WO2019129053A1 - 以抗体Fc区为骨架的融合蛋白二聚体及其应用 - Google Patents

以抗体Fc区为骨架的融合蛋白二聚体及其应用 Download PDF

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WO2019129053A1
WO2019129053A1 PCT/CN2018/123878 CN2018123878W WO2019129053A1 WO 2019129053 A1 WO2019129053 A1 WO 2019129053A1 CN 2018123878 W CN2018123878 W CN 2018123878W WO 2019129053 A1 WO2019129053 A1 WO 2019129053A1
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antibody
fusion protein
polypeptide chain
seq
amino acid
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PCT/CN2018/123878
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English (en)
French (fr)
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李中道
宋立新
张望
张亚峰
汪东亮
柳振宇
章方良
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南京金斯瑞生物科技有限公司
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Priority to US16/958,079 priority Critical patent/US11970537B2/en
Priority to CN201880084110.7A priority patent/CN111527109A/zh
Priority to JP2020555280A priority patent/JP7336457B2/ja
Priority to EP18894894.7A priority patent/EP3733716A4/en
Publication of WO2019129053A1 publication Critical patent/WO2019129053A1/zh

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    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • 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
    • A61K47/6875—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 being a hybrid immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • 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
    • 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
    • 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 present invention relates to a fusion protein, in particular a fusion protein dimer based on the Fc region of an antibody.
  • the immune system is a defense system in mammals that resists the invasion of various pathogens and eliminates tumors.
  • Antibody molecules play an important role in the immune system, such as identifying tumor antigens, activating immune responses, and the like.
  • cytokines as immunoregulators, can simultaneously regulate innate and adaptive immunity, and also have an important role in cancer immunotherapy [1-3]. This type of regulatory factor is involved in almost every part of the immune system and plays a powerful role in regulating and balancing the immune system. Interferon and IL-2 were first selected as cytokines for use in immunotherapy.
  • IFN- ⁇ is used to treat various cancers such as melanoma, renal cancer, follicular lymphoma, and chronic myeloid leukemia; IL-2 was approved by the FDA in 1998 for the treatment of advanced metastatic melanoma and metastasis. Kidney cancer and so on. Other cytokines, such as IL-7, IL-10, IL-12, IL-15 and IL-21, are also in clinical testing [4]. Therefore, cytokines have certain potential for immunotherapy [5,6].
  • cytokine-based immunotherapy is severely impeded in clinical use, mainly due to severe side effects and poorly performing pharmacokinetic properties [5]. Therefore, in order to enhance the therapeutic effect of cytokines, it is often necessary to modify cytokines.
  • a more common method is to use antibody fusion methods to target cytokines to specific sites by antibodies, thereby effectively reducing cytotoxicity and improving pharmacokinetic properties, thereby enhancing immune regulation [7,8]. .
  • some cytokines are fused to an antibody, the activity of the antibody itself is reduced, thereby affecting the overall effect.
  • some cytokines need to be used in combination to achieve their best results [9,10], and existing technologies cannot meet this requirement.
  • the invention provides a fusion protein dimer having an antibody Fc region as a backbone, comprising a first and a second polypeptide chain, wherein the first polypeptide chain comprises a first antibody Fc region and One or more single domain antibodies fused to a first antibody Fc region; the second polypeptide chain comprising a second antibody Fc region and one or more single domain antibodies fused to the second antibody Fc region;
  • the first and/or second polypeptide chain also includes a cytokine fused to the respective antibody Fc region.
  • the single domain antibody is a single domain antibody against an immunological checkpoint molecule.
  • the single domain antibody is an anti-PD-1 single domain antibody or an anti-PD-L1 single domain antibody.
  • the single domain antibody is an anti-PD-1 single domain antibody.
  • the single domain antibody is a PD-L1 single domain antibody.
  • the cytokine is selected from the group consisting of IL-2, IL-12, GM-CSF, IL-2 mutants, and combinations thereof.
  • the two subunits P35 and P40 of IL-12 are joined by a linker sequence to form an IL-12 single chain protein and are present in the fusion protein dimer. In some specific embodiments, the two subunits P35 and P40 of IL-12 are joined by a linker sequence to form an IL-12 single chain protein and are present in the first polypeptide chain and/or the second polypeptide chain.
  • the single domain antibody, antibody Fc region, and cytokine in the fusion protein dimer are joined or directly fused by a linker sequence.
  • the single domain antibody and the antibody Fc region in the fusion protein dimer are joined or directly linked by a linker sequence.
  • the fusion protein dimer is linked between the antibody Fc region and the cytokine by a linker sequence.
  • the linker sequence is selected from the group consisting of (G4S) 1-3, KRVAPELLGGPS, ASTKG, and NSPPAA.
  • the first antibody Fc region and the second antibody Fc region are different (ie, form an antibody Fc fusion protein heterodimer) and have an asymmetric complementary structure therebetween.
  • the asymmetric complementary structure can be formed, for example, by a KiH (knobs-into-holes) technique.
  • the first antibody Fc region has a mutation site combination T366W/S354C
  • the second antibody Fc region has a mutation site combination T366S/L368A/Y407V/Y349C.
  • the antibody Fc region is selected from the group consisting of a human IgG1 mutant and a human IgG4 mutant.
  • the first polypeptide chain and/or the second polypeptide chain comprises two anti-PD-1 single domain antibodies arranged in tandem. In other embodiments, the first polypeptide chain and/or the second polypeptide chain comprises two anti-PD-L1 single domain antibodies arranged in tandem.
  • the cytokine contained in the first polypeptide chain is IL-12 and the cytokine contained in the second polypeptide chain is an IL-2 or IL-2 mutant. In some embodiments, the cytokine contained in the first polypeptide chain is IL-12 and the cytokine is deleted in the second polypeptide chain. In other embodiments, the cytokine contained in the first polypeptide chain is an IL-2 or IL-2 mutant and the cytokine is deleted in the second polypeptide chain.
  • the one or more single domain antibodies are joined to the N-terminus of the Fc region of the antibody by a linker sequence, and the cytokine is linked to the C-terminus of the Fc region of the antibody by a linker sequence.
  • the one or more single domain antibodies are linked to the C-terminus of the Fc region of the antibody by a linker sequence, and the cytokine is linked to the N-terminus of the Fc region of the antibody by a linker sequence.
  • the one or more single domain antibodies are ligated directly to the N-terminus of the Fc region, and the cytokine is linked to the C-terminus of the Fc region of the antibody by a linker sequence.
  • the one or more single domain antibodies are ligated directly to the C-terminus of the Fc region, and the cytokine is linked to the N-terminus of the Fc region of the antibody by a linker sequence.
  • the anti-PD-1 single domain antibody has the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 71. In other embodiments, the anti-PD-L1 single domain antibody has the amino acid sequence set forth in SEQ ID NOs: 72-74.
  • the IL-12 single chain protein has the amino acid sequence set forth in SEQ ID NO:6.
  • the IL-2 has the amino acid sequence set forth in SEQ ID NO:8. In other embodiments, the IL-2 mutant has the amino acid sequence set forth in SEQ ID NO: 10.
  • the first polypeptide chain and the second polypeptide chain are selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40 The amino acid sequence shown at 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 or 62.
  • the first polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 20, and the second polypeptide chain has the amino acid sequence set forth in SEQ ID NOs: 22, 24 or 28.
  • the first polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 26, and the second polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 28;
  • the amino acid sequence set forth in SEQ ID NO: 16 the second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 18; the first polypeptide chain having the amino acid sequence set forth in SEQ ID NO:
  • the second polypeptide chain has the amino acid sequence set forth in SEQ ID NOs: 34, 36; or the first polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 36, the second polypeptide The chain has the amino acid sequence set forth in SEQ ID NO:38.
  • the first polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 40
  • the second polypeptide chain has the amino acid sequence set forth in SEQ ID NOs: 42, 44
  • the first polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 44
  • the second polypeptide chain has the amino acid sequence set forth in SEQ ID NO: 46
  • the first polypeptide chain has SEQ.
  • the first antibody Fc region and the second antibody Fc region are wild type antibody Fc regions, forming an antibody Fc fusion protein homodimer.
  • the invention provides an isolated polynucleotide encoding a first polypeptide chain or a second polypeptide chain of the fusion protein dimer.
  • the polynucleotide has a nucleotide sequence as set forth in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37. In other specific embodiments, the polynucleotide has a nucleotide sequence as set forth in SEQ ID NOs: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, and 61.
  • the invention provides an expression vector comprising the polynucleotide.
  • the invention provides a host cell comprising the expression vector.
  • the invention provides the use of the fusion protein dimer, polynucleotide, expression vector, or host cell for the preparation of an anti-tumor drug.
  • the tumor is melanoma or lung cancer.
  • the invention provides an anti-tumor pharmaceutical composition
  • an anti-tumor pharmaceutical composition comprising the fusion protein dimer and a pharmaceutically acceptable carrier.
  • the invention provides a method of treating a tumor comprising administering to a subject a therapeutically effective amount of the fusion protein dimer or a pharmaceutical composition comprising the fusion protein dimer.
  • the anti-tumor pharmaceutical compositions provided herein can be administered to a subject by at least one route selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intrabronchial, and abdomen.
  • the anti-tumor pharmaceutical composition is administered to a subject by intratumoral or intravenous administration.
  • the invention provides the anti-PD-1 single domain antibody itself having the amino acid sequence set forth in SEQ ID NO:3.
  • the present invention also provides a polynucleotide encoding the anti-PD-1 single domain antibody having the nucleotide sequence shown as SEQ ID NO: 2.
  • the present invention also provides the anti-PD-1 single domain antibody itself having the amino acid sequence shown as SEQ ID NO:71.
  • the invention also provides the anti-PD-L1 single domain antibody itself having the amino acid sequence set forth in SEQ ID NOs: 72-74.
  • the fusion protein of the invention combines the advantages of small molecule, good permeability and high stability of the single domain antibody with the immunomodulation and other activities of the cytokine. More importantly, the present invention uses the antibody Fc fusion protein heterodimer technology to organically fuse the two, which not only enhances the activity of the single domain antibody, but also significantly improves the biological activity of the cytokine; The targeting specificity effectively enhances the targeted transport of cytokines, attenuates cytotoxicity, and thus has better anti-tumor potential and significantly improved pharmacokinetic properties.
  • Figure 1 shows IL-2 activity of the G1-212 fusion protein compared to control IL-2.
  • Figure 2 shows IL-2 activity of G1-716 and G1-717 fusion proteins compared to control IL-2.
  • Figure 3 shows IL-2 activity of G1-723 and G1-405 fusion proteins compared to control IL-2.
  • Figure 4 shows IL-12 activity of the G1-208 fusion protein compared to control IL-12.
  • Figure 5 shows IL-12 activity of G1-716 and G1-719 fusion proteins compared to control IL-12.
  • Figure 6 shows IL-12 activity of G1-723 and G1-405 fusion proteins compared to control IL-12
  • Figure 7 shows the PD-1/PD-L1 pathway blocking activity of the PD-1 single domain antibody compared to the control Keytruda.
  • Figure 8 shows the PD-1/PD-L1 pathway blocking activity of fusion proteins G1-405 and G1-709 compared to the control Keytruda.
  • Figure 9 shows the PD-1/PD-L1 pathway blocking activity of fusion proteins G1-717 and G1-719 compared to the control Keytruda.
  • Figure 10 shows the PD-1/PD-L1 pathway blocking activity of the fusion proteins G1-716 and G1-723 compared to the control Keytruda.
  • Figure 11 shows IL-12 activity of the heterodimeric fusion proteins sPDL1a01, sPDL1a02, sPDL1b01 and sPDL1b02 compared to control IL-12.
  • Figure 12 shows IL-12 activity of the heterodimeric fusion proteins sPDL1c01, sPDL1c02, sPD1a01 and sPD1a02 compared to control IL-12.
  • Figure 13 shows IL-2 activity of heterodimeric fusion proteins sPDL1a01, sPDL1b01, sPDL1c01 and sPD1a01 compared to control IL-2.
  • Figure 14 shows the PD-1/PD-L1 pathway blocking activity of the homodimeric fusion proteins sPDL1a00 and sPDL1b00 without cytokine compared to the control Atezolizumab.
  • Figure 15 shows the PD-1/PD-L1 pathway blocking activity of the heterodimeric fusion proteins sPDL1a01 and sPDL1a02 compared to the control Atezolizumab.
  • Figure 16 shows the PD-1/PD-L1 pathway blocking activity of the heterodimeric fusion proteins sPDL1b01 and sPDL1b02 compared to the control Atezolizumab.
  • Figure 17 shows the PD-1/PD-L1 pathway blocking activity of the heterodimeric fusion proteins sPDL1c01 and sPDL1c02 compared to the control Atezolizumab.
  • Figure 18 shows not only the PD-1/PD-L1 pathway blocking activity of the heterodimeric fusion proteins sPDL1a03, sPDL1b03, sPDL1c03, and the homodimeric fusion protein sPDL1c00 without cytokine compared to the control Atezolizumab.
  • Figure 19 shows the PD-1/PD-L1 pathway blocking activity of the homodimeric fusion protein sPD1a00 without a cytokine compared to the control Keytruda.
  • Figure 20 shows the PD-1/PD-L1 pathway blocking activity of the heterodimeric fusion proteins sPD1a01, sPD1a02 and sPD1a03 compared to the control Keytruda.
  • Figure 21 shows IL-2 activity of the heterodimeric fusion proteins sPD1a03 and sPDL1a03 compared to control IL-2.
  • Figure 22 shows IL-2 activity of the heterodimeric fusion proteins sPDL1b03 and sPDL1c03 compared to control IL-2.
  • Figure 23A shows that the cytokines IL-2 and IL-12 are each optionally linked to the C-terminus or N-terminus of each antibody Fc chain (Fcm, Fcn, respectively) via a linker sequence, correspondingly one or more single domain antibodies (1-n single domain antibodies) are optionally ligated to the other end of each antibody Fc chain by a linker sequence;
  • Figure 23B cytokine IL-12 is optionally fused alone to a single antibody Fc chain (eg, Fcm) C by a linker sequence Terminal or N-terminus, another strand lacks a cytokine, and correspondingly one or more single-domain antibodies (1-n single-domain antibodies) are optionally linked to the other end of each antibody Fc chain by a linker sequence;
  • Figure 23C Cytokine IL-2 is optionally fused alone to the C-terminus or N-terminus of one antibody Fc chain (eg, Fcm) via a linker sequence, and the other strand lacks
  • the present invention provides a fusion protein having anti-tumor activity using an antibody Fc heterodimer technique [13], which includes a single domain antibody and one or more cytokines, and an antibody Fc region as a backbone moiety.
  • antibody Fc heterodimer technique [13] which includes a single domain antibody and one or more cytokines, and an antibody Fc region as a backbone moiety.
  • single domain antibody-cytokine fusion protein “antibody/cytokine fusion protein” and “antibody-cytokine fusion protein” all refer to single domain antibody-cytokine fusion based on antibody Fc heterodimer technology.
  • the protein comprising one or more single domain antibodies and one or more cytokines linked by the Fc region of the antibody, forms a fusion protein.
  • the single domain antibody and cytokine are ligated to the ends of the antibody Fc by a linker sequence, respectively.
  • antibody refers to a large "Y" shaped protein that is secreted by plasma cells (effector B cells) and used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, and the like. Over the past 10 years, more and more monoclonal antibodies have been widely used in cancer therapy. Antibodies referred to in the present invention will include, but are not limited to, the ability to specifically bind to tumor associated antigens, induce antibody-dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) to induce tumor cell immune killing, Or block the immune checkpoint to activate the immune response.
  • the antibody is typically a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds.
  • antibody Fc region refers to a "Y" shaped handle region, ie, fragment crystallizable (Fc), including the second and third constant domains of the heavy chain (CH2 and CH3 domains) ).
  • the antibody Fc region can be obtained by hydrolyzing an antibody molecule by a proteolytic enzyme such as papain.
  • the term "based on the Fc region of an antibody” means that in the fusion protein dimer of the present invention, the single domain antibody and the cytokine are usually located on both sides of the Fc region of the antibody, that is, the single domain antibody and the cytokine are linked by the Fc region of the antibody. Fusion protein. "Based on the Fc region of an antibody” also means that two polypeptide chains (first and second polypeptide chains) are joined together by an Fc region in two polypeptide chains to form a fusion protein dimer, for example, by two Sulfur bond or non-covalent interaction.
  • the dimeric protein referred to in the present invention refers to a protein in the process of formation, if the two subunits/monomers are the same as homodimers (homo-), if they are not identical subunits/monomers Combined, it is called heterodimer (hetero-).
  • antibody Fc heterodimer means that the protein is composed of two different subunits/monomers, and each subunit/monomer contains an antibody Fc fragment. The key is that the two Fc fragments of the two antibodies have different amino acid site mutations to form a complementary protein space structure, so that two different subunits/monomers can be correctly combined.
  • KiH (knobs-into-holes) technique refers to a technique that facilitates assembly between two heterologous antibody heavy chains.
  • a small tyrosine (T) of the heavy chain CH3 region of an antibody can be mutated to a larger tyrosine (Y) to form a prominent "knobs" type structure (T366Y);
  • Another large tyrosine (Y) residue at position 407 of the heavy chain CH3 region is mutated to a smaller threonine (T), forming a recessed "holes" type structure (Y407T); using this "knobs”
  • T threonine
  • Y407T a recessed "holes" type structure
  • the steric hindrance effect of the -into-holes structure ie, the asymmetric complementary structure
  • the present invention achieves a better assembly effect by combining a plurality of site mutations in the Fc region of both antibodies.
  • single domain antibody refers to another form of antibody fragment that contains only one monomeric form of antibody variable region. Like intact antibodies, it also binds antigen specifically, but at a much lower mass than traditional antibodies (approximately 15 kDa). More importantly, thanks to the small size of the single domain antibody, it is more convenient to penetrate tissue or into the interior of the tumor, which is difficult for intact antibodies.
  • cytokine (CK) is a low molecular weight soluble protein that is produced by a variety of cells by an immunogen, mitogen or other stimulant. In vivo, by binding to its specific cell surface receptors, it transmits intracellular signals, thereby changing cell function [4], regulating innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and repair of damaged tissues. Multiple roles. Interleukins (such as IL-2 or IL-12) act as a group of cytokines that regulate the immune response by regulating the immune system. Cytokines can not only function independently, but also can be fused with antibodies to form antibody-cytokine fusion proteins, also known as immune cytokines.
  • This new protein form combines antibody-specific targeting with the immunomodulatory nature of cytokines to enhance the immunotherapeutic effect of antibodies. More importantly, the fused cytokines are transported and enriched to the tumor site by the targeting of the antibody, thereby effectively avoiding the side effects caused by the high dose of cytokines alone.
  • isolated polynucleotide refers to a polynucleotide that is naturally occurring in nature, including polynucleotides isolated from nature (including organisms) by biological techniques, as well as synthetic polynucleotides.
  • the isolated polynucleotide may be genomic DNA, cDNA, mRNA or other synthetic RNA, or a combination thereof.
  • Numerous nucleotide sequences for encoding fusion protein dimers and other polypeptide fragments of the invention are provided herein, and it is noted that one skilled in the art can rely on codon degeneracy based on the amino acid sequences provided herein.
  • a nucleotide sequence that is not identical to the nucleotide sequence provided above is designed, but both encode the same amino acid sequence. These altered nucleotide sequences are also included within the scope of the invention.
  • vector refers to any molecule (eg, nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide-encoding information into a host cell.
  • expression vector refers to a vector suitable for expressing a gene of interest (a nucleotide sequence to be expressed) in a host cell, and generally includes a gene of interest, a promoter, a terminator, a marker gene, and the like.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a gene of interest of interest.
  • the term includes progeny of the parental cell, whether or not the progeny is identical in morphology or genetic composition to the original parental cell, as long as the progeny are present in the selected gene of interest.
  • Common host cells include bacteria, yeast, mammalian cells, and the like, such as CHO cells.
  • the term "pharmaceutically acceptable carrier” as used herein refers to a solid or liquid diluent, filler, antioxidant, stabilizer, etc., which can be administered safely, suitable for humans and/or Animals are administered without excessive adverse side effects while being suitable for maintaining the viability of the drug or active agent located therein.
  • various carriers well known in the art can be administered, including, but not limited to, sugars, starches, celluloses and derivatives thereof, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols. Alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water.
  • the pharmaceutical composition provided by the present invention can be formulated into a clinically acceptable dosage form such as a powder or an injection.
  • the pharmaceutical composition of the invention may be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, and the like. Route administration.
  • terapéuticaally effective amount refers to an amount of active compound that is sufficient to cause a biological or medical response desired by a clinician in a subject.
  • a "therapeutically effective amount" of a fusion protein of the invention can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, condition, and the like. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg of body weight.
  • cytokine In order to utilize the advantages of immunocytokines and single domain antibodies to enhance the efficacy of immunotherapy, we fused a cytokine to a PD-1 single domain antibody and developed a single domain antibody-cytokine fusion protein directed against PD-1, and The function of the fusion protein was assessed by in vitro and in vivo assays.
  • the cytokines mentioned in the present invention include interleukins such as interleukin-2 (IL-2), interleukin-12 (IL-12), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Etc., in which interleukin-2 and interleukin-12 are involved in various aspects of immune regulation, thereby enhancing the immune response.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the cytokine IL-2 or IL-12 can be linked to the N-terminus or C-terminus of the antibody Fc via the linker sequence (Gly4Ser) 3 (ie, (G4S)3).
  • the PD-1 single domain antibody can also be passed through the linker sequence ( Gly4Ser)3 is linked to the N-terminus or C-terminus of the antibody Fc, and the resulting fusion protein maintains the dual effect of antibodies and cytokines.
  • IL-12 Since functional IL-12 is composed of two subunits, p35 and p40, IL-12 will be fused to the antibody Fc alone in the form of two subunits, or the two subunits of IL-12 will first be passed through the linker sequence ( Gly4Ser)3 forms a single-chain protein which is then fused to the antibody Fc.
  • linker sequence Gly4Ser
  • a cytokine In a conventional antibody-cytokine fusion protein structure, a cytokine is simultaneously linked to two heavy chains or two light chains of an antibody to exist as a homodimer.
  • this pharmacokinetic property of immunocytokines based on IgG platform fusion is poor.
  • antibody-based Fc heterodimer technology In order to fuse cytokines into antibodies in monomeric form, while changing the ratio of antibody to cytokines to enhance the targeting of fusion proteins, we used antibody-based Fc heterodimer technology to IL-2 or IL.
  • IL-12 is fused to one Fc chain alone, and the other chain lacks cytokines to form IL-12 or IL-2 monomer fusion proteins, as shown in Figure 23B and Figure 23C, respectively, or IL2 and IL-12 are each linked to On one Fc chain, a fusion protein having both IL-2 and IL-12 proteins was produced, as shown in Figure 23A.
  • the core of the antibody Fc heterodimer technology is to separately engineer the two strands of the antibody Fc to produce an asymmetric complementary structure, thereby combining the two strands of the transformation, thereby avoiding the generation of homodimers.
  • the principle of retrofitting is based on the following aspects: hydrophobic/spatial complementarity (such as KiH and ZW1), electrostatic complementation (such as DD-KK), spatial complementarity + electrostatic interaction (such as EW-RVT), and spatial complementation + hydrogen bond complementation (such as A107) )Wait.
  • hydrophobic/spatial complementation to engineer Fc to fuse with only one cytokine to produce a monomeric immune cytokine.
  • IL-2m Since wild-type IL-2 has some cytotoxicity, in order to further attenuate the toxic side effects, we designed a mutant of IL-2 (IL-2m), which was then fused to the antibody Fc.
  • the antibody Fc-based single domain antibody-cytokine fusion protein constructed by the above method will be expressed in CHO cells, and then purified by Protein A affinity chromatography and molecular sieve to obtain a protein for in vitro and in vivo analysis.
  • the target affinity test of the antibody/cytokine fusion protein to the PD-1 receptor was determined in a cell line expressing PD-1 and activated T cells using Biacore, enzyme-linked adsorption assay, FACS, and the like.
  • Antibody affinity was determined using activated T cells: CD4+ T cells were first isolated from human peripheral blood mononuclear cells using a positive T cell selection kit (BD Bioscience) and then activated with anti-CD3 antibody for 4 days with fluorescently labeled antibodies. The measurement was carried out by the FACS method. Binding parameters of antibody/cytokine fusion protein and PD-1 using human PD-1-FC (R&D system) or FLAG-tagged monkey PD-1 protein, labeling CM5 sensory probe, PD-1 receptor protein flowing through probe Affinity is determined by SPR (Biacore).
  • Antibody affinity was assessed using a cell line expressing the PD-1 antigen: Jurkat cells (Jurkat-PD1) overexpressing the PD-1 antigen were incubated with the PD-1 single domain antibody-cytokine fusion protein at 4 °C for 2 hours, and the cells were washed with PBS. After 3 times, a fluorescently labeled secondary antibody (goat anti-human IgG-FITC) was added, gently mixed, and incubated at 4 ° C for 1 hour. The cells were then washed again three times with PBS, followed by affinity assay using a BD FACSCalibur flow cytometer.
  • Jurkat cells Jurkat-PD1 overexpressing the PD-1 antigen were incubated with the PD-1 single domain antibody-cytokine fusion protein at 4 °C for 2 hours, and the cells were washed with PBS. After 3 times, a fluorescently labeled secondary antibody (goat anti-human IgG-FITC) was added, gently mixed, and incubated at 4 ° C
  • IL-2 and IL-12 can promote the proliferation of human T cells, and can also activate various lymphocytes, thereby promoting T cell proliferation and secretion of ⁇ -interferon.
  • the cytokine activity of the antibody-cytokine fusion protein can be assessed by detecting T cell proliferation or secretion of IFN- ⁇ .
  • the cytokine activity of the antibody-cytokine fusion protein will use the fusion protein to stimulate the release of ⁇ -interferon (IFN- ⁇ ) from NK92 cells to verify the in vitro biological activity of the cytokine in the fusion protein.
  • IFN- ⁇ ⁇ -interferon
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCC refers to NK cells, macrophages, neutrophils, etc., which express IgG Fc receptors, and kills these targets by binding to the Fc fragment of IgG antibodies that have been bound to target cells such as virus-infected cells and tumor cells.
  • target cells such as virus-infected cells and tumor cells.
  • IgG antibodies mediate the role of these cells in ADCC, which is the main cell that can function as ADCC.
  • IgG antibodies specifically bind to antigenic determinants on the surface of target cells; then natural killer cells (NK cells) bind to IgG Fc segments bound to target cells by means of their corresponding receptors on the surface; activated NK cells release perforations Cytotoxic substances such as granules and granzymes kill target cells; target cells undergo apoptosis, and antibodies are treated by the liver.
  • NK cells natural killer cells
  • PBMC cells were isolated from the blood of healthy volunteers according to normal procedures, and the cells were washed twice.
  • ADCC assay The ADCC assay will use the FACS method, live cell and dead cell count to determine its activity.
  • FACS count analysis Identification of target cell (live and dead cells) intervals; identification of effector cell intervals; viable cell (PKH67) count; dead cell (7AAD) cell count.
  • Luciferase assay prepare Bio-GloTM detection reagent, add 80 ⁇ l to 96-well plate containing antibody and cells, and measure cold light in GloMax system for 5-10 minutes at room temperature.
  • In vivo anti-tumor efficacy will be assessed using a human IL-12 receptor and human PD-1 and knock-in mice to establish a tumor model.
  • Humanization of the IL-12 receptor and PD-1, respectively enables direct in vivo pharmacodynamic evaluation of fusion proteins fused with single domain antibodies and cytokines.
  • Tumor models suitable for this evaluation can include, but are not limited to, GL261 or B16 cell lines.
  • the efficacy of the following aspects will be evaluated: (1) inhibition of tumor formation; (2) established tumor growth inhibition; and (3) dose gradient test (three doses).
  • mice 1 x 10 6 GL261 or B16 cells were injected subcutaneously into the abdomen or back of the mice.
  • the mice were randomly divided into 4 groups (8 in each group): control group, anti-PD-1 group, anti-PD-1+IL2 group, anti-PD-1+IL-12 group.
  • anti-PD-1+IL2+IL12 group It was administered once every four days for a total of three times.
  • PBS control
  • PD-1 sdAb-Fc
  • single domain antibody Fc fusion anti-PD-1 group
  • PD-1 sdAb-IL2 fusion protein anti-PD-1+IL2 group
  • PD The -1 sdAb-IL12 fusion protein anti-PD-1+IL12 group
  • PD-1 sdAb-IL2-IL12 fusion protein anti-PD-1+IL2+IL12 group
  • the antibody-cytokine fusion protein of the present invention can selectively transport cytokines to target cells in vivo, and thus cytokines exhibit local biological reactions such as local inflammatory reactions, stimulation of T cell growth and activation.
  • the PD-1 antibody can exert an anticancer effect by binding to receptors on the surface of the cell membrane. Therefore, it is feasible for fusion antibodies and cytokines to treat cancer by targeting the delivery of cytokines to enhance the immune response.
  • This study constructed a series of single domain antibody-cytokine fusion proteins based on antibody Fc heterodimers.
  • the components used to construct the fusion protein are PD-1 single domain antibodies, cytokines IL-12, IL-2 and IL-2 mutants.
  • the anti-PD-1 single domain antibody sequence was inserted after the multiple cloning site EcoRI of the pTT5 expression vector, and the KOZAK sequence GCCGCCACC and signal peptide sequences were added in front of the fusion protein gene to help secrete the fusion protein out of the cell.
  • An SC01 vector expressing a single domain antibody was generated.
  • the antibody Fc fragment was inserted into the pTT5 vector by the multiple cloning site EcoRI, and then the PD-1 single domain antibody sequence was assembled by Gibson, ligated to the N-terminus of the Fc, and ligated by the (G4S)3 linker sequence.
  • the IL-12 sequence is then ligated to the C-terminus of the Fc in a similar manner, with a G4S linker sequence between them.
  • a PD-1 single domain antibody and an IL-12 fusion protein expression vector G1-208 were formed.
  • the KOZAK sequence GCCGCCACC and signal peptide sequences were added in front of the fusion protein gene to help secrete the fusion protein out of the cell.
  • the PD-1 single domain antibody and IL-2 were fused to the N-terminus and C-terminus of Fc, respectively, to produce a PD-1 single domain antibody and an IL-2 fusion protein expression vector G1-212.
  • Both G1-208 and G1-212 are homodimers based on antibody Fc.
  • the knob-into-holes technique was used to perform antibody Fc engineering, in which the mutation site combination of one Fc chain is T366W/S354C, and the mutation site combination of the other Fc chain is T366S/L368A/Y407V/Y349C.
  • the anti-PD-1 single domain antibody sequence is ligated to the N-terminus of Fc by Gibson assembly, and IL-12 and IL-2 are linked to the C-terminus of Fc in a similar manner, thereby producing a single domain fused with PD-1.
  • Expression vector G1-405 of antibody, IL-12 and IL-2 are linked to the C-terminus of Fc in a similar manner, thereby producing a single domain fused with PD-1.
  • G1-716 is based on G1-405, and a PD-1 single domain antibody is ligated to the N-terminus of an existing PD-1 single domain antibody via a (G4S)3 linker sequence.
  • G1-723 is a site mutation modification of the IL-2 gene based on G1-716.
  • G1-717 is based on G1-716 and deletes the IL-12 gene.
  • G1-719 is based on G1-716 and deletes the IL-2 gene.
  • the fusion protein granules constructed above on the pTT5 expression vector were transiently transfected into CHO-3E7 cells by PEI transfection reagent, and then cultured at 37 degrees for 6 days. The culture supernatant was collected by centrifugation, and the fusion protein was first purified by Protein A affinity column, and then the fusion protein was further purified by molecular sieve, and the final purity was over 95%.
  • the signal peptide DNA sequence (SEQ ID NO: 1) used in this study is as follows:
  • the DNA sequence (SEQ ID NO: 2) of the anti-PD-1 single domain antibody SC01 used in this study is as follows:
  • amino acid sequence of SC01 (SEQ ID NO: 3) is as follows:
  • the cytokine IL-12 full-length DNA sequence (SEQ ID NO: 5) used in this study is as follows:
  • the full length amino acid sequence of IL-12 (SEQ ID NO: 6) used is as follows:
  • the DNA sequence (SEQ ID NO: 7) of the cytokine human IL-2 used in this study is as follows:
  • amino acid sequence of human IL-2 (SEQ ID NO: 8) is as follows:
  • the human IL-2 mutant DNA sequence (SEQ ID NO: 9) used in this study is as follows:
  • the human IL-2 mutant amino acid sequence (SEQ ID NO: 10) is as follows:
  • the G1-405 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc1 and Fc2).
  • the Fc1 full-length DNA sequence (SEQ ID NO: 15) is as follows:
  • the G1-405 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc1 and Fc2).
  • Fc1 and Fc2 the full length amino acid sequence of Fc1 (SEQ ID NO: 16) is as follows:
  • the G1-405 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc1 and Fc2).
  • the Fc2 full-length DNA sequence (SEQ ID NO: 17) is as follows:
  • the G1-405 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc1 and Fc2).
  • Fc1 and Fc2 the full length amino acid sequence of Fc2 (SEQ ID NO: 18) is as follows:
  • the G1-716 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc4).
  • the Fc3 full-length DNA sequence (SEQ ID NO: 19) is as follows:
  • the G1-716 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc4).
  • the Fc3 full-length amino acid sequence (SEQ ID NO: 20) is as follows:
  • the G1-716 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc4).
  • the Fc4 full-length DNA sequence (SEQ ID NO: 21) is as follows:
  • the G1-716 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc4).
  • the Fc4 full-length amino acid sequence (SEQ ID NO: 22) is as follows:
  • the G1-723 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc5).
  • the Fc5 full-length DNA sequence (SEQ ID NO: 23) is as follows:
  • the G1-723 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc5).
  • the Fc5 full-length amino acid sequence (SEQ ID NO: 24) is as follows:
  • the G1-717 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc6 and Fc7).
  • the Fc6 full-length DNA sequence (SEQ ID NO: 25) is as follows:
  • the G1-717 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc6 and Fc7).
  • the Fc6 full-length amino acid sequence (SEQ ID NO: 26) is as follows:
  • the G1-717 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc6 and Fc7).
  • the Fc7 full-length DNA sequence (SEQ ID NO: 27) is as follows:
  • the G1-717 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc6 and Fc7).
  • the Fc7 full-length amino acid sequence (SEQ ID NO: 28) is as follows:
  • the G1-719 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc3 and Fc7).
  • the full length DNA and amino acid sequences of Fc3 and Fc7 have been shown above.
  • the G1-709 fusion protein used in this study is a homodimer, and the full-length DNA sequence (SEQ ID NO: 29) is as follows:
  • the G1-709 fusion protein used in this study is a homodimer, and the full length amino acid sequence of the polypeptide chain (SEQ ID NO: 30) is as follows:
  • cytokine was used to stimulate the release of ⁇ -interferon (IFN- ⁇ ) from NK92 cells to verify the in vitro biological activities of the cytokines IL-12 and IL-2 in the fusion protein. Since the fusion protein constructed in this experiment contains cytokines, different fusion proteins were incubated with NK92 cells, and then the release of ⁇ -interferon was detected.
  • IFN- ⁇ ⁇ -interferon
  • the activity of IL-2 was enhanced in all other heterodimers, and the activity of IL-2 of the G1-405 and G1-716 fusion proteins was enhanced by more than 1.5-fold (Figs. 2 and 3), G1-723 and G1-
  • the activity of IL-2 of the 717 fusion protein was enhanced by about 0.5-fold (Figs. 2 and 3).
  • the homodimeric fusion protein fuses two cytokines, IL-2, and should be more potent than the fusion protein that only fuses a heterodimer of IL-2. But the actual result is just the opposite. The possible reason is that the two IL-2s of the homodimer form a steric hindrance with each other, which in turn causes a decrease in IL-2 activity. Therefore, in general, the cytokine activity of a fusion protein fused only to one IL-2 based on the antibody Fc heterodimer technology is superior to that of a fusion protein of two IL-2 homodimers. Cytokine activity. Moreover, the fusion protein of IL-12 and IL-2 was more active than the fusion protein of IL-2 alone, indicating the synergistic effect of IL-12 and IL-2.
  • the cytokine activity of the homodimeric fusion protein G1-208 was reduced by a factor of 9 compared to the biological activity of free IL-12 (Fig. 4), whereas G1- The activity of IL-12 of the 405 fusion protein decreased only by a factor of 0.3 or more (Fig. 6), and the activity of IL-12 of the G1-716 fusion protein was also only 3-fold decreased (Fig. 5).
  • the heterodimeric fusion proteins G1-719 (Fig. 5) and G1-723 Fig.
  • the in vitro activity of IL-12 of G1-723 is decreased, and the activity caused by site mutation of IL-2 is decreased, thereby reducing the synergistic effect with IL-12.
  • the cytokine activity of a fusion protein fused only with one IL-12 based on the antibody Fc heterodimer technology would be superior to the cytokine activity of a fusion protein fused with two IL-12 homodimers. .
  • the fusion protein of IL-12 and IL-2 was stronger than the fusion protein of IL-12 alone, which further proved the synergistic effect of IL-12 and IL-2.
  • Bioassays for PD-1/PD-L1 pathway blockade are assays based on biologically relevant mechanisms of action that can be used to measure antibodies and other organisms that block PD-1/PD-L1 interactions.
  • This detection system includes the following two gene-edited cell lines: cells stably expressing human PD-1 and cells stably expressing human PD-L1.
  • the PD-1/PD-L1 interaction inhibits the T cell receptor (TCR) signaling pathway and NFAT-regulated luciferase activity.
  • TCR T cell receptor
  • the corresponding PD-1 or PD-L1 antibody is added to block the PD-1/PD-L1 interaction, the inhibition signal is released, thereby activating the T cell receptor (TCR) signaling pathway and NFAT-induced luciferase activity.
  • Antibody activity is then analyzed by detecting fluorescent signals.
  • fusion protein in tandem with two single domain antibodies and fused it to the N-terminus of the antibody Fc chain, such as the G1-709 single domain antibody Fc fusion protein, its PD-1 antibody
  • the activity was much enhanced compared to the fusion protein G1-405 without a tandem single domain antibody ( Figure 8).
  • the antibody activities of the fusion proteins G1-717 and G1-719 fused to the antibody and cytokine based on the antibody Fc heterodimer technology were not far from the Keytruda antibody (Fig. 9).
  • the antibody activities of fusion proteins G1-716 and G1-723, which simultaneously fused IL-12 and IL-2, were almost close to the Keytruda antibody (Fig. 10). This not only indicates that the Fc chain enhances the activity of the single domain antibody, but also that the cytokine further promotes antibody activity to some extent.
  • the knob-into-holes technique was used to perform antibody Fc engineering, in which the mutation site combination of one Fc chain was T366W/S354C, and the other The mutation site combination of the Fc chain is T366S/L368A/Y407V/Y349C.
  • the two Fc strands also have other mutation sites S228P and L235E.
  • the Fc fragment of antibody IgG4 was inserted into the pTT5 vector by the multiple cloning site EcoRI, and then the four new single domain antibody sequences were assembled by Gibson and ligated to the N-terminus of the Fc, respectively, without a linker sequence therebetween.
  • the IL-12 or IL-2 sequence is then ligated to the C-terminus of the Fc in a similar manner with a G4S linker sequence between them.
  • a fusion protein expression vector of different single domain antibodies and different cytokines is finally formed.
  • the KOZAK sequence GCCGCCACC and signal peptide sequences were added in front of the fusion protein gene to help secrete the fusion protein out of the cell.
  • the fusion protein granules constructed above on the pTT5 expression vector were transiently transfected into CHO-3E7 cells by PEI transfection reagent, and then cultured at 37 degrees for 6 days. The culture supernatant was collected by centrifugation, and the fusion protein was first purified by Protein A affinity column, and then the fusion protein was further purified by molecular sieve, and the final purity was over 95%.
  • the new anti-PD-1 single domain antibody and the new anti-PD-L1 single domain antibody sequence in this study are as follows:
  • Amino acid sequence of anti-PD1a single domain antibody (SEQ ID NO: 71):
  • Amino acid sequence of anti-PD-L1a single domain antibody (SEQ ID NO: 72):
  • Amino acid sequence of anti-PD-L1b single domain antibody (SEQ ID NO: 73):
  • Amino acid sequence of anti-PD-L1c single domain antibody (SEQ ID NO: 74):
  • the sPD1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc9).
  • the Fc8 full-length DNA sequence (SEQ ID NO: 31) is as follows:
  • the sPD1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc9).
  • the Fc8 full-length amino acid sequence (SEQ ID NO: 32) is as follows:
  • the sPD1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc9).
  • the Fc9 full-length DNA sequence (SEQ ID NO: 33) is as follows:
  • the sPD1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc9).
  • the Fc9 full-length amino acid sequence (SEQ ID NO: 34) is as follows:
  • the sPD1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc10).
  • Fc8 and Fc10 The full length DNA and amino acid sequences of Fc8 have been shown above.
  • the sPD1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc10).
  • the Fc10 full-length DNA sequence (SEQ ID NO: 35) is as follows:
  • the sPD1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc8 and Fc10).
  • the Fc10 full-length amino acid sequence (SEQ ID NO: 36) is as follows:
  • the sPD1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc10 and Fc11).
  • Fc10 and Fc11 The full length DNA and amino acid sequences of Fc10 have been shown above.
  • the sPD1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc10 and Fc11).
  • the Fc11 full-length DNA sequence (SEQ ID NO: 37) is as follows:
  • the sPD1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc10 and Fc11).
  • the Fc11 full-length amino acid sequence (SEQ ID NO: 38) is as follows:
  • the sPDL1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc13).
  • the Fc12 full-length DNA sequence (SEQ ID NO: 39) is as follows:
  • the sPDL1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc13).
  • the Fc12 full-length amino acid sequence (SEQ ID NO: 40) is as follows:
  • the sPDL1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc13).
  • the Fc13 full-length DNA sequence (SEQ ID NO: 41) is as follows:
  • the sPDL1a01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc13).
  • the Fc13 full-length amino acid sequence (SEQ ID NO: 42) is as follows:
  • the sPDL1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc14).
  • Fc12 and Fc14 The full length DNA and amino acid sequences of Fc13 have been shown above.
  • the sPDL1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc14).
  • the Fc14 full-length DNA sequence (SEQ ID NO: 43) is as follows:
  • the sPDL1a02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc12 and Fc14).
  • the Fc14 full-length amino acid sequence (SEQ ID NO: 44) is as follows:
  • the sPDL1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc14 and Fc15).
  • Fc14 and Fc15 The full length DNA and amino acid sequences of Fc14 have been shown above.
  • the sPDL1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc14 and Fc15).
  • the Fc15 full-length DNA sequence (SEQ ID NO: 45) is as follows:
  • the sPDL1a03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc14 and Fc15).
  • the Fc15 full-length amino acid sequence (SEQ ID NO: 46) is as follows:
  • the sPDL1b01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc17).
  • the Fc16 full-length DNA sequence (SEQ ID NO: 47) is as follows:
  • the sPDL1b01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc17).
  • the Fc16 full-length amino acid sequence (SEQ ID NO: 48) is as follows:
  • the sPDL1b01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc17).
  • the Fc17 full-length DNA sequence (SEQ ID NO: 49) is as follows:
  • the sPDL1b01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc17).
  • the Fc17 full-length amino acid sequence (SEQ ID NO: 50) is as follows:
  • the sPDL1b02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc18).
  • Fc16 and Fc18 The full length DNA and amino acid sequences of Fc16 have been shown above.
  • the sPDL1b02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc18).
  • the Fc18 full-length DNA sequence (SEQ ID NO: 51) is as follows:
  • the sPDL1b02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc16 and Fc18).
  • the Fc18 full-length amino acid sequence (SEQ ID NO: 52) is as follows:
  • the sPDL1b03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc18 and Fc19).
  • Fc18 and Fc19 The full length DNA and amino acid sequences of Fc18 have been shown above.
  • the sPDL1b03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc18 and Fc19).
  • the Fc19 full-length DNA sequence (SEQ ID NO: 53) is as follows:
  • the sPDL1b03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc18 and Fc19).
  • the Fc19 full-length amino acid sequence (SEQ ID NO: 54) is as follows:
  • the sPDL1c01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc21).
  • the Fc20 full-length DNA sequence (SEQ ID NO: 55) is as follows:
  • the sPDL1c01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc21).
  • the Fc20 full-length amino acid sequence (SEQ ID NO: 56) is as follows:
  • the sPDL1c01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc21).
  • the Fc21 full-length DNA sequence (SEQ ID NO: 57) is as follows:
  • the sPDL1c01 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc21).
  • the Fc21 full-length amino acid sequence (SEQ ID NO: 58) is as follows:
  • the sPDL1c02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc22).
  • Fc20 and Fc22 The full length DNA and amino acid sequences of Fc20 have been shown above.
  • the sPDL1c02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc22).
  • the Fc22 full-length DNA sequence (SEQ ID NO: 59) is as follows:
  • the sPDL1c02 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc20 and Fc22).
  • the Fc22 full-length amino acid sequence (SEQ ID NO: 60) is as follows:
  • the sPDL1c03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc22 and Fc23).
  • Fc22 and Fc23 The full length DNA and amino acid sequences of Fc22 have been shown above.
  • the sPDL1c03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc22 and Fc23).
  • the Fc23 full-length DNA sequence (SEQ ID NO: 61) is as follows:
  • the sPDL1c03 fusion protein used in this study is a heterodimer formed by two Knb-into-holes structures that have been engineered and fused with other proteins (Fc22 and Fc23).
  • Fc23 the full length amino acid sequence of Fc23 (SEQ ID NO: 62) is as follows:
  • the sPD1a00 fusion protein used in this study is a homodimer, and the full length DNA sequence of the polypeptide chain (SEQ ID NO: 63) is as follows:
  • the sPD1a00 fusion protein used in this study is a homodimer, and the full length amino acid sequence of the polypeptide chain (SEQ ID NO: 64) is as follows:
  • the sPDL1a00 fusion protein used in this study is a homodimer, and the full length DNA sequence of the polypeptide chain (SEQ ID NO: 65) is as follows:
  • the sPDL1a00 fusion protein used in this study is a homodimer, and the full length amino acid sequence of the polypeptide chain (SEQ ID NO: 66) is as follows:
  • the sPDL1b00 fusion protein used in this study is a homodimer, and the full length DNA sequence of the polypeptide chain (SEQ ID NO: 67) is as follows:
  • the sPDL1b00 fusion protein used in this study is a homodimer, and the full length amino acid sequence of the polypeptide chain (SEQ ID NO: 68) is as follows:
  • the sPDL1c00 fusion protein used in this study is a homodimer, and the full length DNA sequence of the polypeptide chain (SEQ ID NO: 69) is as follows:
  • the sPDL1c00 fusion protein used in this study is a homodimer, and the full length amino acid sequence of the polypeptide chain (SEQ ID NO: 70) is as follows:
  • cytokine was used to stimulate the release of ⁇ -interferon (IFN- ⁇ ) from NK92 cells to verify the in vitro biological activities of the cytokines IL-12 and IL-2 in the fusion protein. Since the fusion protein constructed in this experiment contains cytokines, different fusion proteins were incubated with NK92 cells, and then the release of ⁇ -interferon was detected.
  • IFN- ⁇ ⁇ -interferon
  • Bioassays for PD-1/PD-L1 pathway blockade are assays based on biologically relevant mechanisms of action that can be used to measure antibodies and other organisms that block PD-1/PD-L1 interactions.
  • This detection system includes the following two gene-edited cell lines: cells stably expressing human PD-1 and cells stably expressing human PD-L1.
  • the PD-1/PD-L1 interaction inhibits the T cell receptor (TCR) signaling pathway and NFAT-regulated luciferase activity.
  • TCR T cell receptor
  • the corresponding PD-1 or PD-L1 antibody is added to block the PD-1/PD-L1 interaction, the inhibition signal is released, thereby activating the T cell receptor (TCR) signaling pathway and NFAT-induced luciferase activity.
  • Antibody activity is then analyzed by detecting fluorescent signals.

Abstract

提供了一种以抗体Fc区为骨架的融合蛋白二聚体，包括第一和第二多肽链，其中所述第一多肽链包括第一抗体Fc区和与所述第一抗体Fc区融合的一个或多个单域抗体；所述第二多肽链包括第二抗体Fc区和与所述第二抗体Fc区融合的一个或多个单域抗体；所述第一多肽链和/或第二多肽链还包括与各自抗体Fc区融合的细胞因子。还提供了该融合蛋白二聚体在制备***的免疫治疗药物中的应用。所述Fc融合蛋白异源二聚体，不仅提升了单域抗体的活性，而且明显改善了细胞因子的生物学活性；并利用抗体的靶向特异性，有效增强了细胞因子的靶向运输，减弱了细胞毒性，从而具有更佳的抗肿瘤潜力。

Description

以抗体Fc区为骨架的融合蛋白二聚体及其应用 技术领域
本发明涉及一种融合蛋白，尤其是基于抗体Fc区的融合蛋白二聚体。
背景技术
免疫***是哺乳动物体内的一套防卫***，能够抵御各种病原体的入侵和消除肿瘤。抗体分子在免疫***中扮演了十分重要的角色，例如识别肿瘤抗原、激活免疫应答等。同时，细胞因子作为免疫调节因子，能够同时调控固有性免疫和适应性免疫，在癌症免疫治疗中也具有不容忽视的重要性[1-3]。这一类调节因子几乎参与免疫***的各个环节，在调节和平衡免疫***方面行使着强大的功能。干扰素和IL-2最先被选择作为细胞因子应用到免疫治疗中。其中IFN-α被用于治疗各种癌症，比如黑色素瘤、肾癌、滤泡性淋巴瘤、慢性粒细胞白血病；而IL-2在1998年被FDA批准用于治疗晚期转移性黑色素瘤和转移性肾癌等。其他细胞因子，比如IL-7，IL-10，IL-12，IL-15和IL-21等，也正处于临床测试阶段[4]。因此，细胞因子用于免疫治疗具有一定的潜力[5,6]。
但是，基于细胞因子的免疫治疗在临床使用上却受到严重阻碍，主要受制于严重的副作用和表现欠佳的药代动力学性质[5]。因此，为了提升细胞因子的治疗效果，经常需要对细胞因子进行改造。当前，比较常用的方法是采用抗体融合的方法，通过抗体将细胞因子靶向递送到特定部位，从而有效地降低细胞毒性，并改善药代动力学性质，继而增强免疫调节功能[7,8]。尽管如此，有些细胞因子融合到抗体上后，会降低抗体本身的活性，从而影响整体效果。另外，有些细胞因子需要联合使用才能发挥各自最佳的效果[9,10]，而现有的技术不能满足这一要求。
当前，随着PD-1和CTLA-4抗体的成功上市，基于免疫检查点阻断的免疫治疗得到了迅速发展和推广。但是，这些抗体只对一部分病人有效，而且会产生耐药性[11,12]。所以，如果能将细胞因子与抗体分子融合起来，并发挥整体最优的效果，那么就将显著地增强免疫治疗效果，从而使得更多的换着能够得到有效治疗。
发明内容
在一方面，本发明提供了一种以抗体Fc区为骨架的融合蛋白二聚体，包括第一和第二多肽链，其中所述第一多肽链包括第一抗体Fc区和与所述第一抗体Fc区融合的 一个或多个单域抗体；所述第二多肽链包括第二抗体Fc区和与所述第二抗体Fc区融合的一个或多个单域抗体；所述第一和/或第二多肽链还包括与各自抗体Fc区融合的细胞因子。
在一些实施方案中，所述单域抗体为抗免疫检查点分子的单域抗体。优选地，所述单域抗体为抗PD-1单域抗体或抗PD-L1单域抗体。在一些实施方案中，所述单域抗体为抗PD-1单域抗体。在另一些实施方案中，所述单域抗体为PD-L1单域抗体。
在一些实施方案中，所述细胞因子选自IL-2、IL-12、GM-CSF、IL-2突变体以及它们的组合。
在一些实施方案中，所述IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白而存在于所述融合蛋白二聚体中。在一些具体实施方案中，IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白而存在于所述第一多肽链和/或第二多肽链中。
在一些实施方案中，所述融合蛋白二聚体中的单域抗体、抗体Fc区和细胞因子之间通过接头序列连接或直接融合。在一些实施方案中，所述融合蛋白二聚体中的单域抗体和抗体Fc区之间通过接头序列连接或者直接连接。在另一些实施方案中，所述融合蛋白二聚体中抗体Fc区和细胞因子之间通过接头序列连接。优选地，所述接头序列选自(G4S)1-3、KRVAPELLGGPS、ASTKG以及NSPPAA。
在一些实施方案中，所述第一抗体Fc区和所述第二抗体Fc区不同(即形成抗体Fc融合蛋白异源二聚体)，并且它们之间具有非对称互补结构。该非对称互补结构例如可通过KiH(knobs-into-holes)技术形成。优选地，所述第一抗体Fc区具有突变位点组合T366W/S354C，所述第二抗体Fc区具有突变位点组合T366S/L368A/Y407V/Y349C。在一些具体实施方案中，所述抗体Fc区选自人IgG1突变体和人IgG4的突变体。
在一些实施方案中，所述第一多肽链和/或第二多肽链包括两个串联排列的所述抗PD-1单域抗体。在另一些实施方案中，所述第一多肽链和/或第二多肽链包括两个串联排列的所述抗PD-L1单域抗体。
在一些实施方案中，所述第一多肽链中含有的细胞因子为IL-12，第二多肽链中含有的细胞因子为IL-2或IL-2突变体。在一些实施方案中，所述第一多肽链中含有的细胞因子为IL-12，第二多肽链中缺失细胞因子。在另一些实施方案中，所述第一多肽链中含有的细胞因子为IL-2或IL-2突变体，第二多肽链中缺失细胞因子。
在一些实施方案中，所述一个或多个单域抗体通过接头序列连接至所述抗体Fc区的N端，而所述细胞因子通过接头序列连接至所述抗体Fc区的C端。在另一实施方案中，所述一个或多个单域抗体通过接头序列连接至所述抗体Fc区的C端，而所述细胞因子通过接头序列连接至所述抗体Fc区的N端。在另一些实施方案中，所述一个或多 个单域抗体直接连接至所述Fc区的N端，而所述细胞因子通过接头序列连接至所述抗体Fc区的C端。在另一些实施方案中，所述一个或多个单域抗体直接连接至所述Fc区的C端，而所述细胞因子通过接头序列连接至所述抗体Fc区的N端。
在一些实施方案中，所述抗PD-1单域抗体具有如SEQ ID NO：3以及SEQ ID NO:71所示的氨基酸序列。在另一些实施方案中，所述抗PD-L1单域抗体具有如SEQ ID NOs：72-74所示的氨基酸序列。
在一些实施方案中，所述IL-12单链蛋白具有如SEQ ID NO：6所示的氨基酸序列。
在一些实施方案中，所述IL-2具有如SEQ ID NO：8所示的氨基酸序列。在另一些实施方案中，所述IL-2突变体具有如SEQ ID NO：10所示的氨基酸序列。
在一些实施方案中，所述第一多肽链和第二多肽链选自具有如SEQ ID NO：16、18、20、22、24、26、28、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60或62所示的氨基酸序列。
在一些具体实施方案中，所述第一多肽链具有如SEQ ID NO：20所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：22、24或28所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO：26所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：28所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO：16所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：18所示的氨基酸序列；所诉第一多肽链具有如SEQ ID NO：32所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：34、36所示的氨基酸序列；或者所述第一多肽链具有如SEQ ID NO：36所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：38所示的氨基酸序列。
在另一些具体实施方案中，所述第一多肽链具有如SEQ ID NO:40所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：42、44所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：46所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO:48所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：50、52所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO:52所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：54所示的氨基酸序列；所述第一多肽链具有如SEQ ID NO:56所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：58、60所示的氨基酸序列；或者所述第一多肽链具有如SEQ ID NO:60所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：62所示的氨基酸序列。
在另一些实施方案中，所述第一抗体Fc区和第二抗体Fc区为野生型抗体Fc区，形成抗体Fc融合蛋白同源二聚体。
另一方面，本发明提供了分离的多核苷酸，其编码所述融合蛋白二聚体的第一多肽链或第二多肽链。
在一些具体实施方案中，所述多核苷酸具有如SEQ ID NO：15、17、19、21、23、25、27、29、31、33、35或37所示的核苷酸序列。在另一些具体实施方案中，所述多核苷酸具有如SEQ ID NO:39、41、43、45、47、49、51、53、55、57、59及61所示的核苷酸序列。
另一方面，本发明提供了表达载体，其包括所述多核苷酸。
另一方面，本发明提供了宿主细胞，其包括所述表达载体。
另一方面，本发明提供了所述融合蛋白二聚体、多核苷酸、表达载体、或宿主细胞在制备抗肿瘤药物中的应用。优选地，所述肿瘤为黑色素瘤或肺癌。
另一方面，本发明提供了抗肿瘤药物组合物，其包含所述融合蛋白二聚体和药学上可接受的载体。
另一方面，本发明提供了***的方法，包括给予受试者治疗有效量的所述融合蛋白二聚体或含有所述融合蛋白二聚体的药物组合物。
在一些实施方案中，本发明提供的抗肿瘤药物组合物可以通过选自以下的至少一种路径施用至受试者：胃肠外、皮下、肌肉内、静脉内、关节内、支气管内、腹内、囊内、软骨内、腔内、体腔内、小脑内、脑室内、结肠内、宫颈内、胃内、肝内、心肌内、骨内、骨盆内、心包内、腹膜内、胸膜内、***内、肺内、直肠内、肾内、视网膜内、脊柱内、滑膜内、胸腔内、鼓室内、子宫内、膀胱内、玻璃体内、快速注射、结膜下、经***、经直肠、经颊、舌下、鼻内、肿瘤内和经皮肤。优选地，所述抗肿瘤药物组合物通过肿瘤内或静脉内施用至受试者。
另一方面，本发明还提供了所述抗PD-1单域抗体本身，其具有如SEQ ID NO：3所示的氨基酸序列。本发明还提供了所述抗PD-1单域抗体的编码多核苷酸，其具有如SEQ ID NO：2所示的核苷酸序列。本发明还提供了所述抗PD-1单域抗体本身，其具有如SEQ ID NO：71所示的氨基酸序。本发明还提供了所述抗PD-L1单域抗体本身，其具有如SEQ ID NOs：72-74所示的氨基酸序。
本发明的融合蛋白将单域抗体的分子小、渗透性好、稳定性高等优点与细胞因子的免疫调节等活性结合。更重要的是，本发明使用抗体Fc融合蛋白异源二聚体技术，将二者有机融合起来，不仅提升了单域抗体的活性，而且明显改善了细胞因子的生物学活性；并利用抗体的靶向特异性，有效增强了细胞因子的靶向运输，减弱了细胞毒性，从而具有更佳的抗肿瘤潜力以及显著改善的药代动力学性质。
附图简要说明
图1显示与对照IL-2相比，G1-212融合蛋白的IL-2活性。
图2显示与对照IL-2相比，G1-716和G1-717融合蛋白的IL-2活性。
图3显示与对照IL-2相比，G1-723和G1-405融合蛋白的IL-2活性。
图4显示与对照IL-12相比，G1-208融合蛋白的IL-12活性。
图5显示与对照IL-12相比，G1-716和G1-719融合蛋白的IL-12活性。
图6显示与对照IL-12相比，G1-723和G1-405融合蛋白的IL-12活性
图7显示与对照Keytruda相比，PD-1单域抗体的PD-1/PD-L1通路阻断活性。
图8显示与对照Keytruda相比，融合蛋白G1-405和G1-709的PD-1/PD-L1通路阻断活性。
图9显示与对照Keytruda相比，融合蛋白G1-717和G1-719的PD-1/PD-L1通路阻断活性。
图10显示与对照Keytruda相比，融合蛋白G1-716和G1-723的PD-1/PD-L1通路阻断活性。
图11显示与对照IL-12相比，异源二聚体融合蛋白sPDL1a01,sPDL1a02,sPDL1b01和sPDL1b02的IL-12活性。
图12显示与对照IL-12相比，异源二聚体融合蛋白sPDL1c01,sPDL1c02,sPD1a01和sPD1a02的IL-12活性。
图13显示与对照IL-2相比，异源二聚体融合蛋白sPDL1a01,sPDL1b01,sPDL1c01和sPD1a01的IL-2活性
图14显示与对照Atezolizumab相比，没有连接细胞因子的同源二聚体融合蛋白sPDL1a00和sPDL1b00的PD-1/PD-L1通路阻断活性。
图15显示与对照Atezolizumab相比，异源二聚体融合蛋白sPDL1a01和sPDL1a02的PD-1/PD-L1通路阻断活性。
图16显示与对照Atezolizumab相比，异源二聚体融合蛋白sPDL1b01和sPDL1b02的PD-1/PD-L1通路阻断活性。
图17显示与对照Atezolizumab相比，异源二聚体融合蛋白sPDL1c01和sPDL1c02的PD-1/PD-L1通路阻断活性。
图18不仅显示与对照Atezolizumab相比，异源二聚体融合蛋白sPDL1a03,sPDL1b03、sPDL1c03以及没有连接细胞因子的同源二聚体融合蛋白sPDL1c00的PD-1/PD-L1通路阻断活性。
图19显示与对照Keytruda相比，没有连接细胞因子的同源二聚体融合蛋白sPD1a00的PD-1/PD-L1通路阻断活性。
图20显示与对照Keytruda相比，异源二聚体融合蛋白sPD1a01,sPD1a02和sPD1a03的PD-1/PD-L1通路阻断活性。
图21显示与对照IL-2相比，异源二聚体融合蛋白sPD1a03和sPDL1a03的IL-2活性。
图22显示与对照IL-2相比，异源二聚体融合蛋白sPDL1b03和sPDL1c03的IL-2活性。
图23A显示细胞因子IL-2和IL-12可选择地通过接头序列各自连接到每一条抗体Fc链(分别为Fcm、Fcn)上的C端或N端，相应地一个或多个单域抗体(1-n个单域抗体)可选择地通过接头序列连接到每条抗体Fc链另一端；图23B细胞因子IL-12可选择地通过接头序列单独融合到一条抗体Fc链(如Fcm)C端或N端，另外一条链缺失细胞因子，相应地一个或多个单域抗体(1-n个单域抗体)可选择地通过接头序列连接到每条抗体Fc链另一端；图23C细胞因子IL-2可选择地通过接头序列单独融合到一条抗体Fc链(如Fcm)C端或N端，另外一条链缺失细胞因子，相应地一个或多个单域抗体(1-n个单域抗体)可选择地通过接头序列连接到每条抗体Fc另一端。
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有本发明所属领域的普通技术员通常所理解的含义。
本发明利用抗体Fc异源二聚体技术[13]提供了具有抗肿瘤活性的融合蛋白，该融合蛋白包括单域抗体和一个或多个细胞因子，以及作为骨架部分的抗体Fc区。本发明中“单域抗体-细胞因子融合蛋白”、“抗体/细胞因子融合蛋白”及“抗体-细胞因子融合蛋白”均指基于抗体Fc异源二聚体技术的单域抗体-细胞因子融合蛋白，包括一个或多个单域抗体和一个或多个细胞因子通过抗体Fc区连接而形成融合蛋白，可选择地，单域抗体和细胞因子通过接头序列分别连接在抗体Fc的两端。
术语“抗体(antibody)”指由浆细胞(效应B细胞)分泌，被免疫***用来鉴别与中和外来物质如细菌、病毒等的大型“Y”形蛋白质。在过去的10多年里，越来越多的单克隆抗体被广泛地应用到肿瘤治疗中。本发明中提到的抗体将包括但不限于：能够特异性地结合肿瘤相关抗原、通过抗体依赖的细胞毒性作用(ADCC)和补体依赖的细胞毒性作用(CDC)来诱导肿瘤细胞的免疫杀伤，或者阻断免疫检查点，从而活化免疫反应。抗体通常为由2个相同重链和2个相同轻链通过二硫键相互连接组成的四聚体。
术语“抗体Fc区”或“抗体Fc”指“Y”形的柄部区域，即可结晶片段(fragment crystallizable,Fc)，包括重链的第二和第三恒定结构域(CH2和CH3结构域)。可通过蛋白水解酶(如木瓜蛋白酶)水解抗体分子得到抗体Fc区。
术语“以抗体Fc区为骨架”指本发明的融合蛋白二聚体中，单域抗体和细胞因子通常分别位于抗体Fc区的两侧，即单域抗体和细胞因子通过抗体Fc区连接而形成融 合蛋白。“以抗体Fc区为骨架”还意味着两条多肽链(第一和第二多肽链)通过两条多肽链中的Fc区相互作用而结合在一起形成融合蛋白二聚体，例如通过二硫键或者非共价相互作用。
本发明中涉及的二聚体蛋白，是指蛋白在形成的过程中，若两个亚基/单体相同称为同源二聚体(homo-)，若是不完全相同的亚基/单体组合而成，则称为异源二聚体(hetero-)。术语“抗体Fc异源二聚体”是指蛋白由两个不同的亚基/单体组合而成，并且每个亚基/单体均含有一个抗体Fc片段。关键在于这两个抗体Fc片段各有不同的氨基酸位点突变能够形成互补的蛋白质空间结构，从而使得两个不同的亚基/单体能正确的组合在一起。
术语“KiH(knobs-into-holes)技术”指一种促进两种异源抗体重链之间进行装配的技术。例如，可以将一个抗体的重链CH3区366位体积较小的苏氨酸(T)突变为体积较大的酪氨酸(Y)，形成突出的“knobs”型结构(T366Y)；同时将另一个抗体重链CH3区407位较大的酪氨酸(Y)残基突变成较小的苏氨酸(T)，形成凹陷的“holes”型结构(Y407T)；利用这种“knobs-into-holes”结构(即非对称互补结构)的空间位阻效应可以实现两种不同抗体重链间的正确装配。本发明通过在两抗体Fc区中通过多个位点突变组合，实现了更佳的装配效果。
术语“单域抗体(sdAb或nanobody)”指另一种形式的抗体片段，其只含有一个单体形式的抗体可变区。与完整的抗体一样，它也可以特异性地结合抗原，但是质量却比传统抗体小的多(大约15kDa)。更重要的是，正是得益于单域抗体的小尺寸，它能够更便利的穿透组织或者进入肿瘤内部，而这对于完整的抗体来说是很难做到的。
术语“细胞因子(cytokine，CK)”是免疫原、丝裂原或其它刺激剂诱导多种细胞产生的低分子量可溶性蛋白质。在生物体内通过与其特异的细胞表面受体结合，传递细胞内信号，从而改变细胞功能[4]，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种作用。白细胞介素(比如IL-2或IL-12)作为其中的一组细胞因子，通过调控免疫***来调节免疫应答。细胞因子不仅可以单独作用行使功能，也可以与抗体融合，形成抗体-细胞因子融合蛋白，也被称为免疫细胞因子。这种新的蛋白形式将抗体特有的靶向性与细胞因子的免疫调节有机的结合了起来，从而增强了抗体的免疫治疗效果。更重要的是，融合的细胞因子通过抗体的靶向性被运输并富集到肿瘤部位，从而有效的避免了单独使用高剂量的细胞因子引起的副作用。
术语“分离的多核苷酸”指非自然界中天然存在状态的多核苷酸，包括通过生物学技术从自然界(包括生物体内)分离出的多核苷酸，也包括人工合成的多核苷酸。分离的多核苷酸可以是基因组DNA、cDNA、mRNA或合成的其它RNA，或者它们的组 合。本文提供了多个用于编码本发明的融合蛋白二聚体以及其它多肽片段的核苷酸序列，需要指出的是，本领域技术人员可以根据本文所提供的氨基酸序列，基于密码子简并性，设计出与以上提供的核苷酸序列不完全相同的核苷酸序列，但都编码相同的氨基酸序列。这些经改动的核苷酸序列也包括在本发明的范围内。
当涉及多核苷酸时，所用的术语“载体”指用于将核苷酸编码信息转移到宿主细胞内的任一种分子(例如，核酸、质粒、或病毒等)。术语“表达载体”指适于在宿主细胞内表达目的基因(待表达核苷酸序列)的载体，通常包括目的基因、启动子、终止子、标记基因等部分。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等，例如CHO细胞。
提及药物组合物，所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本发明所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明融合蛋白的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。
用语“和/或”指该用语之前和之后的元素可以同时存在，或者仅其中一个元素存在。例如，“A和/或B”可以为A和B、仅A或者仅B。
为了利用免疫细胞因子和单域抗体的优势来提高免疫治疗的效果，我们将细胞因子融合到PD-1单域抗体上，开发出了针对PD-1的单域抗体-细胞因子融合蛋白，并且通过体外和体内的分析评估融合蛋白的功能。本发明中提到的细胞因子包括白细胞介素，例如白细胞介素-2(IL-2)、白细胞介素-12(IL-12)，以及粒细胞-巨噬细胞集落刺激因子(GM-CSF)等，其中白细胞介素-2和白细胞介素-12参与免疫调节的各个环节，从而增强免疫应答。
本发明用于杀伤肿瘤细胞的融合蛋白二聚体的构建以及各种特点可以从下面的描述中进一步理解。
1.融合蛋白的设计构建，以及在CHO细胞的表达和纯化:
细胞因子IL-2或IL-12可通过接头序列(Gly4Ser)3(即(G4S)3)连接到抗体Fc的N端或者C端，类似的，PD-1单域抗体也可以通过接头序列(Gly4Ser)3连接到抗体Fc的的N端或者C端，所形成的融合蛋白保持了抗体和细胞因子的双重效果。由于功能性的IL-12由p35和p40两个亚基构成，IL-12将以两个亚基的形式单独融合到抗体Fc上，或者先将IL-12的两个亚基通过接头序列(Gly4Ser)3形成单链蛋白，然后融合到抗体Fc上。
传统的抗体-细胞因子融合蛋白结构中，细胞因子同时连接到抗体的两条重链或者两条轻链上，从而以同源二聚体的形式存在。但是，这种基于IgG平台融合的免疫细胞因子的药代动力学性质较差。为了将细胞因子以单体形式融合到抗体上，同时改变抗体与细胞因子的比例，从而增强融合蛋白的靶向性，我们采用了基于抗体Fc异源二聚体技术，将IL-2或者IL-12单独融合到一条Fc链上，另外一条链缺失细胞因子，形成IL-12或者IL-2单体融合蛋白，分别如图23B和图23C所示，或者将IL2和IL-12各自连接到一条Fc链上，产生同时具有IL-2和IL-12蛋白的融合蛋白，如图23A所示。
抗体Fc异源二聚体技术的核心是将抗体Fc的两条链分别改造，产生非对称互补结构，从而能将改造的两条链结合起来，避免了同源二聚体的产生。改造的原理基于以下方面：疏水/空间互补(比如KiH和ZW1)、静电互补(比如DD-KK)、空间互补+静电相互作用(比如EW-RVT)、以及空间互补+氢键互补(比如A107)等。本研究中，我们采用疏水/空间互补改造Fc，使其只与一个细胞因子融合，从而产生单体免疫细胞因子。此外，我们也使用野生型的抗体Fc骨架，构建了同源二聚体的免疫因子作为对照。
由于野生型的IL-2具有一定的细胞毒性，为了进一步减弱毒副作用，我们设计了IL-2的突变体(IL-2m)，然后融合到抗体Fc上。
通过以上方法构建的基于抗体Fc的单域抗体-细胞因子融合蛋白将在CHO细胞里面表达，然后通过Protein A亲和层析和分子筛纯化得到用于体内体外分析的蛋白。
2.抗体-细胞因子融合蛋白的体外实验：
2.1：细胞基础的靶点亲和力实验
抗体/细胞因子融合蛋白对PD-1受体的靶点亲和力实验在表达PD-1的细胞系和激活的T细胞中采用Biacore、酶联吸附测定方法、FACS等方法来确定。
实验步骤：
利用激活的T细胞测定抗体亲和力：首先从人周边血液单核细胞中采用阳性T细胞筛选试剂盒(BD Bioscience)分离得到CD4+T细胞，然后用抗CD3抗体激活4天，用荧 光标记的抗体和FACS方法进行测定。抗体/细胞因子融合蛋白和PD-1的结合参数用人的PD-1-FC(R&D***)或FLAG-标签猴PD-1蛋白，标记CM5感觉探针，PD-1受体蛋白流经探针，亲和力通过SPR(Biacore)来确定。
利用表达PD-1抗原的细胞系评估抗体亲和力：将过表达PD-1抗原的Jurkat细胞(Jurkat-PD1)与PD-1单域抗体-细胞因子融合蛋白4℃孵育2小时，经过PBS清洗细胞3次后，加入荧光标记二抗(山羊抗人IgG-FITC)，轻轻混匀，4℃孵育1小时。然后再次使用PBS清洗细胞3次，接着使用BD FACSCalibur流式细胞仪进行亲和力测定。
2.2：功能性测定：
2.2.1：细胞增殖和γ-干扰素以及IL-2分泌实验：
IL-2和IL-12可以促进人T细胞的增殖，也可以激活各种淋巴细胞，从而促进T细胞增殖和γ-干扰素的分泌。通过检测T细胞增殖或者IFN-γ的分泌，就可以评估抗体-细胞因子融合蛋白的细胞因子活性。
本实验中，抗体-细胞因子融合蛋白的细胞因子活性将采用融合蛋白刺激NK92细胞释放γ-干扰素(IFN-γ)来验证融合蛋白中的细胞因子的体外生物活性。具体的做法是，由于本实验构建的融合蛋白中含有细胞因子，于是将不同的融合蛋白与NK92细胞一起孵育，然后检测γ-干扰素的释放。
2.2.2：抗体依赖的细胞介导的细胞毒性作用(antibody-dependent cell-mediated cytotoxicity，ADCC)
概述：ADCC是指表达IgGFc受体的NK细胞、巨噬细胞和中性粒细胞等，通过与已结合在病毒感染细胞和肿瘤细胞等靶细胞表面的IgG抗体的Fc段结合，而杀伤这些靶细胞的作用。IgG抗体可介导这些细胞发挥ADCC作用，其中NK细胞是能发挥ADCC作用的主要细胞。
过程：IgG抗体与靶细胞表面的抗原决定簇特异性地结合；之后自然杀伤细胞(NK细胞)借助其表面相应的受体与结合在靶细胞上的IgG Fc段结合；活化的NK细胞释放穿孔素、颗粒酶等细胞毒物质杀伤靶细胞；靶细胞发生细胞凋亡，抗体被肝处理。
实验步骤：
1.靶细胞的制备：用流感病毒感染Raji细胞48小时，使其达到80-95％的感染率；用生理盐水洗两次，用PKH67标记细胞。
2.效应细胞制备：按照正常的步骤从健康志愿者血液中分离出PBMC细胞，洗涤细胞两次。
3.ADCC测定：ADCC测定将采用FACS的方法，活细胞和死细胞计数来确定其活性。
4.简要步骤：在96孔板上接种标记好的靶细胞；加抗体保温37度15分钟；加未标记的PBMC效应细胞37度保温2小时；加荧光标记的死细胞染料(7AAD)4度20分钟；用FACS做细胞计数。
5.FACS计数分析：鉴定靶细胞(活和死细胞)区间；鉴定效应细胞区间；活细胞(PKH67)计数；死细胞(7AAD)细胞计数。
2.2.3：PD-1抑制(PD-1/PD-L1通路阻断)实验和混合淋巴细胞反应实验
概述：PD-1抑制实验(Promega)，混合淋巴细胞反应实验(Genscript)将按照标准步骤进行。下面是PD-1抑制实验的简要步骤。
实验步骤：
1.融化和接种PD-L1细胞在96孔板，37度保温；
2.准备等倍稀释的基于PD1单域抗体构建的融合蛋白样品和参照品Keytruda。或者准备等倍稀释的基于PDL1单域抗体构建的融合蛋白样品和参照品Atezolizumab。这两个参照品只是作为一个标准品衡量不同批次实验之前的稳定性。；
3.从二氧化碳培养箱中取出96孔培养板，吸取培养液，加入稀释好的抗体到PD-L1细胞孔中。
4.融化和加PD-1效应细胞到含有抗体的96孔板中，37度保温6小时。
5.荧光素酶法检测，准备Bio-GloTM检测试剂，加80μl到含抗体和细胞的96孔板里，室温5-10分钟，在GloMax***检测冷光。
6.数据分析：计算诱导倍数＝RLU抗体稀释/RLU抗体阴性对照；以RLU和Log10以及诱导倍数和Log10；通过GraphPad计算EC50。
3.体内抗肿瘤疗效评估
体内抗肿瘤药效将使用人IL-12受体和人PD-1和敲入小鼠建立肿瘤模型来评估。IL-12受体和PD-1的人源化分别使得融合了单域抗体和细胞因子的融合蛋白的直接体内药效评估成为可能。适用于此评估的肿瘤模型可包括但不限于GL261或B16细胞系。以下方面的药效将被评估：(1)肿瘤形成的抑制；(2)已形成的肿瘤生长抑制；以及(3)剂量梯度测试(三个剂量)。
单独的抗体应该展示出抗肿瘤效果，但是单域抗体-细胞因子融合蛋白会显示出更好的抗肿瘤效果，并且这种肿瘤抑制效果是剂量依赖型的。
3.1小鼠模型的建立和给药处理
在小鼠的腹部或背部皮下注射1×10 6的GL261或B16细胞。等平均肿瘤体积达到100mm 3时，将小鼠随机分为4组(每组8只)：对照组，抗PD-1组、抗PD-1+IL2组、抗PD-1+IL-12组和抗PD-1+IL2+IL12组。四天给药一次，一共三次。每次分别用PBS(对照组)、PD-1(sdAb-Fc)单域抗体Fc融合(抗PD-1组)、PD-1 sdAb-IL2融合蛋 白(抗PD-1+IL2组)、PD-1 sdAb-IL12融合蛋白(抗PD-1+IL12组)和PD-1 sdAb-IL2-IL12融合蛋白(抗PD-1+IL2+IL12组)通过肿瘤内或尾静脉给药。然后每周3次监测肿瘤生长情况。
3.2数据统计学分析
采用SPSS软件分析。数据均以“均数±标准差”的形式表示。使用one-way ANOVA分析，以P<0.05为差异有统计学意义。
本发明的抗体-细胞因子融合蛋白可以在体内选择性的运输细胞因子到靶细胞，因此细胞因子会呈现局部生物学反应，像局部炎症反应，刺激T细胞生长和激活。PD-1抗体更是可以通过结合细胞膜表面的受体来发挥抗癌的作用。因此，融合抗体和细胞因子通过靶向递送细胞因子增强免疫应答来治疗癌症是切实可行的。
实验结果及分析
1.基于抗体Fc异源二聚体的免疫细胞因子融合蛋白的构建
本研究构建了一系列的基于抗体Fc异源二聚体的单域抗体-细胞因子融合蛋白。其中用于构建融合蛋白的组件为PD-1单域抗体，细胞因子IL-12、IL-2以及IL-2突变体。
将抗PD-1单域抗体序列***到pTT5表达载体的多克隆位点EcoRI后面，同时在融合蛋白基因前面还加入KOZAK序列GCCGCCACC和信号肽序列帮助把融合蛋白分泌到细胞外。产生了表达单域抗体的SC01载体。
抗体Fc片段通过多克隆位点EcoRI***到pTT5载体上，然后PD-1单域抗体序列通过Gibson组装，连接到Fc的N端，它们之间通过(G4S)3接头序列连接。接着IL-12序列也通过类似的方式，连接到Fc的C端，它们之间也有G4S接头序列。最终形成了PD-1单域抗体和IL-12融合蛋白表达载体G1-208。同时在在融合蛋白基因前面还加入KOZAK序列GCCGCCACC和信号肽序列帮助把融合蛋白分泌到细胞外。通过类似的方式，PD-1单域抗体和IL-2分别融合到Fc的N端和C端，产生PD-1单域抗体和IL-2融合蛋白表达载体G1-212。
G1-208和G1-212均为基于抗体Fc的同源二聚体。为了产生基于Fc的异源二聚体，将knob-into-holes技术用于进行抗体Fc改造，其中其中一条Fc链的突变位点组合是T366W/S354C，另外一条Fc链的突变位点组合是T366S/L368A/Y407V/Y349C。抗PD-1单域抗体序列通过通过Gibson组装，连接到Fc的N端，IL-12和IL-2通过类似的方式，分别连接到Fc的C端，从而产生了融合有PD-1单域抗体、IL-12和IL-2的表达载体G1-405。
G1-716是在G1-405的基础上，将一个PD-1单域抗体通过(G4S)3接头序列连接到已有的PD-1单域抗体的N端。
G1-723是在G1-716的基础上，对IL-2基因进行位点突变改造而来。
G1-717是在G1-716的基础上，删除IL-12基因改造而来。
G1-719是在G1-716的基础上，删除IL-2基因改造而来。
将两个PD-1单域抗体通过(G4S)3接头序列串联起来，然后再通过(G4S)3接头序列连接到Fc的N端，从而形成同源二聚体G1-709。
本实验中构建的质粒信息见以下表1：
表1 融合蛋白构成
Figure PCTCN2018123878-appb-000001
以上构建到pTT5表达载体上的融合蛋白质粒通过PEI转染试剂瞬转CHO-3E7细胞，然后37度培养6天。通过离心收取培养液上清，首先通过Protein A亲和柱纯化融合蛋白，然后通过分子筛进一步纯化融合蛋白，最终纯度达到95％以上。
本研究中使用的信号肽DNA序列(SEQ ID NO：1)如下：
Figure PCTCN2018123878-appb-000002
本研究中使用的抗PD-1单域抗体SC01的DNA序列(SEQ ID NO：2)如下：
Figure PCTCN2018123878-appb-000003
SC01的氨基酸序列(SEQ ID NO：3)如下：
Figure PCTCN2018123878-appb-000004
本研究中使用的G4S接头的DNA序列(SEQ ID NO：4)如下：
Figure PCTCN2018123878-appb-000005
本研究中使用的细胞因子IL-12全长DNA序列(SEQ ID NO：5)如下：
Figure PCTCN2018123878-appb-000006
所用的IL-12全长氨基酸序列(SEQ ID NO：6)如下：
Figure PCTCN2018123878-appb-000007
本研究中使用的细胞因子人IL-2的DNA序列(SEQ ID NO：7)如下：
Figure PCTCN2018123878-appb-000008
人IL-2的氨基酸序列(SEQ ID NO：8)如下：
Figure PCTCN2018123878-appb-000009
本研究中使用的人IL-2突变体DNA序列(SEQ ID NO：9)如下：
Figure PCTCN2018123878-appb-000010
人IL-2突变体氨基酸序列(SEQ ID NO：10)如下：
Figure PCTCN2018123878-appb-000011
本研究中使用的G1-208融合蛋白全长DNA序列(SEQ ID NO：11)如下：
Figure PCTCN2018123878-appb-000012
Figure PCTCN2018123878-appb-000013
本研究中使用的G1-208融合蛋白全长氨基酸序列(SEQ ID NO：12)如下：
Figure PCTCN2018123878-appb-000014
本研究中使用的G1-212融合蛋白全长DNA序列(SEQ ID NO：13)如下：
Figure PCTCN2018123878-appb-000015
Figure PCTCN2018123878-appb-000016
本研究中使用的G1-212融合蛋白全长氨基酸序列(SEQ ID NO：14)如下：
Figure PCTCN2018123878-appb-000017
本研究中使用的G1-405融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc1和Fc2)通过Knob-into-holes结构形成。其中Fc1全长DNA序列(SEQ ID NO：15)如下：
Figure PCTCN2018123878-appb-000018
Figure PCTCN2018123878-appb-000019
Figure PCTCN2018123878-appb-000020
本研究中使用的G1-405融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc1和Fc2)通过Knob-into-holes结构形成。其中Fc1全长氨基酸序列(SEQ ID NO：16)如下：
Figure PCTCN2018123878-appb-000021
本研究中使用的G1-405融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc1和Fc2)通过Knob-into-holes结构形成。其中Fc2全长DNA序列(SEQ ID NO：17)如下：
Figure PCTCN2018123878-appb-000022
Figure PCTCN2018123878-appb-000023
本研究中使用的G1-405融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc1和Fc2)通过Knob-into-holes结构形成。其中Fc2全长氨基酸序列(SEQ ID NO：18)如下：
Figure PCTCN2018123878-appb-000024
本研究中使用的G1-716融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc3和Fc4)通过Knob-into-holes结构形成。其中Fc3全长DNA序列(SEQ ID NO：19)如下：
Figure PCTCN2018123878-appb-000025
Figure PCTCN2018123878-appb-000026
本研究中使用的G1-716融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc4)通过Knob-into-holes结构形成。其中Fc3全长氨基酸序列(SEQ ID NO：20)如下：
Figure PCTCN2018123878-appb-000027
Figure PCTCN2018123878-appb-000028
本研究中使用的G1-716融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc4)通过Knob-into-holes结构形成。其中Fc4全长DNA序列(SEQ ID NO：21)如下：
Figure PCTCN2018123878-appb-000029
Figure PCTCN2018123878-appb-000030
本研究中使用的G1-716融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc4)通过Knob-into-holes结构形成。其中Fc4全长氨基酸序列(SEQ ID NO：22)如下：
Figure PCTCN2018123878-appb-000031
本研究中使用的G1-723融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc5)通过Knob-into-holes结构形成。其中Fc5全长DNA序列(SEQ ID NO：23)如下：
Figure PCTCN2018123878-appb-000032
Figure PCTCN2018123878-appb-000033
本研究中使用的G1-723融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc5)通过Knob-into-holes结构形成。其中Fc5全长氨基酸序列(SEQ ID NO：24)如下：
Figure PCTCN2018123878-appb-000034
本研究中使用的G1-717融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc6和Fc7)通过Knob-into-holes结构形成。其中Fc6全长DNA序列(SEQ ID NO：25)如下：
Figure PCTCN2018123878-appb-000035
本研究中使用的G1-717融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc6和Fc7)通过Knob-into-holes结构形成。其中Fc6全长氨基酸序列(SEQ ID NO：26)如下：
Figure PCTCN2018123878-appb-000036
本研究中使用的G1-717融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc6和Fc7)通过Knob-into-holes结构形成。其中Fc7全长DNA序列(SEQ ID NO：27)如下：
Figure PCTCN2018123878-appb-000037
Figure PCTCN2018123878-appb-000038
本研究中使用的G1-717融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc6和Fc7)通过Knob-into-holes结构形成。其中Fc7全长氨基酸序列(SEQ ID NO：28)如下：
Figure PCTCN2018123878-appb-000039
本研究中使用的G1-719融合蛋白为异源二聚体，由两条改造过的并融合有其他蛋白的Fc链(Fc3和Fc7)通过Knob-into-holes结构形成。其中Fc3和Fc7全长DNA和氨基酸序列已在上文展示。
本研究中使用的G1-709融合蛋白为同源二聚体，全长DNA序列(SEQ ID NO：29)如下：
Figure PCTCN2018123878-appb-000040
Figure PCTCN2018123878-appb-000041
本研究中使用的G1-709融合蛋白为同源二聚体，多肽链全长氨基酸序列(SEQ ID NO：30)如下：
Figure PCTCN2018123878-appb-000042
2.γ-干扰素释放实验结果分析
本实验采用细胞因子刺激NK92细胞释放γ-干扰素(IFN-γ)来验证融合蛋白中的细胞因子IL-12和IL-2的体外生物活性。由于本实验构建的融合蛋白中含有细胞因子，于是将不同的融合蛋白与NK92细胞孵育，然后检测γ-干扰素的释放。
对于融合了IL-2的融合蛋白来说，与游离的IL-2的生物活性相比较，除了同源二聚体的融合蛋白G1-212的IL-2活性下降0.8倍外(图1)，其它异源二聚体的IL-2的活性均为增强，其中G1-405和G1-716融合蛋白的IL-2的活性增强了1.5倍以上(图2和3)，G1-723和G1-717融合蛋白的IL-2的活性增强了0.5倍左右(图2和3)。理论上，从数量上讲，同源二聚体的融合蛋白融合了2个细胞因子IL-2，应该比只融合了一个IL-2的异源二聚体的融合蛋白的IL-2活性要强，但实际的结果恰好相反。可能的原因是同源二聚体的2个IL-2相互之间形成了空间位阻，反而造成了IL-2活性下降。所以，总体上，基于抗体Fc异 源二聚体技术的只融合了一个IL-2的融合蛋白的细胞因子活性，会优于融合了2个IL-2的同源二聚体的融合蛋白的细胞因子活性。并且，同时融合了IL-12和IL-2的融合蛋白，比只融合了IL-2的融合蛋白的IL-2细胞因子活性要强，说明了IL-12与IL-2的协同效应。
对于融合了IL-12的融合蛋白来说，与游离的IL-12的生物活性相比较，同源二聚体的融合蛋白G1-208的细胞因子活性下降9倍(图4)，而G1-405融合蛋白的IL-12的活性只下降了0.3倍以上(图6)，G1-716融合蛋白的IL-12的活性也只下降了3倍(图5)。这说明与IL-2非对称融合类似，IL-12的非对称融合结构也会在一定程度上提升细胞因子活性。但是，与游离的IL-12的生物活性相比，异源二聚体的融合蛋白G1-719(图5)和G1-723(图6)的细胞因子活性下降8-9倍。对于G1-719的细胞因子活性的急剧下降，可能的原因是IL-12与IL-2具有协同效应，二者的共同存在能促进各自的功能，而G1-719只融合了IL-12，缺乏IL-2，所以比同时融合了IL-12和IL-2的G1-716融合蛋白的细胞因子活性低。G1-723与G1-716的唯一区别是IL-2进行了位点突变，根据已有的研究分析，IL-2突变体会一定程度降低该细胞因子体外生物活性，但显著改善动物体内毒性。所以G1-723的IL-12体外活性下降，与IL-2的位点突变导致的活性下降，从而降低与IL-12的协同效应有关。这也进一步验证了IL-12与IL-2的协同效应。总而言之，基于抗体Fc异源二聚体技术的只融合了一个IL-12的融合蛋白的细胞因子活性，会优于融合了2个IL-12的同源二聚体的融合蛋白的细胞因子活性。并且，同时融合了IL-12和IL-2的融合蛋白，比只融合了IL-12的融合蛋白的IL-12细胞因子活性要强，进一步证明了IL-12与IL-2的协同效应。
3.PD-1抑制实验结果分析
PD-1/PD-L1通路阻断的生物测定是一种基于生物学上相关的作用机制的检测分析，能用于测量那些能阻断PD-1/PD-L1相互作用的抗体和其他生物制剂的效力和稳定性。这个检测***包括以下两个基因编辑的细胞系：能稳定的表达人源PD-1的细胞和稳定的表达人源PD-L1的细胞。
当这两种细胞共培养时，PD-1/PD-L1相互作用会抑制T细胞受体(TCR)信号途径以及NFAT调控的荧光素酶活性。当加入相应的PD-1或PD-L1抗体阻断PD-1/PD-L1相互作用，就会解除抑制信号，从而激活T细胞受体(TCR)信号途径和NFAT诱导的荧光素酶活性。然后通过检测荧光信号分析抗体活性。
本实验中将带有PD-1单域抗体的各种融合蛋白与细胞孵育，从而检测融合蛋白的抗体活性。结果显示，与传统的IgG抗体Keytruda相比较，游离的PD-1单域抗体SC01的活性很弱(图7)。但是，将单域抗体融合到抗体Fc链上后，产生的融合蛋白，比如G1-405，其抗体活性大大增强，虽然比Keytruda抗体相比还有一定差距(图8)。
为了进一步增强融合蛋白的抗体活性，我们构建了串联两个单域抗体的融合蛋白，并将其融合到抗体Fc链的N端，比如G1-709单域抗体Fc融合蛋白，其PD-1抗体活性比没有串联单域抗体的融合蛋白G1-405增强不少(图8)。更进一步的，我们构建的基于抗体Fc异源二聚体技术的融合了抗体和细胞因子的融合蛋白G1-717和G1-719的抗体活性离Keytruda抗体相差也不远(图9)。而同时融合了IL-12和IL-2的的融合蛋白G1-716和G1-723的抗体活性，几乎快要接近Keytruda抗体(图10)。这不仅说明Fc链增强了单域抗体的活性，而且细胞因子在一定程度上进一步促进了抗体活性。
4.基于抗体Fc异源二聚体的其他单域抗体-细胞因子融合蛋白的构建
以上的实验结果显示了基于抗体Fc异源二聚体的单域抗体-细胞因子的优越性。为了进一步验证这种技术平台的适用性和广谱性，我们在接下来的研究中，使用了更多的不同分子的单域抗体，构建了一系列的基于抗体Fc异源二聚体的单域抗体-细胞因子融合蛋白。其中用于构建融合蛋白的细胞因子仍为IL-12、IL-2，单域抗体为1个新的PD-1单域抗体或者3个新的PD-L1单域抗体。与上述的抗体Fc片段是基于人IgG1突变体不同的，新的抗体Fc片段则是基于人的IgG4突变而来。
将新的抗PD-1单域抗体或新的抗PD-L1单域抗体序列分别***到pTT5表达载体的多克隆位点EcoRI后面，同时在融合蛋白基因前面还加入KOZAK序列GCCGCCACC和信号肽序列帮助把融合蛋白分泌到细胞外。产生了表达单域抗体的载体。
与上面的载体构建方式类似，为了产生基于Fc的异源二聚体，将knob-into-holes技术用于进行抗体Fc改造，其中其中一条Fc链的突变位点组合是T366W/S354C，另外一条Fc链的突变位点组合是T366S/L368A/Y407V/Y349C。两条Fc链同时还有其他突变位点S228P和L235E。抗体IgG4的Fc片段通过多克隆位点EcoRI***到pTT5载体上，然后4种新的单域抗体序列通过Gibson组装，分别连接到Fc的N端，它们之间没有接头序列。接着IL-12或者IL-2序列也通过类似的方式，连接到Fc的C端，它们之间有G4S接头序列。最终形成了不同的单域抗体和不同的细胞因子的融合蛋白表达载体。同时在在融合蛋白基因前面还加入KOZAK序列GCCGCCACC和信号肽序列帮助把融合蛋白分泌到细胞外。
同时，在野生型的人IgG4的Fc片段基础上，进行位点突变位点S228P和L235E，然后将不同的单域抗体直接连接到突变后IgG4的Fc的N端，从而形成同源二聚体。
本实验中构建的质粒信息见以下表2：
表1 融合蛋白构成
Figure PCTCN2018123878-appb-000043
Figure PCTCN2018123878-appb-000044
以上构建到pTT5表达载体上的融合蛋白质粒通过PEI转染试剂瞬转CHO-3E7细胞，然后37度培养6天。通过离心收取培养液上清，首先通过Protein A亲和柱纯化融合蛋白，然后通过分子筛进一步纯化融合蛋白，最终纯度达到95％以上。
本研究中新的抗PD-1单域抗体以及新的抗PD-L1单域抗体序列如下所示：
抗PD1a单域抗体的氨基酸序列(SEQ ID NO:71)：
Figure PCTCN2018123878-appb-000045
抗PD-L1a单域抗体的氨基酸序列(SEQ ID NO:72)：
Figure PCTCN2018123878-appb-000046
抗PD-L1b单域抗体的氨基酸序列(SEQ ID NO:73)：
Figure PCTCN2018123878-appb-000047
抗PD-L1c单域抗体的氨基酸序列(SEQ ID NO:74)：
Figure PCTCN2018123878-appb-000048
本研究中使用的sPD1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc9)通过Knob-into-holes结构形成。其中Fc8全长DNA序列(SEQ ID NO：31)如下：
Figure PCTCN2018123878-appb-000049
Figure PCTCN2018123878-appb-000050
本研究中使用的sPD1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc9)通过Knob-into-holes结构形成。其中Fc8全长氨基酸序列(SEQ ID NO：32)如下：
Figure PCTCN2018123878-appb-000051
Figure PCTCN2018123878-appb-000052
本研究中使用的sPD1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc9)通过Knob-into-holes结构形成。其中Fc9全长DNA序列(SEQ ID NO：33)如下：
Figure PCTCN2018123878-appb-000053
本研究中使用的sPD1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc9)通过Knob-into-holes结构形成。其中Fc9全长氨基酸序列(SEQ ID NO：34)如下：
Figure PCTCN2018123878-appb-000054
本研究中使用的sPD1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc10)通过Knob-into-holes结构形成。其中Fc8全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPD1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc10)通过Knob-into-holes结构形成。其中Fc10全长DNA序列(SEQ ID NO：35)如下：
Figure PCTCN2018123878-appb-000055
Figure PCTCN2018123878-appb-000056
本研究中使用的sPD1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc8和Fc10)通过Knob-into-holes结构形成。其中Fc10全长氨基酸序列(SEQ ID NO：36)如下：
Figure PCTCN2018123878-appb-000057
本研究中使用的sPD1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc10和Fc11)通过Knob-into-holes结构形成。其中Fc10全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPD1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc10和Fc11)通过Knob-into-holes结构形成。其中Fc11全长DNA序列(SEQ ID NO：37)如下：
Figure PCTCN2018123878-appb-000058
Figure PCTCN2018123878-appb-000059
本研究中使用的sPD1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc10和Fc11)通过Knob-into-holes结构形成。其中Fc11全长氨基酸序列(SEQ ID NO：38)如下：
Figure PCTCN2018123878-appb-000060
本研究中使用的sPDL1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc13)通过Knob-into-holes结构形成。其中Fc12全长DNA序列(SEQ ID NO：39)如下：
Figure PCTCN2018123878-appb-000061
Figure PCTCN2018123878-appb-000062
Figure PCTCN2018123878-appb-000063
本研究中使用的sPDL1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc13)通过Knob-into-holes结构形成。其中Fc12全长氨基酸序列(SEQ ID NO：40)如下：
Figure PCTCN2018123878-appb-000064
本研究中使用的sPDL1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc13)通过Knob-into-holes结构形成。其中Fc13全长DNA序列(SEQ ID NO：41)如下：
Figure PCTCN2018123878-appb-000065
本研究中使用的sPDL1a01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc13)通过Knob-into-holes结构形成。其中Fc13全长氨基酸序列(SEQ ID NO：42)如下：
Figure PCTCN2018123878-appb-000066
本研究中使用的sPDL1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc14)通过Knob-into-holes结构形成。其中Fc13全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc14)通过Knob-into-holes结构形成。其中Fc14全长DNA序列(SEQ ID NO：43)如下：
Figure PCTCN2018123878-appb-000067
Figure PCTCN2018123878-appb-000068
本研究中使用的sPDL1a02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc12和Fc14)通过Knob-into-holes结构形成。其中Fc14全长氨基酸序列(SEQ ID NO：44)如下：
Figure PCTCN2018123878-appb-000069
本研究中使用的sPDL1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc14和Fc15)通过Knob-into-holes结构形成。其中Fc14全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc14和Fc15)通过Knob-into-holes结构形成。其中Fc15全长DNA序列(SEQ ID NO：45)如下：
Figure PCTCN2018123878-appb-000070
Figure PCTCN2018123878-appb-000071
本研究中使用的sPDL1a03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc14和Fc15)通过Knob-into-holes结构形成。其中Fc15全长氨基酸序列(SEQ ID NO：46)如下：
Figure PCTCN2018123878-appb-000072
本研究中使用的sPDL1b01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc17)通过Knob-into-holes结构形成。其中Fc16全长DNA序列(SEQ ID NO：47)如下：
Figure PCTCN2018123878-appb-000073
Figure PCTCN2018123878-appb-000074
Figure PCTCN2018123878-appb-000075
本研究中使用的sPDL1b01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc17)通过Knob-into-holes结构形成。其中Fc16全长氨基酸序列(SEQ ID NO：48)如下：
Figure PCTCN2018123878-appb-000076
本研究中使用的sPDL1b01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc17)通过Knob-into-holes结构形成。其中Fc17全长DNA序列(SEQ ID NO：49)如下：
Figure PCTCN2018123878-appb-000077
本研究中使用的sPDL1b01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc17)通过Knob-into-holes结构形成。其中Fc17全长氨基酸序列(SEQ ID NO：50)如下：
Figure PCTCN2018123878-appb-000078
本研究中使用的sPDL1b02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc18)通过Knob-into-holes结构形成。其中Fc16全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1b02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc18)通过Knob-into-holes结构形成。其中Fc18全长DNA序列(SEQ ID NO：51)如下：
Figure PCTCN2018123878-appb-000079
Figure PCTCN2018123878-appb-000080
本研究中使用的sPDL1b02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc16和Fc18)通过Knob-into-holes结构形成。其中Fc18全长氨基酸序列(SEQ ID NO：52)如下：
Figure PCTCN2018123878-appb-000081
本研究中使用的sPDL1b03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc18和Fc19)通过Knob-into-holes结构形成。其中Fc18全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1b03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc18和Fc19)通过Knob-into-holes结构形成。其中Fc19全长DNA序列(SEQ ID NO：53)如下：
Figure PCTCN2018123878-appb-000082
Figure PCTCN2018123878-appb-000083
本研究中使用的sPDL1b03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc18和Fc19)通过Knob-into-holes结构形成。其中Fc19全长氨基酸序列(SEQ ID NO：54)如下：
Figure PCTCN2018123878-appb-000084
本研究中使用的sPDL1c01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc21)通过Knob-into-holes结构形成。其中Fc20全长DNA序列(SEQ ID NO：55)如下：
Figure PCTCN2018123878-appb-000085
Figure PCTCN2018123878-appb-000086
Figure PCTCN2018123878-appb-000087
本研究中使用的sPDL1c01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc21)通过Knob-into-holes结构形成。其中Fc20全长氨基酸序列(SEQ ID NO：56)如下：
Figure PCTCN2018123878-appb-000088
本研究中使用的sPDL1c01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc21)通过Knob-into-holes结构形成。其中Fc21全长DNA序列(SEQ ID NO：57)如下：
Figure PCTCN2018123878-appb-000089
本研究中使用的sPDL1c01融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc21)通过Knob-into-holes结构形成。其中Fc21全长氨基酸序列(SEQ ID NO：58)如下：
Figure PCTCN2018123878-appb-000090
本研究中使用的sPDL1c02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc22)通过Knob-into-holes结构形成。其中Fc20全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1c02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc22)通过Knob-into-holes结构形成。其中Fc22全长DNA序列(SEQ ID NO：59)如下：
Figure PCTCN2018123878-appb-000091
Figure PCTCN2018123878-appb-000092
本研究中使用的sPDL1c02融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc20和Fc22)通过Knob-into-holes结构形成。其中Fc22全长氨基酸序列(SEQ ID NO：60)如下：
Figure PCTCN2018123878-appb-000093
本研究中使用的sPDL1c03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc22和Fc23)通过Knob-into-holes结构形成。其中Fc22全长DNA和氨基酸序列已在上文展示。
本研究中使用的sPDL1c03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc22和Fc23)通过Knob-into-holes结构形成。其中Fc23全长DNA序列(SEQ ID NO：61)如下：
Figure PCTCN2018123878-appb-000094
Figure PCTCN2018123878-appb-000095
本研究中使用的sPDL1c03融合蛋白为异源二聚体，由两条改造过的并融合有其它蛋白的Fc链(Fc22和Fc23)通过Knob-into-holes结构形成。其中Fc23全长氨基酸序列(SEQ ID NO：62)如下：
Figure PCTCN2018123878-appb-000096
本研究中使用的sPD1a00融合蛋白为同源二聚体，多肽链全长DNA序列(SEQ ID NO：63)如下：
Figure PCTCN2018123878-appb-000097
Figure PCTCN2018123878-appb-000098
本研究中使用的sPD1a00融合蛋白为同源二聚体，多肽链全长氨基酸序列(SEQ ID NO：64)如下：
Figure PCTCN2018123878-appb-000099
本研究中使用的sPDL1a00融合蛋白为同源二聚体，多肽链全长DNA序列(SEQ ID NO：65)如下：
Figure PCTCN2018123878-appb-000100
Figure PCTCN2018123878-appb-000101
本研究中使用的sPDL1a00融合蛋白为同源二聚体，多肽链全长氨基酸序列(SEQ ID NO：66)如下：
Figure PCTCN2018123878-appb-000102
本研究中使用的sPDL1b00融合蛋白为同源二聚体，多肽链全长DNA序列(SEQ ID NO：67)如下：
Figure PCTCN2018123878-appb-000103
Figure PCTCN2018123878-appb-000104
本研究中使用的sPDL1b00融合蛋白为同源二聚体，多肽链全长氨基酸序列(SEQ ID NO：68)如下：
Figure PCTCN2018123878-appb-000105
本研究中使用的sPDL1c00融合蛋白为同源二聚体，多肽链全长DNA序列(SEQ ID NO：69)如下：
Figure PCTCN2018123878-appb-000106
Figure PCTCN2018123878-appb-000107
本研究中使用的sPDL1c00融合蛋白为同源二聚体，多肽链全长氨基酸序列(SEQ ID NO：70)如下：
Figure PCTCN2018123878-appb-000108
5.广谱性验证：γ-干扰素释放实验结果分析
本实验采用细胞因子刺激NK92细胞释放γ-干扰素(IFN-γ)来验证融合蛋白中的细胞因子IL-12和IL-2的体外生物活性。由于本实验构建的融合蛋白中含有细胞因子，于是将不同的融合蛋白与NK92细胞孵育，然后检测γ-干扰素的释放。
对于融合了IL-2的融合蛋白来说，与游离的IL-2的生物活性相比较，新构建的一系列异源二聚体的IL-2的活性均为增强(图13和21)，或者活性类似(图22)，这与上文的实验结果一致，从而说明了基于抗体Fc异源二聚体技术的融合蛋白能显著性的提高或保持IL-2的活性。
对于融合了IL-12的融合蛋白来说，与游离的IL-12的生物活性相比较，新构建的一系列异源二聚体的IL-2的活性均为增强(图11和12)，再次印证了基于抗体Fc异源二聚体技术的融合蛋白也能显著性的提高IL-12的活性。
6.广谱性验证：PD-1/PD-L1通路阻断实验结果分析
PD-1/PD-L1通路阻断的生物测定是一种基于生物学上相关的作用机制的检测分析，能用于测量那些能阻断PD-1/PD-L1相互作用的抗体和其他生物制剂的效力和稳定性。这个检测***包括以下两个基因编辑的细胞系：能稳定的表达人源PD-1的细胞和稳定的表达人源PD-L1的细胞。
当这两种细胞共培养时，PD-1/PD-L1相互作用会抑制T细胞受体(TCR)信号途径以及NFAT调控的荧光素酶活性。当加入相应的PD-1或PD-L1抗体阻断PD-1/PD-L1相互作用，就会解除抑制信号，从而激活T细胞受体(TCR)信号途径和NFAT诱导的荧光素酶活性。然后通过检测荧光信号分析抗体活性。
上文已有的PD-1抑制实验结果显示，异源二聚体的抗体的活性没有因为融合了细胞因子而受到影响。为了进一步验证这种结构对于其他单域抗体是否也适用，我们又基于新的一批单域抗体序列，构建了一系列新的融合蛋白，包括基于PD-1单域抗体和PD-L1单域抗体的异源二聚体融合蛋白，然后将带有单域抗体的各种融合蛋白与细胞孵育，从而检测融合蛋白的抗体活性，具体实验步骤参见实施例2.2.3。结果显示，与没有融合细胞因子的带有Fc片段的单域抗体同源二聚体相比较，新构建的一系列异源二聚体融合蛋白的抗体的活性与之差异不大(图14-20)。说明基于抗体Fc异源二聚体技术的单域抗体-细胞因子融合蛋白不会影响抗体活性的发挥。
本发明所属领域技术员应理解，以上描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
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Claims (26)

  1. 一种以抗体Fc区为骨架的融合蛋白二聚体，包括第一和第二多肽链，其中所述第一多肽链包括第一抗体Fc区和与所述第一抗体Fc区融合的一个或多个单域抗体；所述第二多肽链包括第二抗体Fc区和与所述第二抗体Fc区融合的一个或多个单域抗体；所述第一多肽链和/或第二多肽链还包括与各自抗体Fc区融合的细胞因子。
  2. 如权利要求1所述的融合蛋白二聚体，其中所述单域抗体为抗免疫检查点分子的单域抗体。
  3. 如权利要求1或2所述的融合蛋白二聚体，其中所述单域抗体为抗PD-1单域抗体或抗PD-L1单域抗体。
  4. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述细胞因子选自IL-2、IL-12、GM-CSF、IL-2突变体以及它们的组合。
  5. 如前述权利要求中任一项所述的融合蛋白二聚体，其中IL-12的两个亚基P35和P40通过接头序列连接形成IL-12单链蛋白而存在于所述第一多肽链和/或第二多肽链中。
  6. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述第一抗体Fc区和所述第二抗体Fc区不同，并且它们之间具有非对称互补结构。
  7. 如权利要求6所述的融合蛋白二聚体，其中所述第一抗体Fc区具有突变位点组合T366W/S354C，所述第二抗体Fc区具有突变位点组合T366S/L368A/Y407V/Y349C。
  8. 如前述权利要求中任一项所述的融合蛋白二聚体，其中第一多肽链和/或第二多肽链包括两个串联排列的所述抗PD-1单域抗体或抗PD-L1单域抗体。
  9. 如前述权利要求中任一项所述的融合蛋白二聚体，其中第一多肽链中含有的细胞因子为IL-12，第二多肽链中含有的细胞因子为IL-2或IL-2突变体或者第一多肽链中含有的细胞因子为IL-12，第二多肽链中缺失细胞因子。
  10. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述抗PD-1单域抗体具有如SEQ ID NO：3或SEQ ID NO:71所示的氨基酸序列。
  11. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述抗PD-L1单域抗体具有如SEQ ID NOs：72-74所示的氨基酸序列。
  12. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述IL-12单链蛋白具有如SEQ ID NO：6所示的氨基酸序列。
  13. 如前述权利要求中任一项所述的融合蛋白二聚体，其中：
    所述第一多肽链具有如SEQ ID NO：20所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：22、24或28所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO：26所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：28所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO：16所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：18所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO：32所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：34、36所示的氨基酸序列；或者
    所述第一多肽链具有如SEQ ID NO：36所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：38所示的氨基酸序列。
  14. 如前述权利要求中任一项所述的融合蛋白二聚体，其中：
    所述第一多肽链具有如SEQ ID NO:40所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：42、44所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO:44所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：46所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO:48所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：50、52所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO:52所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：54所示的氨基酸序列；
    所述第一多肽链具有如SEQ ID NO:56所示的氨基酸序列，所述第二多肽链具有如SEQ ID NOs：58、60所示的氨基酸序列；或者
    所述第一多肽链具有如SEQ ID NO:60所示的氨基酸序列，所述第二多肽链具有如SEQ ID NO：62所示的氨基酸序列。
  15. 如前述权利要求中任一项所述的融合蛋白二聚体，所述融合蛋白二聚体中的单域抗体、抗体Fc区和细胞因子之间通过接头序列连接或直接连接。
  16. 如权利要求15所述的融合蛋白二聚体，其中所述接头序列选自(G4S)1-3、KRVAPELLGGPS、ASTKG以及NSPPAA。
  17. 如前述权利要求中任一项所述的融合蛋白二聚体，其中所述一个或多个单域抗体通过接头序列连接或直接连接至所述抗体Fc区的N端，而所述细胞因子通过接头序列连接至所述所述抗体Fc区的C端；或者所述一个或多个单域抗体通过接头序列连接或直接连接至所述抗体Fc区的C端，而所述细胞因子通过接头序列连接至所述抗体Fc区的N端。
  18. 如权利要求1所述的融合蛋白二聚体，其中所述第一抗体Fc区和第二抗体Fc区为野生型抗体Fc区，从而形成抗体Fc融合蛋白同源二聚体。
  19. 分离的多核苷酸，其编码前述权利要求中任一项所述的融合蛋白二聚体的第一多肽链或第二多肽链。
  20. 如权利要求19所述的多核苷酸，其具有如SEQ ID NO：15、17、19、21、23、25、27、29、31、33、35或37所示的核苷酸序列或者具有如SEQ ID NO:39、41、43、45、47、49、51、53、55、57、59及61所示的核苷酸序列。
  21. 表达载体，其包括所述权利要求19或20所述的多核苷酸。
  22. 宿主细胞，其包括所述权利要求21所述的表达载体。
  23. 权利要求1至18中任一项所述的融合蛋白二聚体、权利要求19或20所述的多核苷酸、权利要求21所述的表达载体或权利要求22所述的宿主细胞在制备抗肿瘤药物中的应用。
  24. 如权利要求23所述的应用，其中所述肿瘤为黑色素瘤或肺癌。
  25. 抗肿瘤药物组合物，其包含权利要求1至18中任一项所述的融合蛋白二聚体和药学上可接受的载体。
  26. ***的方法，包括给予受试者治疗有效量的权利要求1至18中任一项所述的融合蛋白二聚体或含有所述融合蛋白二聚体的药物组合物。
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