disease treatment and an application for non-therapeutic purposes, such as scientific research.
  • the IFN-anti-PD-L1 fusion protein provided by the present invention simultaneously targets PD-L1 and IFN receptors, and the data indicate that activation of IFN signaling in TME can enhance PD against advanced tumors by inducing more powerful T cell activation. -1/PD-L1 treatment.
  • the IFN-anti-PD-L1 fusion protein provided by the present invention, the data shows that the anti-PD-L1 antibody (PD-L1 binding protein) can be used for specifically delivering immunomodulatory molecules into tumor tissues with minimal toxicity.
  • the present invention lays the foundation for the development of novel anti-PD-L1 antibodies for tumor targeting.
  • the IFN-anti-PD-L1 fusion protein provided by the present invention can produce multiple feedforward responses targeting PD-L1 blockade and IFN- ⁇ receptor (IFNAR) activation in the tumor microenvironment. Not only does it increase the targeting effect, but it also enhances the response to IFN treatment, achieving a synergistic effect against PD-L1 and IFN to overcome PD-1/PD-L1 treatment and IFN resistance.
  • IFNAR IFN- ⁇ receptor
  • IFN-anti-PD-L1 fusion protein provided by the present invention is used in combination with PD-1/PD-L1 blocking, IFN-anti-PD-L1 completely eliminates most PD-1/PD-L1 resistance. Tumors are induced to produce a memory T cell immune response.
  • the fusion protein provided by the invention has two structures of homodimer and heterodimer, and has high IFN receptor binding affinity, more effective antiviral activity and excellent tumor targeting in vivo and in vitro, respectively. Sex, serum half-life and tumor control in vivo.
  • the fusion protein provided by the present invention comprises a weak affinity mutant IFN ⁇ , which has more specific target cell binding ability and avoids the peripheral off-target effect of IFN.
  • FIG. 1 Topical administration of type I IFN overcomes the resistance of advanced tumors to PD-1/PD-L1 blockade.
  • mice bearing advanced A20 tumors >100 mm 3
  • the survival curve is displayed.
  • FIG. 1 Construction and properties of IFN-anti-PD-L1 fusion protein.
  • (a) Schematic representation of homodimers or heterodimers of the IFN-anti-PD-L1 fusion protein. scFv, single-chain variable region fragment.
  • (bc) shows a flow cytogram of the binding of the proteins shown in IFNAR1 -/- A20 cells (b) and PD-L1 -/- A20 cells (c). The numbers represent the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • the biological activity of the IFN-anti-PD-L1 protein was measured by an antiviral infection bioassay. L929 cells were incubated overnight with each protein prior to infection with VSV-GFP virus.
  • mice were injected intravenously with 25 ⁇ g of the indicated protein. The protein concentration in tumor tissue (h) or serum (i) was measured by ELISA at different time points. Data represent mean ⁇ SEM and represent at least two independent experiments. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • IFN-anti-PD-L1 fusion protein has less toxic side effects and good in vivo anti-tumor effects.
  • (a-b) Tumor-bearing mice were intravenously injected with 100 ⁇ g of IFN-anti-PD-L1 heterodimer or IFN-anti-HBs protein on day 0 and day 4. The mouse survival curve (a) and body weight change (b) are shown.
  • (c) 100 ⁇ g of IFN-anti-PD-L1 heterodimer or IFN-anti-HBs protein was injected intravenously into MC38 tumor-bearing mice. Serum was collected at 6 or 24 hours after injection. Cytokine levels in serum were measured by cytokine microsphere detection technique (CBA).
  • CBA cytokine microsphere detection technique
  • PD-L1 expressed in host or tumor cells is effective in mediating the anti-tumor effect of the IFN-anti-PD-L1 fusion protein.
  • (a) PD-L1 expression in WT A20, PD-L1 -/- A20, WT MC38, and PD-L1 -/- MC38 cells was measured by flow cytometry.
  • IFNAR expressed by host cells is essential for tumor control.
  • PD-1 blockade further ensures that IFN-anti-PD-L1 induces a feed-forward anti-tumor response.
  • (a) Treatment of A20 tumor-bearing Balb/c mice with 20 ⁇ g of IFN-anti-PD-L1 on day 15 and/or with 100 ⁇ g of anti-PD-1 antibody on days 14 and 17 (n 4-5 ). Tumor growth was measured twice a week.
  • (b) Treatment of B16 tumor-bearing C57BL/6 mice with 25 ⁇ g of IFN-anti-PD-L1 on days 11 and 14 and/or on days 12 and 15 with 100 ⁇ g of anti-PD-1 antibody (n 3) -5).
  • mice (c) after (a) combination therapy complete tumor regression in mice (n 4) with 2.5 ⁇ 10 7 cells were reseeded th A20 tumor.
  • the first experimental mice inoculated with A20 cells were used as controls.
  • mice Treat mice with IFN-anti-PD-L1 and/or anti-PD-1 as described in (a). After 12 days of treatment, the tumor draining lymph nodes were isolated to prepare a single cell suspension.
  • Tumor infiltrating DC (CD11c+) and T (CD8+) cells were isolated from A20 tumor-bearing mice and co-cultured in the presence of irradiated A20 cells. IFN ⁇ or anti-PD-L1 antibody was added to the medium. After three days, the supernatant was collected and IFN ⁇ levels were measured by CBA.
  • IFN-anti-PD-L1 heterodimer specifically targets tumor tissue and induces less toxicity.
  • 30 ⁇ g of IFN-anti-PD-L1 was injected intravenously into MC38 tumor-bearing mice. Tissues were collected on days 1, 3 and 5 after injection. The concentration of the fusion protein was determined by ELISA.
  • MC38 tumor-bearing mice were treated as in Figure 3c and serum was collected 6 or 24 hours after injection. Cytokine levels in serum were measured by CBA. Data indicate mean ⁇ SEM and represents two experimental replicates.
  • IFN-anti-PD-L1 upregulates the expression of CD80 in tumor infiltrating DCs. Two days after IFN-anti-PD-L1 treatment, MC38 tumor tissues were isolated. Flow cytometry was used to detect the expression of CD80 in tumor infiltrating DC (CD11c+MHCII+).
  • NK and CD4+ T cells have no significant effect on the anti-tumor response mediated by anti-PD-1 antibody and IFN-anti-PD-L1 combination therapy.
  • mice were treated according to Figure 6e, and the spleens were isolated to prepare a single cell suspension. Cells were co-cultured with irradiated or non-irradiated A20. An IFN ⁇ ELISPOT assay was performed.
  • FIG. 10 Schematic model of anti-tumor effects mediated by anti-PD-L1 antibody (IFN-anti-PD-L1) with IFN.
  • Anti-PD-L1 specifically carries IFN to tumor tissue (1).
  • Interferon-mediated up-regulation of PD-L1 enhances tumor-specific targeting (2 and 3).
  • the antibody blocks the PD-L1/PD-1 signaling pathway to release the immune brake signal (3).
  • IFN-anti-PD-L1 up-regulates IFNAR expression (4), which further makes tumors sensitive to treatment (5). Taken together, these factors (re)activate T cell responses to control tumor growth (6).
  • FIG. 12 Detection of IFN ⁇ -Fc mutant activity.
  • the biological activity of the IFN ⁇ -Fc mutant was measured by an antiviral infection bioassay. L929 cells were incubated overnight with each protein prior to infection with VSV-GFP virus. After a further 30 hours of culture, the percentage of virus-infected cells was determined by flow cytometry, and the inhibition rate and EC50 value of different concentrations of protein on cell infection were calculated. Both R144A and A145G mutants have weaker viability and are potentially preferred targets.
  • FIG. 13 Mutant IFN-anti-PD-L1 in vitro targeting assay.
  • the ratio of EC50 of PD-L1-A20 cells to PD-L1+A20 cells can reflect the targeting of this bispecific protein. The ratio can be found by normalizing the ratio of wt-mIFNa4-Fc. R144A and A145G are the two most targeted mutations.
  • mice Female (6-8 weeks old) BALB/c mice and C57BL/6 mice were purchased from Vitallihua (Beijing, China). All mice were housed in the animal house of the Institute of Biophysics under specific pathogen free (SPF) conditions. Animal care and experiments were conducted in accordance with the guidelines of the Institute of Biophysics of the Chinese Academy of Sciences, following the approval of the ICCUC (Institutional Laboratory Animal Care and Use Committee). PD-L1 -/- and IFNAR1 -/- mice were housed under SPF conditions at the UT Southwestern Medical Center. The animal program complies with NIH guidelines. The study was approved by the Animal Care and Use Committee of the UT Southwest Medical Center.
  • SPPF pathogen free
  • 293F cells were supplied by Dr. Xu Wei (Jining Jerry, Suzhou, Jiangsuzhou, China) and cultured in SMM 293-TI medium (M293TI, Sino Biological).
  • A20, MC38 and L929 cell lines were purchased from ATCC (Manassas, VA).
  • the anti-PD-1 blocking antibody (4H2) was from Bristol-Myers Squibb (Redwood City, CA).
  • Anti-PD-L1 antibody (10F.9G2) and anti-IFNAR1 antibody (MAR1-5A3) were purchased from BioXCell (West Riverside, NH).
  • Anti-CD8 (TIB210) and anti-CD4 deletion antibody (GK1.5) were prepared by the laboratory.
  • Anti-sialic acid GM1 antibody was purchased from Biolegend (San Diego, CA).
  • Heterodimer The variable regions of the light and heavy chains of the PD-L1 binding protein (YW243.55.S70) sequence were synthesized according to the patent (Patent No.: US8217149B2). The light and heavy chain sequences were ligated through the GGGGSGGGGSGGGGS linker, and human IgG1 Fc (SEQ ID NO. 15) was inserted into the C-terminus of the heavy chain, designated ScFv (PD-L1)-Fc (SEQ ID NO. 1), to obtain a different The first polypeptide of the source dimer.
  • the coding nucleic acid sequence of ScFv(PD-L1)-Fc SEQ ID NO.
  • Heterodimerization of PD-L1 binding protein and IFN ⁇ was generated using the previously reported knob-to-holes technique.
  • the plasmid was transiently transfected into 293F cells at a ratio of 1:2. The supernatant was collected on the 7th day after transfection.
  • the fusion protein was purified using a Protein A-Sepharose column according to the operating manual (Repligen).
  • heterodimer In the preparation of the heterodimer, different type I interferons are also used to prepare the second polypeptide mIFNb-Fc (encoding nucleic acid as shown in SEQ ID NO. 7), hIFN ⁇ 2-Fc (coding nucleic acid such as SEQ ID) NO.8), hIFNb-Fc (encoding nucleic acid as shown in SEQ ID NO. 9), mIFN ⁇ -Fc (encoding nucleic acid as shown in SEQ ID NO. 10), the heterodimers produced are all Good inhibition of tumor cell proliferation; especially in the above heterodimeric fusion protein, IFN ⁇ -Fc is more effective; and mIFN ⁇ 4-Fc and Anti-PD-L1 constitute the most heterodimeric fusion protein. Excellent, relevant detailed comparison data is not shown here.
  • the IFN- ⁇ 4 C-terminus of mouse is ligated to the N-terminus of ScFv (PD-L1)-Fc, and the first polypeptide and the second polypeptide of the homodimeric fusion protein are obtained (SEQ ID NO. 3).
  • the nucleotide sequence (SEQ ID NO. 6) was cloned into the pEE12.4 vector, and after transfection, a homodimeric protein was spontaneously formed by dimerization of Fc.
  • the binding of the fusion protein was detected using PE-anti-human IgG Fc (eBioscience).
  • Specific antibodies anti-PD-L1 antibody (10F.9G2), anti-IFNAR1 antibody (MAR1-5A3), anti-CD45 antibody (30-F11), anti-CD80 antibody (16-10A1), anti-CD86 antibody (GL1) from BioLegend or eBioscience.
  • the cells were suspended in FACS buffer (1% bovine serum albumin and 0.05% NaN3), blocked with anti-CD16/32 antibody (anti-Fc ⁇ III/II receptor, clone 2.4G2) for 30 minutes, then plated with specific antibodies on ice. Dye for 30 minutes. Samples were analyzed on a FACSCalibur or Fortessa flowcytometer (BD Biosciences). Data was analyzed using FlowJo software (TreeStar).
  • L929 mouse fibroblasts sensitive to VSV infection were used to quantify the biological activity of IFN.
  • Cells were incubated overnight with serial dilutions of IFNa-Fc or IFN-anti-PD-L1 at 37 °C.
  • A20 cells (3 x 10 6 ) were injected subcutaneously (sc) into the right side of Balb/c mice. Mice were intravenously injected with 30 ⁇ g of IFN ⁇ -Fc or IFN-anti-PD-L1 on day 15. Different mouse tissues were collected after perfusion on day 3, and the levels of human Fc in homogenate extracts of different organs were determined by ELISA.
  • mice were injected subcutaneously into the right side of Balb/c mice. 20 ⁇ g of IFN-anti-PD-L1 was injected intravenously. To block PD-1 signaling, mice were treated intravenously with 100 ⁇ g of anti-PD-1 antibody (4H2) one day prior to IFN-anti-PD-L1 treatment, once every two weeks. For CD8+ T cell deletion, 200 ⁇ g of anti-CD8 antibody (TIB210) was intraperitoneally injected one day before IFN-anti-PD-L1 treatment. To block type I IFN signaling, 100 ⁇ g of anti-IFNAR1 antibody (MAR1-5A3) was injected one day prior to IFN-anti-PD-L1 treatment.
  • MAR1-5A3 anti-IFNAR1 antibody
  • mice C57BL/6 mice were injected subcutaneously with 5 x 10 5 MC38 cells on the right side. Mice were injected intravenously with 25 ⁇ g of IFN-anti-PD-L1 twice. Tumor volume was measured twice a week and calculated as (length x width x height/2). In order to block lymphocyte trafficking, mice were intraperitoneally injected with 25 ⁇ g of FTY720, and 20 ⁇ g of FTY720 was given to block every other day.
  • ELISPOT detects tumor antigen-specific T cells
  • Single cell suspensions were prepared by isolating lymph nodes (LNs) or spleens from tumor-bearing mice.
  • LNs lymph nodes
  • A20 tumor cells were irradiated with a single dose of 60 Gys (10 Gys/min, 6 minutes).
  • Splenocytes or LN cells were co-cultured with irradiated tumor cells for 4 hours at a ratio of 4:1.
  • IFN- ⁇ production was determined using the IFN- ⁇ ELISPOT assay kit according to the manufacturer's protocol (BD Biosciences). Cytokine spots were counted using an immunospot analyzer (CTL).
  • CTL immunospot analyzer
  • Tumor tissues were collected, cut into small pieces, and resuspended in digestion buffer (RPMI-1640 medium containing 1 mg/ml collagenase IV and 100 ⁇ g/ml DNase I). After digestion at 37 ° C for 45 min, a single cell suspension was prepared through a 70 ⁇ m cell strainer, and CD8+ T cells and DCs (MHCII + CD11c+) were sorted by FACS. T cells, DCs and irradiated tumor cells were cultured in a ratio of 10:1:2.5 at IFN ⁇ (2 ng/ml) or anti-PD-L1 antibody (10 ⁇ g/ml). After three days, the supernatant was collected and the IFN- ⁇ level was measured by CBA.
  • digestion buffer RPMI-1640 medium containing 1 mg/ml collagenase IV and 100 ⁇ g/ml DNase I.
  • Example 1 Topical administration of type I IFN overcomes tumor resistance to PD-1/PD-L1 blockade therapy
  • Type I IFN a potent cytokine that enhances cross-presentation of cytotoxic T cells, could improve PD-1/PD-L1 blockade.
  • Advanced tumors were treated with PD-L1 blockade and IFN ⁇ .
  • IFN ⁇ 4 anti-PD-L1 antibody nor IFN ⁇
  • combination therapy induced a stronger anti-tumor effect, resulting in complete eradication of tumors in all treated mice (Fig. 1d).
  • a similar synergistic effect was found in another tumor model, MC38 (Fig. 1e).
  • Type I IFN signaling acts locally in the tumor microenvironment (TME) because intratumoral delivery of IFNa effectively controls tumor growth (Fig. 1f). In contrast, when IFN is delivered systemically, these effects completely disappear.
  • type I IFN has a synergistic effect with PD-1/PD-L1 blocking therapy and is capable of controlling advanced tumors. This also indicates that interferon needs to be targeted to TME to achieve an optimized anti-tumor effect.
  • Example 2 Construction of an IFN-anti-PD-L1 fusion protein for specific delivery of IFN to tumor tissues
  • PD-L1 has been reported to be highly expressed in tumor tissues. Recent studies have shown that anti-PD-L1 antibodies specifically accumulate in PD-L1-positive tumor tissues. Moreover, in addition to their anti-tumor function, IFNs strongly induce PD-L1 expression, thus inhibiting T cell responses to tumors.
  • the present invention proposes to use IFN and anti-PD-L1 antibody to construct fusion protein IFN-anti-PD-L1, which can further up-regulate PD-L1 in tumor Expression in the tissue, resulting in an increase in antibody accumulation.
  • the present invention produces a fusion protein containing a single-chain variable region fragment of anti-PD-L1 antibody [scFv(PD-L1)] and IFN ⁇ in the form of a homodimer or a heterodimer (Fig. 2a) ).
  • scFv(PD-L1) single-chain variable region fragment of anti-PD-L1 antibody
  • IFN ⁇ IFN- ⁇ receptor 1
  • IFNAR1 -/- A20 cells expressing PD-L1 the fusion protein had similar affinity to the anti-PD-L1 antibody (Fig. 2b).
  • PD-L1 -/- A20 cells expressing IFNAR the binding of heterodimers was reduced compared to IFN-Fc or homodimer (Fig. 2c).
  • the fusion protein effectively protected L929 cells from vesicular stomatitis virus (VSV) infection, indicating that the antiviral activity of IFN remained unchanged (Fig. 2d).
  • VSV vesicular stomatitis virus
  • Example 3 Targeted delivery of IFN by anti-PD-L1 to control advanced tumors
  • IFN-anti-PD-L1 IFN- ⁇ 4
  • fusion protein i.t.
  • anti-PD-L1 antibodies failed to control tumor growth
  • heterodimers and homodimers of IFN-anti-PD-L1 fusion proteins overcame anti-PD-L1 resistance in most treated mice.
  • induced complete tumor regression (2e) 2e.
  • Example 4 Tumor-targeting IFN-anti-PD-L1 showed less toxicity and strong anti-tumor activity
  • type I IFN is limited by the severe side effects of system delivery.
  • IFN- ⁇ 4 high doses of heterodimer (IFN-anti-PD-L1) or non-targeting control IFN-anti-HBs (anti-targeting) Hepatitis B virus surface protein) fusion protein.
  • IFN-anti-PD-L1 heterodimer
  • IFN-anti-PD-L1 non-targeting control IFN-anti-HBs (anti-targeting) Hepatitis B virus surface protein
  • the tumor-bearing mice developed severe weight loss, decreased activity, wrinkled skin, and all died within one day (Fig. 3a).
  • none of the mice treated with IFN-anti-PD-L1 died and recovered after mild weight loss (Figs. 3a and 3b).
  • the IFN-anti-PD-L1 fusion protein accumulates in tumors but does not accumulate in normal tissues (Fig. 7a).
  • Tumor-specific targeting is critical for the anti-tumor effect of the fusion protein, as a simple mixture of IFN-Fc and anti-PD-L1 antibody does not produce a synergistic effect like the IFN-anti-PD-L1 fusion protein (Fig. 3d) .
  • IFN-anti-PD-L1 can target tumor tissue to inhibit tumor growth and has less toxic side effects.
  • IFN-anti-PD-L1 IFN- ⁇ 4 heterodimer
  • IFN-anti-PD-L1 significantly increased PD-L1 expression in tumor tissues (Fig. 3e). Increased PD-L1 expression can enhance tumor-specific accumulation of the fusion protein.
  • the level of IFN receptors in CD45 + cells (leukocytes) also increased (Fig. 3f), further sensitizing cells to IFN therapy.
  • Example 6 Anti-tumor effect of PD-L1 on interferon fusion protein in tumor cells is not required
  • PD-L1 which is expressed in tumor cells or non-tumor cells
  • the present invention uses the CRISPR/Cas9 technology to knock out PD-L1 in tumor cells.
  • PD-L1 expression was completely abolished in knockout A20 and MC38 tumor cells (Fig. 4a). IFNs can induce PD-L1 expression.
  • WT wild-type
  • the fusion protein IFN-anti-PD-L1 IFN- ⁇ 4 heterodimer
  • WT or PD-L1 knockout PD-L1 -/ - tumor-bearing mice.
  • the level of protein in the tumor tissue is measured.
  • the fusion protein accumulates in tumor tissue regardless of whether the tumor cells express PD-L1 (Fig. 4b).
  • Both PD-L1 knockout tumors and WT tumors were able to be effectively controlled when tumors were treated with IFN-anti-PD-L1 (Fig. 4c and 4d).
  • IFN-anti-PD-L1 created a feedforward loop that up-regulated PD-L1 expression in TME (Fig. 3e). Since both tumor and stromal cells express PD-L1, we examined the expression levels of PD-L1 in different cell subpopulations in tumor tissues. IFN-anti-PD-L1 treatment significantly up-regulated PD-L1 expression in tumor and stromal cells (Fig. 4e). Since PD-L1 in tumor cells is not required, we would like to know if PD-L1 is required in the host cell. Interestingly, IFN-anti-PD-L1 well controlled tumor growth in PD-L1-deficient mice (Fig. 4f). Taken together, these data indicate that PD-L1 expressed in host cells or tumor cells is sufficient to mediate tumor targeting and anti-tumor effects of the fusion protein.
  • Example 7 IFN-anti-PD-L1 fusion protein promotes activation of APC and T cells in TME
  • mice were treated with anti-IFNAR blocking antibody during treatment with IFN-anti-PD-L1 fusion protein (heterodimer).
  • the anti-IFNAR antibody completely abolished the anti-tumor effect of the fusion protein, suggesting an important role for type I IFN signaling (Fig. 5a).
  • IFNAR is expressed in both tumor and host cells. To test whether IFN receptors in tumor cells are necessary, we knocked out IFNAR1 in tumor cells (Fig. 5b). Interestingly, IFN-anti-PD-L1 effectively controlled tumor growth in mice bearing A20.IFNAR1 - /- tumors (Fig. 5c).
  • IFN receptors in tumor cells are not required, we investigated whether receptors expressed on host cells are required. MC38 tumors were inoculated into WT or IFNAR -/- mice and treated with the fusion protein. It was observed that the anti-tumor effect disappeared in the deficient mice, suggesting that the interferon receptor plays a more important role in the host (Fig. 5d).
  • CD8+ T cells are required for anti-tumor effects, and when anti-CD8 antibodies are used to block CD8+ T cells, anti-tumor effects are completely eliminated (Fig. 5e).
  • type I IFN enhances DC cross-presentation in TME, resulting in better T cell activation.
  • IFN-anti-PD-L1 treatment increased the expression of DC-activated marker molecules CD86 and CD80 (Fig. 5f and Fig. 8).
  • Fig. 5f and Fig. 8 As a control, no significant activation of DC was observed in IFN-Fc treated tumors, indicating an important role for tumor specific targeting.
  • Example 8 Targeted delivery of IFN by PD-L1 antibody overcomes tumor resistance to PD-1 blocking therapy
  • B16F10 melanoma is a well-known mouse tumor model that is resistant to PD-1/PD-L1 blockade therapy. Consistent with previous reports, PD-1/PD-L1 blockade had no effect on tumor growth in the B16F10 model (Fig. 6b). IFN-anti-PD-L1 treatment only partially controls the tumor. Interestingly, the combination of IFN-anti-PD-L1 and PD-1 blockade significantly improved the anti-tumor effect.
  • mice were vaccinated with complete tumor regression after combination therapy with lethal doses of A20 cells. All mice resisted the re-challenged tumor, confirming that the fusion protein induced a memory adaptive immune response (Fig. 6c).
  • mice undergoing combination therapy are treated with a deleterious antibody against NK, CD4+ or CD8+ T cells.
  • NK, CD4+ or CD8+ T cells In the absence of CD8+ T cells, the anti-tumor effect completely disappeared (Fig. 6d).
  • deletion of NK or CD4+ T cells has a limited effect ( Figures 9a-9b).
  • To detect whether tumor-specific T cells were produced after treatment cells were isolated from lymph nodes or spleen tissues and co-cultured with irradiated A20 tumor cells. An IFN-[alpha] ELISPOT assay was performed to assess tumor-specific T cell responses.
  • the PD-1/PD-L1 blockade alone has limited effect on T cell activation (Fig. 6e and Fig.
  • IFN-anti-PD-L1 IFN- ⁇ 4 heterodimer
  • IFN- ⁇ 4 heterodimer IFN- ⁇ 4 heterodimer
  • TME TME
  • DCs and T cells can be isolated from tumors established in vivo.
  • the cells were co-cultured for three days in the presence of IFN, anti-PD-L1 antibody or a combination of both. Although monotherapy was limited, combination therapy with IFN and anti-PD-L1 antibodies significantly increased IFNy production by T cells (Fig. 6f).
  • Example 9 Targeted delivery of IFN activates tumor-resident T cells for tumor control
  • T cells play an important role in the anti-tumor immune response.
  • T cells may come from two main sources. Some are existing T cells in tumor tissue, while others are newly activated T cells that migrate from the periphery to the tumor tissue. IFN can not only stimulate DC to activate TIL, but also increase chemokines that attract T cells.
  • FTY720 to block the transport of peripheral lymphocytes to tumor tissue. Interestingly, even in the case of blocked lymphocyte trafficking, similar to the control group, IFN and PD-1/PD-L1 blocking combination therapy could control tumors (Fig. 6g).
  • the biological activity of the IFN ⁇ -Fc mutant was measured by an antiviral infection bioassay.
  • L929 cells were incubated overnight with each protein mix prior to infection with VSV-GFP virus. After 30 hours of culture, the percentage of virus-infected cells was determined by flow cytometry, and the inhibition rate and EC50 value of the protein infection by different concentrations of protein were calculated (Fig. 12). The results showed that the constructed mutants had some activity diminished, and the two mutants R144A and A145G had the weakest viability and were potential potential targets.
  • the proliferation of cells treated with different concentrations of protein was detected by CCK8 kit.
  • the results showed that the mutant IFN-anti-PD-L1 reduced the EC50 value of the proliferation inhibition of the targeted deletion PD-L1 -/- A20 cell compared to the wild-type fusion protein.
  • the EC50 values of mutant IFN-anti-PD-L1 and wild-type fusion protein for cell proliferation inhibition were not significantly different (Fig. 13).
  • the amino acid sequence of the first polypeptide Anti-PD-L1 (ScFv(PD-L1)-Fc) of the mutant IFN-anti-PD-L1 fusion protein is shown in SEQ ID NO. 1, and the second polypeptide mutant mIFNa4
  • the amino acid sequence of -Fc is shown in the table below.
  • the ratio of EC50 of PD-L1 -/- A20 cells to PD-L1 +/+ A20 cells can reflect the targeting of this bispecific protein, and the ratio is normalized with the data of wt-mIFNa4-Fc. It was found that R144A and A145G are the two most targeted mutations, suggesting that these two mutant fusion proteins can specifically target PD-L1-positive target cells while avoiding induction of IFNAR signaling pathways on other cells. activation.
  • the reduced viability IFN ⁇ mutant when fused to a targeting protein, is capable of inducing IFNAR activation only on target cells. Peripheral off-target when IFN is used is avoided.
  • the mutant IFN-anti-PD-L1 constructed by the present invention has greater potential for treating tumors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Endocrinology (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Oncology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明涉及一种IFN-anti-PD-L1融合蛋白，包含融合蛋白的药物组合物和试剂盒，以及所述融合蛋白在肿瘤治疗中的应用。本发明所述融合蛋白能够同时靶向PD-L1和IFN受体，肿瘤微环境中IFN信号的激活可以通过诱导更强大的T细胞活化来增强针对肿瘤的PD-1/PD-L1治疗；同时，抗PD-L1抗体可以用于将免疫调节分子特异性递送到肿瘤组织中；融合蛋白作用产生多重前馈应答，不仅增加靶向效应，降低毒性，而且增强对IFN治疗的应答，从而使抗肿瘤作用最大化。

Description

一种IFN与抗PD-L1抗体的融合蛋白及其应用 技术领域
本发明属于基因工程和生物医学医疗技术领域，具体涉及IFN与抗PD-L1抗体的融合蛋白，包含融合蛋白的药物组合物和试剂盒，以及融合蛋白在治疗肿瘤疾病中的应用。
背景技术
程序性细胞死亡蛋白1(PD-1)是一个关键的免疫检查点分子，它可抑制T细胞的TCR信号活化，从而减弱免疫反应的强度和时长。其配体PD-L1在肿瘤细胞中普遍上调，成为肿瘤逃避免疫应答的机制之一。PD-1/PD-L1阻断疗法(简称PD疗法)可以诱导各种癌症患者对肿瘤细胞产生持久的免疫反应。然而在实践中，客观有效的免疫反应仅在小部分实施PD疗法的患者中观察到；另外，获得性PD治疗的耐药性现象也越来越受到重视，而且其机制尚不明确。因此，研究分析为何某些肿瘤不能对PD疗法产生反应或产生抗药性成为PD疗法相关研究的重中之重。
部分学者提出，PD疗法能够有效的控制肿瘤是由于T细胞会释放免疫抑制信号。据报道，PD疗法的优良疗效与充足的肿瘤浸润淋巴细胞(TILs)数量相关。然而，即使是在大量淋巴细胞浸润的情况下，单纯的PD疗法可能也不能有效地(重新)激活肿瘤特异性T细胞。在这些情况下，可能需要对其他的负协同抑制剂进行阻断或上调刺激信号，以诱导T细胞(重新)激活。然而，能够有效促进T细胞免疫的信号分子至今仍存在较多争论。
I型干扰素包含IFNα和IFNβ，通过促进树突状细胞(DC)的成熟，处理和递呈抗原来活化T细胞，扮演天然免疫和适应性免疫之间桥梁的角色。早期研究发现I型干扰素有抑制肿瘤细胞增殖和促进凋亡作用，因此被批准用于治疗临床特定的肿瘤,包括淋巴瘤、黑色素瘤、肾细胞肿瘤等。最近的研究表明I型干扰素信号通路介导的APC和T细胞活化在肿瘤放疗和化疗过程中起着至关重要的作用。但肿瘤组织内I型干扰素的表达非常低。给与外源的I型干扰素治疗既能抑制肿瘤的增殖和存活，又能激活抗肿瘤免疫应答，因此成为重要的研究靶点。
临床上高剂量的I型干扰素治疗会带来较好的肿瘤控制效果。但是由于IFNAR受体在正常组织表达广泛，高剂量的I型干扰素会诱发病人严重的副反应，包括流感样症状(发烧、头痛等)、呕吐、白细胞减少、贫血、血小板减少等症状。此外，I型干扰素会上调免疫抑制性分子PD-L1的表达，反过来抑制抗肿瘤免疫反应降低治疗效果。如何克服I型干扰素的全身毒性及带来的免疫抑制，是待解决的重要问题。
发明内容
尽管存在肿瘤浸润淋巴细胞，大多数患者仍然对强化PD-1/PD-L1治疗不应答。发明人认为，天然免疫抗原提呈细胞活化缺陷可能会限制PD-1/PD-L1阻断后肿瘤特异性T细胞的完全激活。I型干扰素(IFN)的局部递送恢复抗原呈递，但也上调PD-L1，抑制随后的T细胞活化。为了克服上述现有技术的局限和不足，本发明提供了一种IFN-抗PD-L1抗体(IFN-anti-PD-L1)融合蛋白，其可以同时靶向PD-L1和IFN受体，在实验中观察到，IFN-anti-PD-L1融合蛋白能够在肿瘤组织聚集，显著增加抗原交叉提呈并克服PD-L1介导的免疫抑制。IFN-anti-PD-L1融合蛋白能够同时释放免疫抑制信号和提供共刺激信号来(重新)激活T细胞，可作为新一代的抗PD-L1抗体用于肿瘤疾病的治疗。
本发明目的通过以下技术方案来实现：
本发明提供了一种融合蛋白，所述融合蛋白由干扰素(IFN)与PD-L1结合蛋白融合连接构成IFN-anti-PD-L1，所述融合蛋白为同源二聚体蛋白或异源二聚体蛋白。
本发明所述同源二聚体蛋白包含第一多肽和第二多肽，所述第一多肽和第二多肽相同；所述第一多肽和第二多肽自N端至C端顺序包含IFN、PD-L1结合蛋白、和免疫球蛋白Fc区。
本发明所述异源二聚体蛋白包含第一多肽和第二多肽，所述第一多肽与第二多肽不同；所述第一多肽包含PD-L1结合蛋白，所述第二多肽包含IFN和免疫球蛋白Fc区，IFN位于Fc区的N端；所述第一多肽包含的Fc区与第二多肽包含的Fc区源自具有相同或不同亚型的免疫球蛋白。
本发明所述干扰素(IFN)可选自I型干扰素、I型干扰素突变体、II型干扰素和/或III型干扰素，例如IFN-α、IFN-β、IFN-γ、IFN-λ1(IL-29)、IFN-λ2(IL-28a)、IFN-λ(IL-28b)和IFN-ω等，优选I型干扰素，优选IFN-α4，更优选IFN-α4突变体，所述IFN可来自人源或鼠源；优选IFN-α4(SEQ ID NO.13)，更优选IFN-α4突变体；更优选突变体mIFN-α4(L30A)(SEQ ID NO.25)、mIFN-α4(R144A)(SEQ ID NO.27)、mIFN-α4(A145G)(SEQ ID NO.29)、mIFN-α4(R149A)(SEQ ID NO.31)、mIFN-α4(S152A)(SEQ ID NO.33)、hIFN-α2(Q124R)(SEQ ID NO.35)。
本发明所述免疫球蛋白Fc区可选自IgG1、IgG2、IgG3和/或IgG4的恒定区氨基酸序列，优选IgG1。其中IgG1具有较强的诱导ADCC和CDC效应的能力和较长的血清半衰期，是抗体药最常见的抗体亚型；IgG2、IgG4具有较弱的诱导ADCC和CDC效应的能力，但血清半衰期较长。
本发明所述PD-L1结合蛋白可选自抗PD-L1抗体(完整抗体)、单链抗体(ScFv)、Fab片段、F(ab’) 2片段；所述抗PD-L1抗体优选自：Tecentriq、Bavencio、Imfinzi、KN035、CS1001、KL-A167、SHR-1316和/或YW243.55.S70；更优选结合PD-L1的单链抗体(ScFv)；更优选YW243.55.S70。
本发明所述同源二聚体蛋白的第一多肽和第二多肽优选包含SEQ ID NO.3所示氨基酸序列；所述异源二聚体第一多肽优选包含SEQ ID NO.1所示氨基酸序列，所述第二多肽包含SEQ ID NO.2、37、39、41、43、45、47所示氨基酸序列。
本发明还提供了融合蛋白的应用，将IFN-anti-PD-L1施用于肿瘤细胞，其能够抑制肿瘤细胞生长和/或迁移。
本发明提供了一种IFN-anti-PD-L1融合蛋白在肿瘤疾病治疗中的应用；以及IFN-anti-PD-L1融合蛋白在制备药物或试剂盒中的应用。所述肿瘤优选为对PD-1/PD-L1阻断单独治疗无效的肿瘤或晚期肿瘤，更优选适用对抗PD-1/PD-L1抗体单独治疗产生抗性或无效的肿瘤；上述肿瘤优选B细胞淋巴瘤、结肠癌和黑色素瘤。
本发明提供了一种药物制剂或药物组合物，所述药物制剂的活性成分包含本发明所述融合蛋白IFN-anti-PD-L1。
本发明提供了一种试剂盒，所述试剂盒包含本发明所述融合蛋白IFN-anti-PD-L1。
本发明提供了一种核酸分子，所述核酸分子编码本发明所述融合蛋白IFN-anti-PD-L1。
所述核酸分子：1)编码所述同源二聚体的核酸分子优选SEQ ID NO.6所示核苷酸序列；
2)编码所述异源二聚体的核酸分子可选自SEQ ID NO.4、5、7、8、9、10、38、40、42、44、46、48所示核苷酸序列；优选SEQ ID NO.4和SEQ ID NO.5，SEQ ID NO.4和SEQ ID NO.40，或SEQ ID NO.4和SEQ ID NO.42。
本发明提供了一种载体，所述载体包含上述核酸分子。
本发明提供了一种细胞，所述细胞包含本发明所述融合蛋白IFN-anti-PD-L1或编码融合蛋白的核酸分子，用于生产融合蛋白。所述细胞选自非人哺乳动物细胞，优选CHO和HEK293细胞。
本发明提供一种治疗肿瘤的方法，其包括向癌症患者施用有效量的融合蛋白IFN-anti-PD-L1。所述肿瘤优选PD-1/PD-L1阻断单独治疗无效的肿瘤。
本发明提供一种治疗PD-1/PD-L1阻断单独治疗无效或晚期肿瘤的方法，其包括向患者施用有效量的IFN-α和抗PD-L1抗体；同时，本发明提供了一种IFN-α和抗PD-L1抗体共同用于制备药物组合物、药物制剂或试剂盒的用途；本发明还提供了一种包含IFN-α和抗PD-L1抗体的药物组合物、药物制剂或试剂盒；上述所述IFN-α需向肿瘤内施用。
在此基础上，本发明提供了一种抗PD-1/PD-L1抗体和IFN-anti-PD-L1的联合疗法，以及包含抗PD-1/PD-L1抗体和IFN-anti-PD-L1融合蛋白的药物组合物或试剂盒。所述疗法包括向患者施用有效量的抗PD-1/PD-L1抗体和IFN-anti-PD-L1，所述施用为顺序施用或同时施用。所述肿瘤为对PD-1/PD-L1阻断单独治疗产生抗性或无效的肿瘤，或晚期肿瘤；优选对抗PD-1/PD-L1抗体单独治疗产生抗性或无效的肿瘤。或所述肿瘤的患者患有外周淋巴细胞运输缺陷/障碍相关疾病，所述患者的外周淋巴细胞无法迁移至肿瘤组织。
本发明提供一种融合蛋白IFN-anti-PD-L1与抗PD-1/PD-L1抗体共同用于制备治疗肿瘤的药物组合物或试剂盒的用途。所述肿瘤为对PD-1/PD-L1阻断单独治疗无效的肿瘤，或晚期肿瘤；优选对抗PD-1/PD-L1抗体单独治疗产生抗性或无效的肿瘤。或所述肿瘤的患者患有外周淋巴细胞运输缺陷/障碍相关疾病，所述患者的外周淋巴细胞无法迁移至肿瘤组织。
本发明提供一种IFN-α和抗PD-L1抗体共同用于制备治疗对PD-1/PD-L1阻断单独治疗无效的肿瘤，或晚期肿瘤的药物组合物、药物制剂或试剂盒的用途，所述IFN-α需向肿瘤内施用。
本发明提供一种包含IFN-α和抗PD-L1抗体的药物组合物、药物制剂或试剂盒，所述IFN-α需向肿瘤内施用。
本发明提供一种融合蛋白IFN-anti-PD-L1在上调白细胞中IFN受体表达中的用途；所述白细胞优选为CD45+细胞；所述IFN受体优选为IFNAR。同时，提供一种融合蛋白IFN-anti-PD-L1在制备上调白细胞中IFN受体表达的组合物中的用途。
本发明提供一种融合蛋白IFN-anti-PD-L1在激活DC细胞或TIL细胞中的用途。同时，提供一种融合蛋白IFN-anti-PD-L1在制备激活DC细胞或TIL细胞的组合物中的用途。
本发明提供一种融合蛋白IFN-anti-PD-L1在激活肿瘤驻留T细胞中的用途。同时，提供一种融合蛋白IFN-anti-PD-L1在制备激活肿瘤驻留T细胞的组合物中的用途。
上述“用途”既可表示治疗目的的用途，也可表示非治疗目的的用途。
本发明提供一种IFN-α4突变体，所述突变体为与受体亲和力减弱突变，包括mIFN-α4(L30A)(SEQ ID NO.25)、mIFN-α4(R144A)(SEQ ID NO.27)、mIFN-α4(A145G)(SEQ ID NO.29)、mIFN-α4(R149A)(SEQ ID NO.31)、mIFN-α4(S152A)(SEQ ID NO.33)、和/或hIFN-α2(Q124R)(SEQ ID NO.35)。
本发明提供编码上述IFN-α4突变体的分离的核酸分子，所述核酸分子的核苷酸序列如SEQ ID NO.26、28、30、32、34、36所示。
本发明提供一种IFN-α4突变体在制备治疗肿瘤的融合蛋白或药物中的用途。
术语及定义
除非特别说明，本申请中所使用的术语和定义均是本领域中惯常使用的含义并且为本领域技术人员所知晓。
如本申请中所使用的，术语“肿瘤位点”是指含有或被怀疑含有肿瘤细胞的体内或离体位置。所述肿瘤位点包括固体肿瘤以及接近或邻近肿瘤生长处的位置。
如本申请中所使用的，术语“施用”是指全身性和/或局部施用。术语“全身性施用”是指非局部地施用，从而所施用的物质可能影响整个身体中的若干器官或组织；或者从而所施用的物质可能穿越整个身体中的数个器官或组织而到达靶位点。例如，向受试者的循环系统施用可引起治疗性产物在多于一个组织或器官中从所施用的载体表达，或者可引起治疗性产物在特异性位点处由所施用的载体表达，例如，这是由于天然的趋向性或由于与组织特异性启动子元件的可操作连接。本领域技术人员将理解，所述全身性施用涵盖各种形式的施用，这包括但不限于：肠胃外施用、静脉内施用、肌内施用、皮下施用、经皮施用、肿瘤内施用、口服等。
术语“局部施用”是指在特异性位点处或其周围施用。本领域技术人员将理解，局部施用涵盖各种形式的施用，例如直接注射到特定位点处或注射到其周围(例如肿瘤内施用)。
如本文中所使用的，术语“治疗有效量”是指达到治疗目的疾病或病况(例如肿瘤/癌症，例如用于使肿瘤消退或减小肿瘤的大小)所需的本发明的干扰素，或者本发明试剂盒中组分的量。可以通过实践、按照常规的方式来关于特定的目的而确定所述有效量。特别地，所述治疗有效量可以是达到下述目的所需的量：减少癌细胞的数目；减少肿瘤大小；抑制(即减缓或停止)癌细胞浸润到外周器官中；抑制(即减缓或停止)肿瘤转移；抑制肿瘤生长；和/或缓解与癌症相关的一种或多种症状。
术语“抗体”涵盖例如，单克隆抗体、多克隆抗体、单链抗体、抗体片段(其显示出所需的生物学或免疫学活性)。在本申请中，术语“免疫球蛋白”(Ig)与抗体可互换地使用。所述抗体可特异性地靶向肿瘤抗原，例如表面肿瘤抗原，例如EGFR，CD4，CD8、Neu等。
本发明所述“肿瘤”可选自B细胞淋巴瘤、肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤。
本发明所述“肿瘤细胞”可选自B细胞淋巴瘤、肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌 或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤的癌症产生的细胞。
本发明所述“应用”或“用途”，既可表示以疾病治疗为目的的应用，也可表示非治疗目的的应用，例如，科学研究等。
本发明的有益效果：
1、本发明提供的IFN-anti-PD-L1融合蛋白同时靶向PD-L1和IFN受体，数据表明TME中IFN信号的激活可以通过诱导更强大的T细胞活化来增强针对晚期肿瘤的PD-1/PD-L1治疗。
2、本发明提供的IFN-anti-PD-L1融合蛋白，数据显示抗PD-L1抗体(PD-L1结合蛋白)可以用于将免疫调节分子特异性递送到肿瘤组织中，毒性最小。本发明为开发用于肿瘤靶向的抗PD-L1新型抗体奠定了基础。
3、本发明提供的IFN-anti-PD-L1融合蛋白可以在肿瘤微环境内产生靶向PD-L1阻断和IFN-α受体(IFNAR)激活的多重前馈应答。不仅增加靶向效应，而且增强对IFN治疗的应答，实现了抗PD-L1和IFN的协同效应，以克服PD-1/PD-L1治疗和IFN抵抗。实验数据表明，IFN-anti-PD-L1能够同时实现靶向肿瘤组织活化抗原递呈细胞和阻断PD-L1抑制性信号的作用，可作为新一代的抗PD-L1抗体用于肿瘤疾病的治疗。
4、本发明提供的IFN-anti-PD-L1融合蛋白与PD-1/PD-L1阻断联合使用时，IFN-anti-PD-L1完全消除了大多数PD-1/PD-L1抗性肿瘤并诱导产生记忆性T细胞免疫应答。
5、本发明提供的融合蛋白具有同源二聚体和异源二聚体两种结构，分别在体内和体外具有高效的IFN受体结合亲和力、更有效的抗病毒活性和优异的肿瘤靶向性、血清半衰期和体内肿瘤控制。
6、本发明提供的融合蛋白包含亲和力减弱突变型IFNα，具有更特异的靶细胞结合能力，避免IFN的外周脱靶效应。
附图说明
图1.I型IFN的局部用药克服了晚期肿瘤对PD-1/PD-L1阻断的抗性。(a)用3x10 6个A20细胞皮下接种Balb/c小鼠(n＝3)；携带早期阶段肿瘤(<50mm 3)的小鼠在11天和15天使用200μg抗PD-L1抗体处理，腹腔内给药(intraperitoneally(i.p.))。(b)携带晚期A20肿瘤(>100mm 3)的小鼠(n＝4)在第15天和第19天用200μg抗PD-L1抗体处理。每周两次测量肿瘤生长。(c)按照(a)和(b)处理小鼠。治疗三天后，分离引流淋巴结的细胞，并与使用或不用辐照的A20细胞共培养两天。进行IFNγELISPOT测定。(d)将A20荷瘤小 鼠(n＝5)在11天和15天使用200μg抗-PD-L1抗体处理(i.p.)，和/或在11天使用25μg IFNα-Fc处理(瘤内注射intratumorally(i.t.))。(e)用5×10 5个MC38细胞接种C57BL/6小鼠(n＝5)。在第9，12和15天用200μg抗PD-L1抗体处理(i.p.)，和/或在第9天用25μgIFNα-Fc处理。显示存活曲线。(f)A20荷瘤小鼠(n＝5)在第11天用25μgIFNα-Fc处理(i.t.或静脉注射(i.v.))。黑色箭指示用IFNα-Fc处理。数据表示平均值±SEM并且代表至少两个独立的实验。*，p<0.05；**，p<0.01；n.s.，不显著。
图2.IFN-anti-PD-L1融合蛋白的构建及其性质。(a)IFN-anti-PD-L1融合蛋白的同源二聚体或异源二聚体示意图。scFv，单链可变区片段。(b-c)显示IFNAR1 -/-A20细胞(b)和PD-L1 -/-A20细胞(c)中所示蛋白质的结合的流式细胞图。数字表示平均荧光强度(MFI)。(d)通过抗病毒感染生物测定法测量IFN-anti-PD-L1蛋白的生物活性。在感染VSV-GFP病毒之前，将L929细胞与每种蛋白一起培养过夜。再培养30小时后，通过流式细胞术测定病毒感染细胞的百分比。(e-f)用3×10 6个A20细胞接种Balb/c小鼠(n＝5)。肿瘤建立后，通过瘤内注射(e，第18和22天处理)或静脉注射(f，第11和15天)20μg相应的蛋白，每周测量两次肿瘤大小。(g)用5×10 5个MC38细胞接种C57BL/6小鼠(n＝4-8)。在第14天和第18天用25μg对照或融合蛋白进行静脉注射处理。(h-i)用25μg所示蛋白质静脉注射小鼠。通过ELISA测量不同时间点肿瘤组织(h)或血清(i)中的蛋白质浓度。数据表示平均值±SEM并且代表至少两个独立的实验。*，p<0.05；**，p<0.01。
图3.IFN-anti-PD-L1融合蛋白具有较小的毒副作用和良好的体内抗肿瘤效果。(a-b)在第0天和第4天用100μgIFN-anti-PD-L1异源二聚体或IFN-anti-HBs蛋白质静脉注射荷瘤小鼠。图示小鼠存活曲线(a)和体重变化(b)。(c)100μg IFN-anti-PD-L1异源二聚体或IFN-anti-HBs蛋白质静脉注射MC38荷瘤小鼠。注射后第6或24小时收集血清。通过细胞因子微球检测技术(CBA)测量血清中的细胞因子水平。(d)在第11和15天，用IFNα-Fc(12.5μg)，抗-PD-L1抗体(12.5μg)，IFNα-Fc和抗PD-L1抗体的混合物(12.5μg+12.5μg)，或IFN-anti-PD-L1融合蛋白异源二聚体(25μg)静脉注射处理A20荷瘤小鼠(n＝5)。每周测量两次肿瘤大小。(e)使用25μg对照蛋白或IFN-anti-PD-L1异源二聚体处理MC38荷瘤小鼠。两天后，收集肿瘤组织并通过流式细胞术测定PD-L1水平。FMO，fluorescence minus one。(f)按照(e)处理小鼠。两天后收获肿瘤组织。通过流式细胞术测定CD45阴性和CD45阳性细胞中的IFNAR水平。数据表示平均值±SEM并且代表至少两个独立的实验。*，p<0.05；**，p<0.01；***，p<0.001；n.d.，不可检测；n.s.，不显著。
图4.宿主或肿瘤细胞中表达的PD-L1均能有效介导IFN-anti-PD-L1融合蛋白的抗肿瘤作 用。(a)通过流式细胞术测量WT A20，PD-L1 -/-A20，WT MC38，以及PD-L1 -/-MC38细胞中的PD-L1表达。(b)30μg的IFN-anti-PD-L1异源二聚体静脉注射WT或PD-L1 -/-荷瘤小鼠。在注射后的不同时间点收集肿瘤组织。通过ELISA测量融合蛋白的浓度。(c)在第11和15天用对照Ig或IFN-anti-PD-L1处理WT或PD-L1 -/-A20荷瘤小鼠(n＝4-5)。每周两次测量肿瘤生长。(d)在第8天和第12天，用对照Ig或IFN-anti-PD-L1处理WT或PD-L -/-MC38荷瘤小鼠(n＝5-6)。(e)用IFN-anti-PD-L1处理MC38荷瘤小鼠。两天后，收集肿瘤组织。通过流式细胞术评估CD45阴性与CD45阳性细胞中的PD-L1水平。(f)用MC38细胞接种PD-L1 -/-小鼠(n＝4-5)。在第14天和第18天用25μg对照Ig或IFN-anti-PD-L1处理小鼠。每周测量两次肿瘤生长。数据表示平均值±SEM，并且代表至少两个独立的实验。*，p<0.05；**，p<0.01；***，p<0.001；n.s.，不显著。
图5.宿主细胞表达的IFNAR对于肿瘤控制是必需的。(a)在第11天用IFN-anti-PD-L1处理A20荷瘤小鼠(n＝5)。为了阻断IFNAR信号通路，在第11和14天使用100μg抗IFNAR阻断抗体瘤内注射(i.t.)处理小鼠。(b)通过流式细胞术评估体内(B220+)WT或IFNAR - /-A20肿瘤细胞中的IFNAR表达。(c)在第16天和第19天用IFN-anti-PD-L1处理IFNAR -/-A20荷瘤小鼠(n＝6)。每周两次测量肿瘤生长。(d)5×10 5个MC38细胞接种WT或IFNAR1 -/-小鼠。在第10天和第13天用25μgIFN-anti-PD-L1处理小鼠(n＝4-5)。(e)在第11天和第14天用IFN-anti-PD-L1处理荷瘤小鼠(n＝5-6)。在第9、12和16天施用了抗CD8删除抗体。(f)在IFN-anti-PD-L1治疗两天后，分离MC38肿瘤组织。通过流式细胞术测量CD86在肿瘤浸润性DC中的表达(CD11c+MHCII+)。左图为代表性图样，右图显示MFI。数据表示平均值±SEM并且代表至少两个独立的实验。*，p<0.05；**，p<0.01；***，p<0.001；n.s.，不显著。
图6.PD-1阻断进一步确保IFN-anti-PD-L1诱导前馈抗肿瘤应答。(a)在第15天用20μg IFN-anti-PD-L1，和/或在第14天和第17天用100μg抗PD-1抗体处理A20荷瘤Balb/c小鼠(n＝4-5)。每周测量两次肿瘤生长。(b)在第11和14天用25μgIFN-anti-PD-L1，和/或在第12天和第15天用100μg抗-PD-1抗体处理B16荷瘤C57BL/6小鼠(n＝3-5)。(c)在(a)联合治疗后肿瘤完全消退的小鼠(n＝4)中用2.5×10 7个A20细胞重新接种肿瘤。使用接种了A20细胞的首次实验小鼠作为对照。(d)用(a)中相同的IFN-anti-PD-L1和anti-PD-1处理小鼠(n＝4)。为使细胞耗尽(depletion)，从抗PD-1抗体治疗前一天向小鼠注射200μg抗-CD8抗体。(e)如(a)中所述用IFN-anti-PD-L1和/或anti-PD-1处理小鼠。治疗12天后，分离肿瘤引流淋巴结，制备单细胞悬液。将细胞与经辐照或不经辐照的A20共同培养。进行 IFNγELISPOT测定。(f)从A20荷瘤小鼠中分离肿瘤浸润性DC(CD11c+)和T(CD8+)细胞，并在经辐照的A20细胞存在下共培养。将IFNα或抗PD-L1抗体加入到培养基中。三天后，收集上清液并通过CBA测量IFNγ水平。(g)如(a)中所述用IFN-anti-PD-L1和/或anti-PD-1处理A20荷瘤小鼠(n＝5-6)。FTY720从第14天开始每隔一天给药一次。为了删除肿瘤内CD8 +T细胞，在第14天和第17天将30μg抗-CD8抗体注射入肿瘤内。黑色和蓝色箭头分别表示用抗PD-1和IFN-anti-PD-L1治疗。数据表示平均值±SEM并且代表至少两个独立的实验。*，p<0.05；**，p<0.01；***，p<0.001。
图7.IFN-anti-PD-L1异源二聚体特异性靶向肿瘤组织并诱导较小的毒性。(a)30μg的IFN-anti-PD-L1静脉内注射MC38荷瘤小鼠。注射后第1，3和5天收集组织。ELISA测定融合蛋白浓度。(b)如图3c处理MC38荷瘤小鼠，注射后6或24小时收集血清。通过CBA测量血清中的细胞因子水平。数据指示平均值±SEM并且代表两个实验重复。
图8.IFN-anti-PD-L1上调肿瘤浸润DC中CD80的表达。IFN-anti-PD-L1治疗2天后，分离MC38肿瘤组织。流式细胞术检测肿瘤浸润DC(CD11c+MHCII+)中CD80的表达。
图9.NK和CD4+T细胞在由抗PD-1抗体和IFN-anti-PD-L1联合治疗介导的抗肿瘤反应中没有明显作用。如图6a处理A20荷瘤小鼠(n＝4-5)。(a)为删除NK细胞，从第13天开始每周两次腹膜内注射20μg抗唾液酸GM1抗体。(b)为删除CD4 +T细胞，从第13天开始每周两次腹膜内注射200μg抗-CD4抗体。肿瘤生长每周测量两次。数据表示平均值±SEM，并且是两个实验重复的代表。黑色和蓝色箭头分别表示用抗PD-1和IFN-anti-PD-L1处理。(c)按照图6e处理小鼠，分离脾脏，制备单细胞悬液。将细胞与经辐照或不经辐照的A20共同培养。进行IFNγELISPOT测定。
图10.带有IFN的抗PD-L1抗体(IFN-anti-PD-L1)介导的抗肿瘤作用的示意图模型。抗PD-L1特异性地将IFN运载到肿瘤组织(1)。干扰素介导的PD-L1上调增强肿瘤特异性靶向(2和3)。抗体阻断PD-L1/PD-1信号通路以释放免疫刹车信号(3)。此外，IFN-anti-PD-L1上调IFNAR表达(4)，这进一步使肿瘤对治疗敏感(5)。总之，这些因素(重新)激活了T细胞应答来控制肿瘤生长(6)。
图11.IFNα亲和力降低突变体构建。针对一型干扰素与受体相互作用的关键位点，做单个定点选择性突变。
图12.IFNα-Fc突变体活性检测。通过抗病毒感染生物测定法测量IFNα-Fc突变体的生物活性。在感染VSV-GFP病毒之前，将L929细胞与每种蛋白质一起培养过夜。再培养30小 时后，通过流式细胞术测定病毒感染细胞的百分比，并计算不同浓度的蛋白对细胞感染的抑制率和EC50值。R144A和A145G两个突变体具有较弱的活力，是潜在的优选靶点。
图13.突变型IFN-anti-PD-L1体外靶向性测试。利用WT的PD-L1 +A20细胞及实验室先前利用CRISPR-Cas9技术敲除的PD-L1 -/-A20细胞，加入不同浓度的双特异性蛋白孵育72小时，通过CCK8试剂盒检测不同浓度蛋白处理下细胞的增殖结果。PD-L1-A20细胞与PD-L1+A20细胞的EC50的比值可以反映该种双特异性蛋白的靶向性，将该比值与wt-mIFNa4-Fc的数据进行归一化比较后可以发现，R144A和A145G是靶向性最好的两种突变。
具体实施方式
通过以下实施例对本发明作进一步的详细描述，但应理解本发明并不受以下内容所限制。
材料和方法：
小鼠：
雌性(6-8周龄)BALB/c小鼠和C57BL/6小鼠购自维通利华(中国北京)。所有小鼠在生物物理研究所的动物房中饲养在无特定病原体(SPF)条件下。动物护理和实验按照中国科学院生物物理研究所的指导方针进行，遵循IACUC(Institutional Laboratory Animal Care and Use Committee)批准的方案。PD-L1 -/-和IFNAR1 -/-小鼠在UT西南医学中心的SPF条件下饲养。动物方案符合NIH指导方针。研究得到了UT西南医学中心动物护理和使用委员会的批准。
细胞系和试剂：
293F细胞由徐霆博士(中国江苏省苏州市康宁杰瑞)提供，并在SMM 293-TI培养基(M293TI，Sino Biological)中培养。A20，MC38和L929细胞系购自ATCC(Manassas，VA)。抗PD-1阻断抗体(4H2)来自百时美施贵宝Bristol-Myers Squibb(Redwood City，CA)。抗-PD-L1抗体(10F.9G2)和抗-IFNAR1抗体(MAR1-5A3)购自BioXCell(West Lebanon，NH)。Anti-CD8(TIB210)和anti-CD4删除抗体(GK1.5)由实验室自行制备。抗脱唾液酸GM1抗体购自Biolegend(San Diego，CA)。
IFN-anti-PD-L1融合蛋白的构建：
异源二聚体：根据专利(专利号：US8217149B2)合成了PD-L1结合蛋白(YW243.55.S70)序列的轻链和重链的可变区。将轻链和重链序列通过GGGGSGGGGSGGGGS接头连接，并 将人IgG1Fc(SEQ ID NO.15)插入重链的C端，命名为ScFv(PD-L1)-Fc(SEQ ID NO.1)，获得异源二聚体的第一多肽。然后将ScFv(PD-L1)-Fc的编码核酸序列(SEQ ID NO.4)克隆到pEE12.4载体(Lonza)中。将鼠IFN-α4(SEQ ID NO.13)的cDNA序列(SEQ ID NO.14)克隆并通过(G4S) 4连接子插入到人IgG1Fc的N端，表达获得异源二聚体的第二多肽mIFNα4-Fc(SEQ ID NO.2)。将编码mIFNα4-Fc的核苷酸序列(SEQ ID NO.5)克隆到pEE6.4载体(Lonza)中。PD-L1结合蛋白和IFNα的异源二聚化使用之前报道的knob-to-holes技术产生。质粒以1：2的比例瞬时转染到293F细胞中。转染后第7天收集上清液。根据操作手册(Repligen公司)使用蛋白A-琼脂糖凝胶柱纯化融合蛋白。
在异源二聚体的制备过程中，还使用了不同的I型干扰素制备第二多肽mIFNb-Fc(编码核酸如SEQ ID NO.7所示)、hIFNα2-Fc(编码核酸如SEQ ID NO.8所示)、hIFNb-Fc(编码核酸如SEQ ID NO.9所示)、mIFNγ-Fc(编码核酸如SEQ ID NO.10所示)，所产生的异源二聚体均具有较好的抑制肿瘤细胞增殖的效果；在上述异源二聚体融合蛋白中，尤以IFNα-Fc效果更佳；而mIFNα4-Fc与Anti-PD-L1构成的异源二聚体融合蛋白效果最优，相关详细比较数据此处未展示。
同源二聚体：小鼠IFN-α4C端与ScFv(PD-L1)-Fc的N端相连接，获得同源二聚体融合蛋白的第一多肽和第二多肽(SEQ ID NO.3)。将其核苷酸序列(SEQ ID NO.6)克隆到pEE12.4载体上，转染后通过Fc的二聚化自发形成同源二聚体蛋白。
流式细胞术
使用PE-抗人IgG Fc(eBioscience)检测融合蛋白的结合。特异性抗体：抗PD-L1抗体(10F.9G2)，抗-IFNAR1抗体(MAR1-5A3)，抗CD45抗体(30-F11)，抗CD80抗体(16-10A1)，抗CD86抗体(GL1)来自BioLegend或eBioscience。细胞悬浮于FACS缓冲液(1％牛血清白蛋白和0.05％NaN3)，用抗CD16/32抗体(抗-FcγIII/II受体，克隆2.4G2)封闭30分钟，然后用特异性抗体在冰上染色30分钟。样品在FACSCalibur或Fortessa flowcytometer(BD生物科学)上进行分析。使用FlowJo软件(TreeStar)分析数据。
IFNα抗病毒活性
使用对VSV感染敏感的L929小鼠成纤维细胞来定量IFN的生物学活性。将细胞与连续稀释的IFNα-Fc或IFN-anti-PD-L1在37℃孵育过夜。第二天，用MOI＝5的VSV-GFP感染细胞，再培养30小时。然后收集细胞并用4％PFA固定。使用FACSFotassa流式细胞仪(BD Biosciences)获取数据并使用FlowJo软件(TreeStar)分析数据。GFP阳性细胞被定义为病 毒感染的细胞。
蛋白体内分布定量研究
将A20细胞(3×10 6)皮下(s.c.)注射到Balb/c小鼠的右侧。第15天给小鼠静脉注射30μgIFNα-Fc或IFN-anti-PD-L1。第3天灌流后收集不同的小鼠组织，用ELISA法测定不同器官组织匀浆提取物中人Fc的水平。
肿瘤生长和治疗
将A20细胞(3×10 6)皮下注射到Balb/c小鼠的右侧。静脉注射20μg IFN-anti-PD-L1。为了阻断PD-1信号传导，在IFN-anti-PD-L1治疗前一天，用100μg抗PD-1抗体(4H2)静脉内处理小鼠，两周一次。为使CD8+T细胞删除，IFN-anti-PD-L1治疗前一天腹膜内注射200μg抗-CD8抗体(TIB210)。为了阻断I型IFN信号传导，在IFN-anti-PD-L1处理前一天注射100μg抗-IFNAR1抗体(MAR1-5A3)。将C57BL/6小鼠右侧皮下注射5×10 5个MC38细胞。小鼠静脉注射25μgIFN-anti-PD-L1两次。每周测量两次肿瘤体积，计算为(长×宽×高/2)。为了阻断淋巴细胞运输，小鼠腹腔内注射25μgFTY720，每隔一天给予20μgFTY720维持阻断。
ELISPOT检测肿瘤抗原特异性T细胞
从荷瘤小鼠中分离淋巴结(LNs)或脾脏，制备单细胞悬浮液。用单剂量的60Gys(10Gys/min，6分钟)照射A20肿瘤细胞。将脾细胞或LN细胞与照射的肿瘤细胞以4：1的比例共培养48小时。根据制造商的操作方案(BD Biosciences)，用IFN-γELISPOT测定试剂盒测定IFN-γ产生。用免疫斑点分析仪(CTL)计算细胞因子斑点。
离体培养和功能分析
收集肿瘤组织，切成小块，重悬于消化缓冲液(含有1mg/ml胶原酶IV和100μg/ml DNA酶I的RPMI-1640培养基)中。37℃消化45min后，通过70μm细胞过滤器制成单细胞悬液，用FACS分选CD8+T细胞和DCs(MHCII+CD11c+)。T细胞，DCs和辐照后的肿瘤细胞以10:1:2.5比例在IFNα(2ng/ml)或抗PD-L1抗体(10μg/ml)的条件下培养。三天后，收集上清液并通过CBA测量IFN-γ水平。
统计分析
数据显示为平均值±SEM。使用不成对的Student's双尾t检验比较统计学分析。使用GraphPad Prism 5.0版(GraphPad Software)进行分析。用*，**和***分别表示p<0.05，p<0.01 和p<0.001的统计学显著性差异。
实施例1：I型IFN的局部用药克服了对PD-1/PD-L1阻断治疗的肿瘤抗性
最近的一项研究表明，用免疫检查点阻断治疗的患者的临床反应与T细胞活化状态和肿瘤负荷相关。与之一致的，本发明发现抗PD-L1抗体在小A20肿瘤(<50mm 3)中显示有效的肿瘤控制(图1a)。相反，当肿瘤变大(>100mm 3)时，抗肿瘤效果显著降低(图1b)。晚期肿瘤可能已经形成了多种抑制抗肿瘤免疫反应的机制。事实上，当比较小肿瘤和大肿瘤中的T细胞激活时，观察到PD-L1阻断在小肿瘤中诱导强烈的T细胞激活，而同样的治疗对晚期肿瘤中的T细胞的作用有限(图1c)，该数据提示了T细胞活化不足可能是晚期肿瘤对免疫检查点阻断疗法不能有效应答的原因。
为了验证这一假设，设计实验探讨了是否提供I型IFN(一种增强对细胞毒性T细胞的交叉呈递的强效细胞因子)能够改善PD-1/PD-L1阻断治疗。晚期肿瘤采用PD-L1阻断和IFNα联合治疗。单独的抗PD-L1抗体和IFNα(IFNα4)都不能够控制肿瘤，并且所有肿瘤最终发生进展(图1d)。令人印象深刻的是，联合治疗诱导更强的抗肿瘤作用，导致所有治疗小鼠完全根除肿瘤(图1d)。在另一种肿瘤模型MC38(图1e)中发现了类似的协同效应。I型IFN信号在肿瘤微环境(TME)局部起作用，因为IFNα的瘤内递送有效地控制肿瘤生长(图1f)。相反，当全身性递送IFN时，这些作用完全消失。
综上，上述数据表明I型IFN与PD-1/PD-L1阻断治疗具有协同作用，能够控制晚期肿瘤。这也表明干扰素需要靶向到TME以实现优化的抗肿瘤作用。
实施例2：构建IFN-anti-PD-L1融合蛋白用以将IFN特异性递送至肿瘤组织中
大多数患者不能够实施干扰素的肿瘤局部用药。此外，I型IFN的全身性递送通常具有有限的抗肿瘤活性和严重的副作用。具有细胞因子的靶向性抗体已被证明是免疫调节分子局部递送的有效策略。然而，鉴别用于治疗靶向的肿瘤特异性分子相当困难。已报道PD-L1在肿瘤组织中高度表达。最近的研究显示，抗PD-L1抗体特异性累积在PD-L1阳性的肿瘤组织中。而且，除了它们的抗肿瘤功能之外，IFNs能强力诱导PD-L1表达，因此抑制了T细胞对肿瘤的应答。为了克服这种抵消效应，实现在TME中免疫(再)激活的相互促进，本发明提出使用IFN与抗PD-L1抗体构建融合蛋白IFN-anti-PD-L1，可以进一步上调PD-L1在肿瘤组织中的表达，从而导致抗体累积增加。
为了验证这个假设，本发明以同源二聚体或异源二聚体形式产生了含有抗PD-L1抗体单链可变区片段[scFv(PD-L1)]和IFNα的融合蛋白(图2a)。为了评价所得IFN-anti-PD-L1 融合蛋白(使用IFN-α4)，测试了其与PD-L1或IFN-α受体1(IFNAR1)的亲和力。A20细胞对于PD-L1和IFN受体都是阳性的。因此，我们敲除了A20细胞中的一个受体，并测试了融合蛋白与另一个受体的结合。在表达PD-L1的IFNAR1 -/-A20细胞中，融合蛋白与抗PD-L1抗体具有相似的亲和力(图2b)。在表达IFNAR的PD-L1 -/-A20细胞中，与IFN-Fc或同源二聚体相比，异源二聚体的结合减少(图2c)。
Figure PCTCN2019082360-appb-000001
注，“+”表示结合强度，“-”表示未结合，空格表示未实验。
融合蛋白有效地保护了L929细胞免受水泡性口炎病毒(VSV)的感染，表明IFN的抗病毒活性保持不变(图2d)。
综上，这些数据表明IFN-anti-PD-L1的同源二聚体和异源二聚体形式融合蛋白可以与PD-L1结合，同时保持有效的IFN生物活性。
实施例3：通过抗PD-L1靶向递送IFN，控制晚期肿瘤
鉴于IFN-anti-PD-L1(IFN-α4)融合蛋白在体外的强效活性，进一步研究了它是否可以控制体内肿瘤生长。将具有晚期A20肿瘤的小鼠用融合蛋白(i.t.)处理。虽然抗PD-L1抗体未能控制肿瘤生长，但IFN-anti-PD-L1融合蛋白的异源二聚体和同源二聚体在大部分处理的小鼠中克服了抗PD-L1抗性并诱导了完全肿瘤消退(2e)。
为了测试融合蛋白的靶向效果，将A20荷瘤小鼠用融合蛋白进行系统性处理。令人吃惊的是，尽管同源二聚体对IFN受体具有更高的结合亲和力，并且在体外具有更有效的抗病毒活性(图2c和2d)，但是当体内递送时，仅异源二聚体而不是同源二聚体能有效抑制肿瘤生长(图2f)。在MC38模型中观察到类似的作用(图2g)。我们想知道这种差异是否是由于它们的体内动力学不同。进一步实验结果显示与同源二聚体相比，异源二聚体在肿瘤组织中积累的水平要高得多(图2h)。此外，异源二聚体的血清半衰期显著增长(图2i)。这些数据同样提示异源二聚体具有更好的抗肿瘤作用，同时表示异源二聚体形式是体内研究的较好候选者。
综上，这些数据显示抗PD-L1抗体靶向递送IFN可诱导有效的抗肿瘤作用，导致改善的 肿瘤控制。
实施例4：肿瘤靶向性IFN-anti-PD-L1显示出较小的毒性和强的抗肿瘤活性
I型IFN的应用由于系统递送时严重的副作用而受到限制。为了测试带有IFN(IFN-α4)的抗PD-L1抗体的体内毒性，用高剂量的异源二聚体(IFN-anti-PD-L1)或非靶向对照IFN-anti-HBs(抗乙型肝炎病毒表面蛋白)融合蛋白。IFN-anti-HBs第二次注射后，荷瘤小鼠出现严重的体重减轻，活动减少，皮毛皱褶，并在一天内全部死亡(图3a)。相反，用IFN-anti-PD-L1处理的小鼠没有一只死亡，并在轻度体重减轻后恢复(图3a和3b)。IFN-anti-PD-L1融合蛋白在肿瘤中积累而不在正常组织中累积(图7a)。
为了更好地评价副作用，首次注射后测定血清中的细胞因子水平。令人印象深刻的是，非靶向的IFN-anti-HBs诱导了炎性细胞因子TNF，IFN，MCP-1，IL-6和IL-10等的高表达(图3c和图7b)。
肿瘤特异性靶向对于融合蛋白抗肿瘤作用是至关重要的，因为IFN-Fc与抗PD-L1抗体的简单混合物不产生如IFN-anti-PD-L1融合蛋白那样的协同作用(图3d)。
综上所述，这些数据表明IFN-anti-PD-L1可靶向肿瘤组织抑制肿瘤生长，并且具有较小的毒副作用。
实施例5：IFN-anti-PD-L1上调TME中的PD-L1和IFNAR受体
由于I型IFN是诱导PD-L1表达的最有效的细胞因子，在用IFN-anti-PD-L1(IFN-α4异源二聚体)全身治疗后测量TME中的PD-L1水平。IFN-anti-PD-L1显著增加了肿瘤组织中的PD-L1表达(图3e)。增加的PD-L1表达可以增强融合蛋白的肿瘤特异性积累。有趣的是，CD45 +细胞(白细胞)中IFN受体的水平也增加(图3f)，进一步使细胞对IFN治疗敏感。综上所述，数据显示，IFN-anti-PD-L1产生多个前馈反应，这可能进一步增强肿瘤靶向和抗肿瘤作用。
实施例6：肿瘤细胞中PD-L1对干扰素融合蛋白的抗肿瘤作用是非必需的
许多肿瘤细胞过度表达PD-L1作为逃避免疫反应的策略。PD-L1在肿瘤外的许多细胞中可被炎性细胞因子进一步诱导。关于PD-1/PD-L1阻断治疗作用于肿瘤细胞还是非肿瘤细胞表达的PD-L1至今没有定论。为了确定肿瘤细胞中的PD-L1是否对于带有IFN的抗PD-L1抗体是必需的，本发明使用CRISPR/Cas9技术敲除肿瘤细胞中的PD-L1。在敲除后的A20和MC38肿瘤细胞中完全消除了PD-L1表达(图4a)。IFNs可以诱导PD-L1表达。当被IFNα刺激时，野生型(WT)细胞上调PD-L1，而敲除细胞仍不表达PD-L1(数据未显示)。
为了测试肿瘤细胞上的PD-L1对靶向作用是否必需，使用融合蛋白IFN-anti-PD-L1(IFN-α4异源二聚体)处理WT或PD-L1敲除(PD-L1 -/-)的荷瘤小鼠。测量肿瘤组织中的蛋白质水平。令我们惊讶的是，无论肿瘤细胞是否表达PD-L1，融合蛋白均在肿瘤组织中累积(图4b)。当使用IFN-anti-PD-L1治疗肿瘤时，PD-L1敲除肿瘤和WT肿瘤均能够被有效地控制(图4c和4d)。这些数据表明肿瘤细胞中的PD-L1对于抗肿瘤效果是非必需的。
之前的数据显示IFN-anti-PD-L1创建了一个前馈环，以上调TME中的PD-L1表达(图3e)。由于肿瘤和基质细胞均可表达PD-L1，我们检测了肿瘤组织内不同细胞亚群的PD-L1表达水平。IFN-anti-PD-L1治疗显著上调肿瘤和基质细胞中的PD-L1表达(图4e)。由于肿瘤细胞中的PD-L1不是必需的，因此我们想知道PD-L1在宿主细胞中是否需要。有趣的是，IFN-anti-PD-L1在PD-L1缺陷型小鼠中良好的控制肿瘤生长(图4f)。综上，这些数据表明，在宿主细胞或肿瘤细胞中表达的PD-L1都足以介导融合蛋白的肿瘤靶向和抗肿瘤作用。
实施例7：IFN-anti-PD-L1融合蛋白促进TME中APC和T细胞的活化
接下来，研究了I型IFN信号是否是抗肿瘤作用必需的。在IFN-anti-PD-L1融合蛋白(异源二聚体)处理期间用抗-IFNAR阻断抗体处理小鼠。抗-IFNAR抗体完全消除了融合蛋白的抗肿瘤作用，提示了I型IFN信号传导的重要作用(图5a)。
IFNAR在肿瘤和宿主细胞中均有表达。为了检测肿瘤细胞中的IFN受体是否是必需的，我们敲除了肿瘤细胞中的IFNAR1(图5b)。有趣的是，IFN-anti-PD-L1在携带A20.IFNAR1 - /-肿瘤的小鼠中有效控制肿瘤生长(图5c)。
由于肿瘤细胞中的IFN受体不是必需的，因此我们研究了在宿主细胞上表达的受体是否是必需的。将MC38肿瘤接种到WT或IFNAR -/-小鼠中并用融合蛋白处理。实验观察到在缺陷型小鼠中抗肿瘤作用消失了，提示干扰素受体在宿主中起更重要的作用(图5d)。
CD8+T细胞是抗肿瘤作用所必需的，当使用抗CD8抗体对CD8+T细胞进行阻断时，完全消除了抗肿瘤作用(图5e)。之前的研究表明，I型IFN增强TME中的DC交叉呈递，导致更好的T细胞激活。事实上，IFN-anti-PD-L1处理增加了DC活化的标志分子CD86和CD80的表达(图5f和图8)。作为对照，在IFN-Fc处理的肿瘤中未观察到DC的显著激活，这表明肿瘤特异性靶向的重要作用。
综上，这些数据表明IFN融合蛋白主要通过宿主细胞中的IFN信号传导来介导其抗肿瘤作用。
实施例8：通过PD-L1抗体靶向递送IFN克服了对PD-1阻断疗法的肿瘤抗性
晚期肿瘤常对PD-1/PD-L1阻断治疗有抗药性。事实上，抗PD-1抗体和抗PD-L1抗体治 疗都不能控制晚期A20肿瘤的生长(图6a)。与PD-1/PD-L1阻断相比，IFN-anti-PD-L1融合蛋白(IFN-α4异源二聚体)显示更好的抗肿瘤效果。然而，一些肿瘤最初控制后最终复发(图2f-2g)。免疫治疗后PD-L1过度表达可能进一步限制了T细胞介导的肿瘤控制。因此，我们假设抗PD-1抗体和IFN-anti-PD-L1的联合疗法可以克服对IFN或PD-1/PD-L1阻断疗法的肿瘤抗性。实际上，联合治疗导致更好的肿瘤控制，晚期肿瘤几乎完全消退(图6a)。
B16F10黑色素瘤是一种众所周知的对PD-1/PD-L1阻断治疗具有抗性的小鼠肿瘤模型。与先前的报道一致，PD-1/PD-L1阻断对B16F10模型中的肿瘤生长没有影响(图6b)。IFN-anti-PD-L1治疗仅部分控制肿瘤。有趣的是，IFN-anti-PD-L1与PD-1阻断联合治疗显著改善了抗肿瘤作用。
为了检测IFN-anti-PD-L1介导的抗肿瘤反应是否导致延长的保护性T细胞免疫，我们再次向联合治疗后经历完全肿瘤消退的小鼠接种致死剂量的A20细胞。所有的小鼠均抵制住了再次攻击的肿瘤，证实了融合蛋白诱导了记忆性的适应性免疫应答(图6c)。
为了确定组合疗法需要哪个或哪些细胞群，对经历联合疗法的小鼠用抗NK，CD4+或CD8+T细胞的删除性抗体进行处理。在没有CD8+T细胞的情况下，抗肿瘤作用完全消失(图6d)。相反，NK或CD4+T细胞的删除具有有限的效应(图9a-9b)。为了检测治疗后是否产生肿瘤特异性T细胞，从淋巴结或脾脏组织中分离细胞，并与经照射的A20肿瘤细胞共培养。进行IFN-αELISPOT测定以评估肿瘤特异性T细胞应答。单独的PD-1/PD-L1阻断对T细胞激活的作用有限(图6e和图9c)。相反，IFN-anti-PD-L1(IFN-α4异源二聚体)诱导更好的反应。重要的是，IFN-anti-PD-L1与PD-1阻断联合治疗显著增加了肿瘤特异性T细胞的数量(图6e和图9c)。
为了在确定的体外系统中测试IFN，PD-L1和肿瘤细胞的效果，可以更好地重现TME，从体内建立的肿瘤中分离DC和T细胞。在IFN，抗PD-L1抗体或两者的组合的存在下共培养细胞三天。虽然单一治疗效果有限，但IFN和抗PD-L1抗体的联合治疗显著增加了T细胞的IFNγ产生(图6f)。
综上，这些数据表明，联合干扰素和PD-1/PD-L1阻断治疗协调诱导强大的肿瘤特异性T细胞反应，可以克服肿瘤对晚期肿瘤的免疫检查点阻断的抗性。
实施例9：IFN的靶向递送激活肿瘤驻留T细胞用于肿瘤控制
数据显示，肿瘤特异性T细胞在抗肿瘤免疫应答中发挥重要作用。这些T细胞可能来自两个主要来源。一些是肿瘤组织内已有的T细胞，而另一些是从外周迁移到肿瘤组织中的新激活的T细胞。IFN不仅可以刺激DC激活TIL，还可以增加吸引T细胞的趋化因子。为了 测试哪些T细胞群体是必需的，我们使用FTY720来阻断外周淋巴细胞向肿瘤组织的运输。有趣的是，即使在淋巴细胞运输受阻的情况下，与对照组相似，IFN和PD-1/PD-L1阻断联合治疗也能控制肿瘤(图6g)。这些数据表明通过IFN-anti-PD-L1(IFN-α4异源二聚体)重新激活预先存在的T细胞足以用于肿瘤控制。一致地，CD8 +T细胞的局部删除减少了所有的抗肿瘤作用(图6g)。综上所述，这些数据表明，PD-L1阻断可以逆转T细胞耗竭状态，IFN有效地重新激活部分恢复的驻留T细胞用于肿瘤控制。
实施例10：IFNα亲和力降低突变体构建
针对已知的与受体IFNAR结合的IFNα关键氨基酸位点，做定点突变，具体突变位点请参见图11。筛选亲和力降低的IFNα突变体。IFNα突变体的氨基酸序列及编码核酸序列参见说明书序列表SEQ ID NO.25-36。
名称 氨基酸序列 编码核酸序列
Mouse IFNα4(L30A) SEQ ID NO.25 SEQ ID NO.26
Mouse IFNα4(R144A) SEQ ID NO.27 SEQ ID NO.28
Mouse IFNα4(A145G) SEQ ID NO.29 SEQ ID NO.30
Mouse IFNα4(R149A) SEQ ID NO.31 SEQ ID NO.32
Mouse IFNα4(S152A) SEQ ID NO.33 SEQ ID NO.34
humanIFNα2(Q124R) SEQ ID NO.35 SEQ ID NO.36
实施例11：IFNα-Fc突变体活性检测
通过抗病毒感染生物测定法测量IFNα-Fc突变体的生物活性。在感染VSV-GFP病毒之前，将L929细胞与每种蛋白混合一起培养过夜。培养30小时后，通过流式细胞术测定病毒感染细胞的百分比，并计算不同浓度的蛋白对细胞感染的抑制率和EC50值(图12)。结果显示构建的突变体都有一定的活性减弱，其中R144A和A145G两个突变体具有最弱的活力，是潜在的优选靶点。
实施例12：突变型IFN-anti-PD-L1体外靶向性测试
利用WT的PD-L1 +A20细胞和PD-L1 -/-A20细胞，通过CCK8试剂盒检测不同浓度蛋白处理下细胞的增殖结果。结果显示突变型IFN-anti-PD-L1相较于野生型融合蛋白，对靶向性缺失的PD-L1 -/-A20细胞增殖抑制的EC50值降低。而在靶向性存在的PD-L1 +/+A20细胞上，突变型IFN-anti-PD-L1与野生型融合蛋白对细胞增殖抑制的EC50值差别不大(图13)。突变型IFN-anti-PD-L1融合蛋白的第一多肽Anti-PD-L1(ScFv(PD-L1)-Fc)的氨基酸序列如SEQ ID NO.1所示，第二多肽突变型mIFNa4-Fc的氨基酸序列如下表所示。
名称 氨基酸序列 编码核酸序列
mIFNa4(L30A)-Fc SEQ ID NO.37 SEQ ID NO.38
mIFNα4(R144A)-Fc SEQ ID NO.39 SEQ ID NO.40
mIFNα4(A145G)-Fc SEQ ID NO.41 SEQ ID NO.42
mIFNα4(R149A)-Fc SEQ ID NO.43 SEQ ID NO.44
mIFNα4(S152A)-Fc SEQ ID NO.45 SEQ ID NO.46
hIFNα2(Q124R)-Fc SEQ ID NO.47 SEQ ID NO.48
PD-L1 -/-A20细胞与PD-L1 +/+A20细胞的EC50的比值可以反映该种双特异性蛋白的靶向性，将该比值与wt-mIFNa4-Fc的数据进行归一化比较后可以发现，R144A和A145G是靶向性最好的两种突变，提示这两个突变体融合蛋白可以较特异的靶向PD-L1阳性的靶细胞，同时避免在其他细胞上诱导IFNAR信号通路活化。
活力降低的IFNα突变体与靶向蛋白融合后，能够只在靶细胞上诱导IFNAR活化。避免了IFN使用时的外周脱靶。本发明构建的突变型IFN-anti-PD-L1治疗肿瘤具有更大的潜力。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (22)

  1. 一种融合蛋白，其特征在于，所述融合蛋白由干扰素(IFN)与PD-L1结合蛋白融合连接构成IFN-anti-PD-L1，所述融合蛋白为同源二聚体蛋白或异源二聚体蛋白；
    优选的，所述同源二聚体蛋白包含第一多肽和第二多肽，所述第一多肽和第二多肽相同；所述第一多肽和第二多肽自N端至C端顺序包含IFN、PD-L1结合蛋白、和免疫球蛋白Fc区；
    优选的，所述异源二聚体蛋白包含第一多肽和第二多肽，所述第一多肽与第二多肽不同；所述第一多肽包含PD-L1结合蛋白，所述第二多肽包含IFN和免疫球蛋白Fc区，IFN位于Fc区的N端；所述第一多肽包含的Fc区与第二多肽包含的Fc区源自具有相同亚型的免疫球蛋白。
  2. 根据权利要求1所述融合蛋白，其特征在于，所述干扰素(IFN)可选自I型干扰素、I型干扰素突变体、II型干扰素和/或III型干扰素，例如IFN-α、IFN-β、IFN-γ、IFN-λ1(IL-29)、IFN-λ2(IL-28a)、IFN-λ(IL-28b)和IFN-ω；所述IFN可来自人源或鼠源；优选I型干扰素，优选IFN-α4(SEQ ID NO.13)，更优选IFN-α4突变体；更优选突变体mIFN-α4(L30A)(SEQ ID NO.25)、mIFN-α4(R144A)(SEQ ID NO.27)、mIFN-α4(A145G)(SEQ ID NO.29)、mIFN-α4(R149A)(SEQ ID NO.31)、mIFN-α4(S152A)(SEQ ID NO.33)、hIFN-α2(Q124R)(SEQ ID NO.35)。
  3. 根据权利要求1所述融合蛋白，其特征在于，所述免疫球蛋白Fc区可选自IgG1、IgG2、IgG3和/或IgG4的恒定区氨基酸序列，优选IgG1。
  4. 根据权利要求1所述融合蛋白，其特征在于，所述PD-L1结合蛋白可选自抗PD-L1抗体、单链抗体(ScFv)、Fab片段、F(ab’) 2片段；所述抗PD-L1抗体优选自：Tecentriq、Bavencio、Imfinzi、KN035、CS1001、KL-A167、SHR-1316和/或YW243.55.S70；更优选单链抗体(ScFv)；更优选YW243.55.S70。
  5. 根据权利要求1所述融合蛋白，其特征在于：
    1)所述同源二聚体的第一多肽和第二多肽包含SEQ ID NO.3所示氨基酸序列；
    2)所述异源二聚体第一多肽包含SEQ ID NO.1所示氨基酸序列，第二多肽包含SEQ ID NO.2、37、39、41、43、45、47所示氨基酸序列。
  6. 编码权利要求1-5所述融合蛋白的分离的核酸分子，其特征在于：
    1)编码所述同源二聚体的核酸分子优选SEQ ID NO.6所示核苷酸序列；
    2)编码所述异源二聚体的核酸分子可选自SEQ ID NO.4、5、7、8、9、10、38、40、42、44、46、48所示核苷酸序列；优选SEQ ID NO.4和SEQ ID NO.5，SEQ ID NO.4和SEQ ID NO.40，或SEQ ID NO.4和SEQ ID NO.42。
  7. 一种载体，其特征在于，所述载体包含权利要求6所述核酸分子。
  8. 权利要求1-5任一项所述融合蛋白和/或权利要求6所述核酸分子在制备药物组合物或试剂盒中的应用，所述药物组合物优选为治疗肿瘤的药物组合物；所述肿瘤优选为对PD-1/PD-L1阻断单独治疗无效的肿瘤或晚期肿瘤；更优选为治疗B细胞淋巴瘤、黑色素瘤和结肠癌的药物组合物。
  9. 一种药物制剂、药物组合物或试剂盒，其特征在于，所述药物制剂、药物组合物或试剂盒包含权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1。
  10. 一种细胞，其特征在于，所述细胞包含权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1或编码所述融合蛋白的核酸分子，用于生产融合蛋白；所述细胞选自非人哺乳动物细胞，优选CHO和HEK293细胞。
  11. 权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1与抗PD-1/PD-L1抗体共同用于制备治疗肿瘤的药物组合物或试剂盒的用途。
  12. 根据权利要求11所述用途，其特征在于，所述肿瘤为对PD-1/PD-L1阻断单独治疗无效的肿瘤或晚期肿瘤；优选对抗PD-1/PD-L1抗体单独治疗产生抗性或无效的肿瘤。
  13. 根据权利要求11所述用途，其特征在于，所述肿瘤的患者患有外周淋巴细胞运输缺陷/障碍相关疾病，所述患者的外周淋巴细胞无法迁移至肿瘤组织。
  14. 一种IFN-α和抗PD-L1抗体共同用于制备治疗肿瘤的药物组合物、药物制剂或试剂盒的用途，其特征在于，所述IFN-α需向肿瘤内施用。
  15. 根据权利要求14所述用途，其特征在于，所述肿瘤为对PD-1/PD-L1阻断单独治疗无效的肿瘤、对IFN单独治疗无效的肿瘤或晚期肿瘤；优选对抗PD-1/PD-L1抗体单独治疗产生抗性或无效的肿瘤。
  16. 一种包含IFN-α和抗PD-L1抗体的药物组合物、药物制剂或试剂盒，其特征在于，所述IFN-α需向肿瘤内施用。
  17. 权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1在上调白细胞中IFN受体表达中的用途；所述白细胞优选为CD45+细胞；所述IFN受体优选为IFNAR。
  18. 权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1在激活DC细胞或TIL细胞中的用途。
  19. 权利要求1-5任一项所述融合蛋白IFN-anti-PD-L1在激活肿瘤驻留T细中的用途。
  20. 一种IFN-α4突变体，其特征在于，所述突变体为与受体亲和力减弱突变，包括mIFN-α4(L30A)(SEQ ID NO.25)、mIFN-α4(R144A)(SEQ ID NO.27)、mIFN-α4(A145G)(SEQ ID NO.29)、mIFN-α4(R149A)(SEQ ID NO.31)、mIFN-α4(S152A)(SEQ ID NO.33)、和/或hIFN-α2(Q124R)(SEQ ID NO.35)。
  21. 编码权利要求20所述突变体的分离的核酸分子，其特征在于，所述核酸分子的核苷酸序列如SEQ ID NO.26、28、30、32、34、36所示。
  22. 权利要求20所述IFN-α4突变体在制备治疗肿瘤的融合蛋白或药物中的用途。
PCT/CN2019/082360 2018-04-16 2019-04-12 一种ifn与抗pd-l1抗体的融合蛋白及其应用 WO2019201161A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2020557311A JP7133241B2 (ja) 2018-04-16 2019-04-12 Ｉｆｎと抗ｐｄ－ｌ１抗体の融合タンパク質およびその使用
US17/048,106 US20210147548A1 (en) 2018-04-16 2019-04-12 Fusion protein of interferon (ifn) and anti-pd-l1 antibody and use thereof
EP19789504.8A EP3783035A4 (en) 2018-04-16 2019-04-12 IFN AND ANTI-PD-L1 ANTIBODY FUSION PROTEIN AND USE THEREOF

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810336178.1A CN108727504B (zh) 2018-04-16 2018-04-16 一种ifn与抗pd-l1抗体的融合蛋白及其应用
CN201810336178.1 2018-04-16

Publications (1)

Publication Number Publication Date
WO2019201161A1 true WO2019201161A1 (zh) 2019-10-24

Family

ID=63939644

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/082360 WO2019201161A1 (zh) 2018-04-16 2019-04-12 一种ifn与抗pd-l1抗体的融合蛋白及其应用

Country Status (5)

Country Link
US (1) US20210147548A1 (zh)
EP (1) EP3783035A4 (zh)
JP (1) JP7133241B2 (zh)
CN (1) CN108727504B (zh)
WO (1) WO2019201161A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022015711A1 (en) * 2020-07-14 2022-01-20 Immune Targeting Inc. Fusion proteins of anti-pd-l1 and attenuated interferon, and compositions and therapeutic methods thereof

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108727504B (zh) * 2018-04-16 2021-08-27 泉州向日葵生物科技有限公司 一种ifn与抗pd-l1抗体的融合蛋白及其应用
CN111423510B (zh) * 2019-01-10 2024-02-06 迈威(上海)生物科技股份有限公司 重组抗人pd-1抗体及其应用
CN111100211B (zh) * 2019-01-30 2022-04-08 武汉九州钰民医药科技有限公司 一种Fc融合蛋白及其应用
CN110151999A (zh) * 2019-05-22 2019-08-23 中国人民解放军第四军医大学 用于抑制恶性黑色素瘤进展的靶向药物
CN117050182A (zh) * 2019-06-27 2023-11-14 启愈生物技术(上海)有限公司 抗PD-L1纳米抗体及其Fc融合蛋白和应用
CN116867805A (zh) * 2021-09-27 2023-10-10 盛禾(中国)生物制药有限公司 一种异源二聚体蛋白质及其应用
CN116023503B (zh) * 2021-10-25 2024-05-31 上海交通大学 一种融合蛋白及其制备方法和用途
CN114106198B (zh) * 2021-11-11 2023-04-07 武汉大学 一种pd1抗体与ccl7融合蛋白及其制备方法与应用
CN114853909A (zh) * 2022-05-13 2022-08-05 南京吉盛澳玛生物医药有限公司 新型IL-2与INFα和Fc融合蛋白的设计、制备及用途
WO2024008039A1 (zh) * 2022-07-08 2024-01-11 盛禾(中国)生物制药有限公司 一种异源二聚体融合蛋白及其应用
WO2024067785A1 (zh) * 2022-09-29 2024-04-04 中国科学院生物物理研究所 基于干扰素协同免疫检查点阻断抗体治疗慢性乙型肝炎
WO2024104412A1 (en) * 2022-11-16 2024-05-23 I-Mab Biopharma Co., Ltd. Attenuated interferon proteins and fragments and multifunctional polypeptides and conjugates

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN104403004A (zh) * 2014-11-24 2015-03-11 苏州丁孚靶点生物技术有限公司 抗体-干扰素异二聚体的制备和用途
WO2017134302A2 (en) * 2016-02-05 2017-08-10 Orionis Biosciences Nv Targeted therapeutic agents and uses thereof
CN108727504A (zh) * 2018-04-16 2018-11-02 中国科学院生物物理研究所 一种ifn与抗pd-l1抗体的融合蛋白及其应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU728146B2 (en) * 1996-03-14 2001-01-04 Immune Response Corporation, The Targeted delivery of genes encoding interferon
BR0010725A (pt) * 1999-05-19 2002-02-19 Lexigen Pharm Corp Expressão e exportação de proteìnas de interferon-alfa como proteìnas de fusão de fc
WO2012171541A1 (en) * 2011-06-15 2012-12-20 Scil Proteins Gmbh Human fusion proteins comprising interferons and hetero-dimeric modified ubiquitin proteins
CA2861927C (en) * 2012-01-20 2021-01-26 Vib Vzw Targeted mutant alpha-helical bundle cytokines
US10668165B2 (en) * 2015-01-16 2020-06-02 Immunwork Inc. Molecular constructs for treating tumors
DK3909972T3 (da) * 2015-06-19 2024-04-29 Sebastian Kobold Pd1-cd28-fusionsproteiner og disses anvendelse i medicin
WO2017044487A1 (en) * 2015-09-09 2017-03-16 Seattle Children's Hospital (dba Seattle Children's Research Institute) Genetic engineering of macrophages for immunotherapy
WO2017091611A1 (en) * 2015-11-23 2017-06-01 Immungene, Inc Enhanced cancer immunotherapy using antibody-interferon fusion molecules
RU2758007C2 (ru) * 2016-06-30 2021-10-25 Онкорус, Инк. Доставка терапевтических полипептидов посредством псевдотипированных онколитических вирусов
US20210024631A1 (en) * 2018-03-28 2021-01-28 Orionis Biosciences, Inc. Bi-functional proteins and construction thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN104403004A (zh) * 2014-11-24 2015-03-11 苏州丁孚靶点生物技术有限公司 抗体-干扰素异二聚体的制备和用途
WO2017134302A2 (en) * 2016-02-05 2017-08-10 Orionis Biosciences Nv Targeted therapeutic agents and uses thereof
CN108727504A (zh) * 2018-04-16 2018-11-02 中国科学院生物物理研究所 一种ifn与抗pd-l1抗体的融合蛋白及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3783035A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022015711A1 (en) * 2020-07-14 2022-01-20 Immune Targeting Inc. Fusion proteins of anti-pd-l1 and attenuated interferon, and compositions and therapeutic methods thereof

Also Published As

Publication number Publication date
JP7133241B2 (ja) 2022-09-08
CN108727504B (zh) 2021-08-27
JP2021521789A (ja) 2021-08-30
US20210147548A1 (en) 2021-05-20
EP3783035A1 (en) 2021-02-24
CN108727504A (zh) 2018-11-02
EP3783035A4 (en) 2022-07-20

Similar Documents

Publication Publication Date Title
WO2019201161A1 (zh) 一种ifn与抗pd-l1抗体的融合蛋白及其应用
JP7288405B2 (ja) ヒトドメインを有する抗ｂ細胞成熟抗原キメラ抗原受容体
JP2020200346A (ja) 腫瘍療法のためのｉｌ−１２とｔ細胞阻害分子遮断薬とを含む医薬組成物
EP0751781B1 (en) Use of monoclonal antibodies or soluble oligomeric ligands in the manufacture of a medicament for the prevention or treatment of neoplastic disorders
EP4032552B1 (en) Method and compositions for enhanced anti-tumor effector functioning of t cells
US20180147257A1 (en) Btn3a ectodomain proteins and methods of use
JP7423607B2 (ja) インターロイキン ２突然変異タンパク質およびｉ型インターフェロンで構成される融合タンパク質
US20220041724A1 (en) Plasmid constructs for treating cancer and methods of use
Zhang et al. Biological effects of IL-15 on immune cells and its potential for the treatment of cancer
KR20080112232A (ko) 포유동물 환자의 면역기능의 개선 방법 및 질병의 예방 또는 치료 방법
JP2021513570A (ja) サイトカインをコードするｒｎａを用いた治療
WO2021238904A1 (zh) Fc-CD80融合蛋白和其缀合物以及它们的用途
EP3247368A1 (en) Cytokine receptor genes and the use thereof to enhance therapy
KR20230070256A (ko) 이중특이성 재조합 단백질 및 이의 용도
WO2022089601A1 (zh) 一种il-2与抗体亚单位构成的双功能融合蛋白
WO2021068196A1 (en) Methods of using il-33 protein in treating cancers
CN108495865A (zh) 具有细胞因子受体激活或阻断结构域的嵌合抗原受体
JP2023514152A (ja) Ｉｌ－１０およびその使用
US20230134725A1 (en) Fusion Protein Extensions
JP2022511286A (ja) Ｓｉｒｐアルファ系キメラタンパク質を含む併用療法
JP7514231B2 (ja) 癌を治療するためのプラスミド構築体および使用方法
WO2023143308A1 (zh) 一种pd1抗体与白介素2融合的双功能分子
TWI734027B (zh) 用於治療癌症之組合物
WO2024060140A1 (zh) EGFRvIII嵌合抗原受体及其用途
KR20240074772A (ko) 항-pd-l1/il-10 융합 단백질을 이용한 질병 치료 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19789504

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020557311

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019789504

Country of ref document: EP

Effective date: 20201116