WO2022223001A1 - 双特异性多功能融合多肽 - Google Patents

双特异性多功能融合多肽 Download PDF

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WO2022223001A1
WO2022223001A1 PCT/CN2022/088198 CN2022088198W WO2022223001A1 WO 2022223001 A1 WO2022223001 A1 WO 2022223001A1 CN 2022088198 W CN2022088198 W CN 2022088198W WO 2022223001 A1 WO2022223001 A1 WO 2022223001A1
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antigen
antibody
fusion polypeptide
receptor
amino acid
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PCT/CN2022/088198
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English (en)
French (fr)
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芦迪
霍永庭
路力生
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广东菲鹏制药股份有限公司
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Priority to KR1020237036723A priority Critical patent/KR20230160389A/ko
Priority to JP2023561150A priority patent/JP2024514802A/ja
Priority to IL307899A priority patent/IL307899A/en
Priority to AU2022261268A priority patent/AU2022261268A1/en
Priority to BR112023021886A priority patent/BR112023021886A8/pt
Priority to EP22791107.0A priority patent/EP4317184A1/en
Priority to CA3217520A priority patent/CA3217520A1/en
Priority to MX2023012489A priority patent/MX2023012489A/es
Publication of WO2022223001A1 publication Critical patent/WO2022223001A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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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]
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    • 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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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
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    • C07K14/55—IL-2
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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
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    • 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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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
    • 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 the field of biomedicine, in particular, to a bispecific fusion polypeptide and/or a multifunctional fusion polypeptide comprising a ligand and a receptor.
  • Bispecific antibodies are currently the most popular new biomacromolecular drug structures in clinical practice.
  • Bispecific Antibodies refer to antibodies that can simultaneously bind to two different antigens or different epitopes of one antigen, and can exert biological functions that cannot be achieved by monoclonal antibodies through a unique mode of action.
  • bispecific antibodies With the advancement of recombinant protein expression and genetic engineering technology, the forms of bispecific antibodies are becoming more and more diverse. Up to now, more than 20 kinds of bispecific antibody forms have been developed into technology platforms.
  • the core value of the double antibody technology platform is to solve the problem of heavy chain and heavy chain mismatch, light chain and heavy chain mismatch.
  • ART-Ig Strand Exchange Engineering Domain (SEED) technology, XmAb.
  • SEED Strand Exchange Engineering Domain
  • the technical platforms to solve the mismatch between heavy chain and light chain mainly include:
  • the technology for solving the mismatch between the heavy chain and the heavy chain is relatively mature, and the technology for solving the mismatch between the light chain and the heavy chain still has room for improvement.
  • the present invention is specially proposed.
  • the present invention aims to solve one of the technical problems in the related art to a certain extent.
  • the inventors have proposed a new idea for the development of bispecific antibodies, using the specific affinity of ligands and receptors to replace CH1 and CL in antibodies or their functional fragments, thereby avoiding or reducing errors in heavy and light chains. Further, the substitutions may be simultaneously or independently selected from CH2, CH3, and optionally CH4, thereby promoting the formation of heavy chain heterodimers.
  • the bispecific antibody proposed in the present invention is a multifunctional fusion protein, and the multifunctional fusion protein can not only exert dual target specificity, but also exert the biological activity of ligand receptor conduction.
  • the invention provides a bispecific fusion polypeptide comprising a first antigen binding moiety comprising:
  • a first polypeptide comprising, from the N-terminus to the C-terminus, the first heavy chain variable domain VH1 of the first antibody operably linked to the first conjugated fragment
  • a second polypeptide comprising, from the N-terminus to the C-terminus, the first light chain variable domain VL1 of the first antibody operably linked to the second conjugated fragment
  • the first conjugated fragment and the second conjugated fragment are capable of specific binding
  • the first conjugate fragment is a receptor
  • the second conjugated fragment is a ligand
  • the first conjugate fragment is a ligand
  • the second conjugated fragment is a receptor
  • a second antigen-binding portion is further included, the second antigen-binding portion being different from the first antigen-binding portion;
  • the second antigen binding moiety includes:
  • a third polypeptide comprising, from the N-terminus to the C-terminus, the second heavy chain variable domain VH2 of the second antibody operably linked to the third conjugated fragment, and
  • a fourth polypeptide comprising, from the N-terminus to the C-terminus, the second light chain variable domain VL2 of the second antibody operably linked to the fourth conjugated fragment;
  • the third conjugated fragment and the fourth conjugated fragment can specifically bind; the third conjugated fragment is a receptor, and the fourth conjugated fragment is a ligand; or the third conjugated fragment is a ligand;
  • the triconjugate fragment is a ligand and the fourth conjugate fragment is a receptor;
  • the third conjugate fragment and/or the fourth conjugate fragment and the first conjugate fragment and/or the second conjugate fragment are selected from different receptors and ligands.
  • a second antigen-binding portion is further included, the second antigen-binding portion being different from the first antigen-binding portion;
  • the second antigen binding moiety includes:
  • a third polypeptide comprising, from the N-terminus to the C-terminus, the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain constant region CH1, and
  • a fourth polypeptide comprising, from the N-terminus to the C-terminus, the second light chain variable domain VL2 of the second antibody operably linked to the antibody light chain constant region CL.
  • the receptor contains only an active site that recognizes and binds a ligand, and does not contain a functionally active site that generates a response.
  • At least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments, the non-natural interchain bond is formed between a first mutated residue contained in the receptor and a second mutated residue contained in the ligand; in some embodiments, the first and the second mutated residue At least one of the groups is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
  • At least one native glycosylation site is absent in the receptor and/or ligand.
  • the receptor and its ligand are selected from interleukins and their receptors.
  • the interleukin and its receptor steric conformation are Totite, selected from IL15/IL15R, IL2/IL2R, IL4/IL-4R ⁇ +R ⁇ , IL-6/IL-6R, IL -11/IL-11R, IL-13/IL-13R1, IL-20/IL20R ⁇ +IL20R ⁇ and/or IL24/IL20R ⁇ +IL20R ⁇ .
  • the interleukin and its receptor are in a clamp-type stereo conformation and are selected from IL7/IL7R, IL21/IL21R, IL23A/IL12B. In some embodiments, the ligand and receptor are selected from IL15 and IL15R ⁇ .
  • the E at position 90 of the IL15 is mutated to a C
  • the P at position 67 of the IL15R ⁇ is mutated to C.
  • the first heavy chain variable domain VH1 and the first light chain variable domain VL1 comprises any combination of the following mutations:
  • the D at position 61 of the IL15 is mutated to N
  • the E at position 64 is mutated to Q
  • the N at position 65 is mutated to D.
  • At least one N-glycosylation site of the IL15 is absent, preferably, the N-glycosylation site is selected from N71, N79 and/or N112; preferably, the IL15 comprises the following amino acids Mutations: N71Q, N79Q and/or N112Q.
  • At least one O-glycosylation site of the IL15R ⁇ is absent, preferably, the O-glycosylation site is selected from T2, T81 and/or T86; preferably, the IL15R ⁇ comprises the following amino acids Mutations: T2A, T81A and/or T86A.
  • the ligands and receptors are selected from IL2 and IL2R ⁇ .
  • the S at position 75 of the IL2 is mutated to a C, and the N-terminus of the IL2R ⁇ is extended by two or three amino acids.
  • the extended amino acid at position 2 is cysteine
  • the extended amino acid at position 1 is a non-polar fatty acid amino acid, an aromatic amino acid, or an uncharged R group. Any of an amino acid, an amino acid whose R group is positively charged, or an amino acid whose R group is negatively charged.
  • the extended amino acid at position 2 is cysteine
  • the extended amino acids at positions 1 and 3 are non-polar fatty acid amino acids, aromatic amino acids, and R groups. Any of an uncharged amino acid, a positively charged R group, or a negatively charged R group.
  • the bispecific fusion polypeptide comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments, the antibody Fc constant region To associate into heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions and/or increased flexibility; in some embodiments, the antibody Fc constant region comprises CH2, CH3 and any The selected CH4, the CH2, CH3 and/or optionally CH4 are replaced by the receptor and its ligand.
  • the first antigen-binding moiety and the second antigen-binding moiety bind to different antigens or to different epitopes of the same antigen; in some embodiments, the first antigen-binding moiety targets immunization cells, the second antigen binding moiety targets tumor cells; in some embodiments, both the first antigen binding moiety and the second antigen binding moiety target tumor cells; in some embodiments, the first antigen binding moiety Both an antigen binding moiety and the second antigen binding moiety target immune cells.
  • the combination of the first antigen and the second antigen can engage T cells and tumor antigens; in some embodiments, the first antigen and the second antigen can engage after binding NK cells and tumor antigens; in some embodiments, the combination with the first antigen and the second antigen can synergistically inhibit signaling pathways; in some embodiments, with the first antigen and the second antigen Upon binding, protein complexes can be formed.
  • the present invention also relates to isolated nucleic acids encoding bispecific fusion polypeptides as described above.
  • the present invention also relates to vectors containing nucleic acids as described above.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • the present invention also relates to a method for preparing a bispecific fusion polypeptide, comprising:
  • the bispecific fusion polypeptides expressed in the host cells are collected.
  • the present invention also relates to pharmaceutical compositions comprising a bispecific fusion polypeptide as described above, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • the present invention also relates to the use of the bispecific fusion polypeptide as described above in the preparation of a medicament for treating diseases.
  • Figure 1 shows four classic double antibody platforms: Figure 1A shows the KiH heterodimeric Fc transformation technology; Figure 1B shows the CrossMab bispecific antibody technology; Figure 1C shows the Wuhan Youzhiyou YBody double antibody technology (asymmetric scFv double antibody technology). Anti-); Figure 1D is a symmetrical scFv double antibody;
  • Figure 2 is a novel dual-characteristic antibody FiBody provided by the present invention, in which CH1 and CL of one side Fab are replaced by ligand receptors with specific affinity;
  • Figure 3 is an exemplary display of 4 possible solutions of FiBody:
  • Figure 3-1 is an engineered ligand receptor with non-naturally occurring interchain bonds between the ligand receptors;
  • Figure 3-2 is the CH1, CL is replaced by receptors and ligands, and the two sides are selected from different ligands;
  • Figure 3-3 shows that the CH1 and CL of the Fab on one side of the antibody are replaced by ligands, and the CH3 segment in the Fc dimer is also liganded.
  • Receptor replacement Figure 3-4 shows that the CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and CH2 in the Fc dimer is also replaced by ligand receptors; there are many other feasible transformation methods;
  • Figure 4 is exemplary when the bispecific antibody of the present invention is used to treat tumors, the targeted binding of the antigen binding moiety of the bispecific antibody includes exemplary 3 types: Figure 4-A The first antigen binding moiety target To T cells, the second antigen binding moiety targets tumor cells; Figure 4-B Both the first antigen binding moiety and the second antigen binding moiety target tumor cells; Figure 4-C The first antigen binding moiety and the second antigen binding moiety Both target T cells; Figure 4-D exemplarily shows the bi-specific antibody of the present invention, which can be optionally a trifunctional fusion protein. In addition to different antigen binding, it can also activate the ligand receptor pathway and stimulate the biological activity of the ligand receptor. ;
  • Figure 5 is a stereoscopic image of interleukins and their receptors, which can be divided into four types: type A is a lift type, type B is a bow-tie type, type C is a baseball player type, and type D is a clamp type;
  • Figure 6 is an example of four types of interleukins and their receptors in three-dimensional conformation.
  • Class A holds IL2/IL2R
  • class B bows IL22/IL22R
  • class C bows IL18/IL18R
  • class D clamps IL21/IL21R;
  • Figure 7 shows the FiBody design based on IL15 (ligand) and IL15RA (receptor) in the embodiment of the present invention, the second antigen-binding region VH is connected to IL15RA, and the second antigen-binding region VL is connected to IL15;
  • Figure 8 is a schematic diagram of the optimized structure of disulfide bond modification
  • Figure 9 is a schematic diagram of the three-dimensional structure of the interaction between IL15/IL15RA and IL2/15R ⁇ / ⁇ C complex;
  • Figure 10 is a schematic structural diagram of the mismatch molecule R1042/R1124 in the embodiment of the present invention.
  • Fig. 11 is the HPLC-SEC detection result of sample R0951 in the embodiment of the present invention.
  • Fig. 12 is the HPLC-SEC detection result of sample R1042 in the embodiment of the present invention.
  • Fig. 13 is the HPLC-SEC detection result of sample R0809 in the embodiment of the present invention.
  • Fig. 14 is the HPLC-SEC detection result of sample R1110 in the embodiment of the present invention.
  • Figure 15 is the HPLC-SEC detection result of the sample R1262 in the embodiment of the present invention.
  • Figure 16 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end to CHO-Tigit cells (R0950, R0951, R0952, R0954, R0955, R0960);
  • Figure 17 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end to CHO-Tigit cells (R1123/R1119/R1120/R1124);
  • Figure 18 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end to CHO-Tigit cells (R1042/R1043);
  • Figure 19 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R0810);
  • Figure 20 is the FCM method in the embodiment of the present invention to detect the binding activity of the TIGIT end of the double antibody to CHO-Tigit cells (R1262); wherein, hIgG1 is the subtype control antibody.
  • Figure 21 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R0950, R0951, R0952, R0954, R0955, R0960);
  • Figure 22 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1115-R1120, R1123-R1124);
  • Figure 23 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R0950, R1042, R1043);
  • Figure 24 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1081-R1086);
  • Figure 25 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1109-R1111);
  • Figure 26 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody PD-L1 end to CHO-PD-L1 cells (R1262);
  • Figure 27 is the blocking detection result of R1262 on the binding force of the ligand and the targeting region in the embodiment of the present invention.
  • Figure 28 is the binding activity (R0950, R0951, R0952, R0954, R0955, R0960) of the sample receptor-ligand complex (IL15/IL15R) in the example of the present invention
  • Figure 29 is the binding activity (R1042, R1043) of the sample receptor ligand complex (IL15/IL15R) in the example of the present invention.
  • Fig. 30 is the gel electrophoresis detection result (R1072, R1081, R1082, R0954, R1084-R1086) of samples optimized for disulfide bond modification in the embodiment of the present invention
  • Figure 31 is the gel electrophoresis of the FiBody samples prepared in Example 9 of the present invention (the disulfide bond-modified IL2/IL2R ⁇ complexes 1 and 2, IL2/IL2R ⁇ complex 3 constructed in Example 8) and R1115 in Example 2 Test results; wherein, complexes 1 and 2 are disulfide bond modified IL2/IL2R ⁇ complexes, complex 3 is an existing reported IL2/IL2R ⁇ complex, and complex 4 is a disulfide bond unmodified IL2/IL2R ⁇ complex ( Sample R1115).
  • Figure 32 is the gel electrophoresis detection results of the purified disulfide-modified IL2/IL2R ⁇ complexes 5, 6, 7, 8 and complex 1 prepared in Example 14 of the present invention, and the disulfide-bond-unmodified IL2/IL2R ⁇ complexed 4 was used as a control.
  • Figure 33 is the gel electrophoresis detection results of the purified disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, 13 and complex 1 prepared in Example 14 of the present invention, and the disulfide bond is not modified IL2/IL2R ⁇ IL2R ⁇ complex 4 served as a control.
  • antigen-binding portion or "antigen-binding domain” means the portion of an antigen-binding molecule that confers its binding specificity to an antigenic determinant.
  • the "antigen-binding portion” is a functional fragment of an antibody.
  • amino acid means one of the 20 naturally occurring amino acids encoded by DNA and RNA.
  • the one-letter abbreviations for non-polar fatty acid amino acids are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine ( M); the single-letter abbreviations for aromatic amino acids are phenylalanine (F), tryptophan (W), and tyrosine (Y); the single-letter abbreviations for amino acids with uncharged R groups are serine ( S), Threonine (T), Cysteine (C), Proline (P), Aspartic acid (N), Glutamine (Q);
  • One-letter abbreviation for R-group positively charged amino acid They are lysine (K), arginine (R), histidine (H), respectively; the one-letter abbreviations for amino acids with negative charges in the R group are aspartic acid (D) and glutamic acid (E).
  • wild-type or WT means the amino acid sequence or nucleotide sequence found in nature, including allelic variation. WT proteins have amino acid sequences or nucleotide sequences that have not been intentionally modified.
  • antibody encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody or bispecific fusion polypeptide that binds a particular antigen.
  • a native intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and first, second and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region (“LCVR”) ) and a constant region (CL).
  • HCVR variable region
  • CH1, CH2, CH3, respectively first, second and third constant regions
  • LCVR variable region
  • Mammalian heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇
  • mammalian light chains can be classified as ⁇ or ⁇ .
  • Antibodies are "Y” shaped, with a backbone consisting of the second (CH2), third (CH3) and optionally fourth constant regions (CH4) of two heavy chains, which are bound by disulfide bonds.
  • Each arm of the "Y"-shaped structure contains the variable (VH) and first constant (CH1) domains of one of the heavy chains, which bind to the variable (VL) and constant (CL) domains of one of the light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light (L) chain include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy (H) chain include HCDR1, HCDR2, and HCDR3 where the three CDRs are separated by a lateral contiguous portion called the framework region (FR), which is more highly conserved than the CDRs and forms a scaffold to support the hypervariable loop.
  • the HCVR and LCVR each contain 4 FRs, and the CDRs and The FRs are arranged from the amino terminus to the carboxy terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Antibodies can be divided into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms, depending on whether they contain alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • the hypervariable region typically comprises amino acid residues 24-34 (LCDR1; “L” for light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and in the heavy chain variable region Amino acid residues about 31-35B (HCDR1; “H” for heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3); Kabat et al., "SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST), 5th ed. Bethesda, Maryland Public Health Service, National Institutes of Health, Bethesda, Md.
  • Residues eg residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53- 55 (HCDR2) and 96-101 (HCDR3); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
  • the antibody is a bispecific antibody (BiAb).
  • BiAb bispecific antibody
  • the term “bispecific” refers herein to two different antigens, or even when the two are the same antigen, each having binding specificities for different epitopes.
  • the epitopes can be derived from different antigens or the same antigen.
  • the terms "bispecific fusion polypeptide” and “bispecific antibody” are used herein to refer to all products made that have either a full-length antibody or a fragment with an antigen-binding site.
  • the antibody can be a human antibody, a non-human antibody (eg, a mouse derived antibody), a humanized antibody, or a chimeric antibody (eg, a human-mouse chimeric antibody or a chimera of different subtypes of antibodies).
  • variants of antibodies result from conservative modifications or conservative substitutions or substitutions in the antibody sequences provided herein.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Fab is a Fab fragment of an immunoglobulin that contains no or a small portion of residual Fc fragments, eg, a Fab fragment comprising the variable regions of the heavy and light chains, and all or part of the first constant domain.
  • Fab hereinafter may also refer to fragments such as F(ab)2.
  • Fc or "Fc region” or “Fc domain” means a constant region comprising an antibody, in some cases excluding all or a portion of a first constant region immunoglobulin domain (eg, CH1) or a portion thereof, and Polypeptides of all or a portion of the hinge are further excluded in some cases.
  • Fc can refer to the last two constant region immunoglobulin domains of IgA, IgD and IgG (eg CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM, and optionally these domains All or part of the N-terminal of the flexible hinge.
  • the Fc may comprise the J chain.
  • the Fc domain comprises the immunoglobulin domains CH2 and CH3 (C ⁇ 2 and C ⁇ 3) and a lower hinge region located between CH1 (C ⁇ 1) and CH2 (C ⁇ 2).
  • a human IgG heavy chain Fc region is generally defined to include residues E216, C226 or A231 at its carboxy terminus, where numbering is according to the EU index as in Kabat.
  • the Fc region is subjected to amino acid modifications, eg, the Fc is a heterodimer.
  • Modification herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence or changes in the portion chemically linked to the protein.
  • Amino acid modification herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are always amino acids encoded by DNA, eg, 20 amino acids with codons in DNA and RNA.
  • Epitope as used herein means a determinant that interacts with a particular antigen-binding domain, eg, the variable region (called a paratope) of an antibody molecule.
  • Epitopes are groupings of molecules such as amino acids or sugar side chains, and typically have specific structural characteristics as well as specific charge characteristics. A single molecule can have more than one epitope.
  • Epitopes may comprise amino acid residues that are directly involved in binding (also referred to as immunodominant components of epitopes) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; In other words, the amino acid residues are within the coverage area of the specific antigen-binding peptide.
  • Epitopes can be conformational or linear.
  • a conformational epitope results from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain.
  • Linear epitopes are epitopes that arise from adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished by loss of binding to the former but not the latter in the presence of denaturing solvents.
  • Epitopes typically include at least 3, and more typically at least 5 or 8-10 amino acids in unique spatial conformations.
  • Antigen-binding molecules that recognize the same epitope can be validated in simple immunoassays, showing the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to the target antigen.
  • the present invention includes not only the antigen-binding molecules and antigen-binding domains recited herein, but also antigen-binding molecules and antigen-binding structures that compete for binding with the epitopes bound by the recited antigen-binding molecules or antigen-binding domains area.
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to a directed ligand that can be competitively blocked by a corresponding substance in vitro or in vivo with a specific ligand.
  • the biological binding process of structural site interactions Such as the binding between an antigen and an antibody or a receptor and a ligand.
  • KD dissociation constant
  • Binding properties can be determined by methods well known in the art, such as biolayer interferometry and surface plasmon resonance-based methods.
  • One such method entails measuring the rates of association and dissociation of antigen binding site/antigen or receptor/ligand complexes, where the rates depend on the concentration of the complex partner, the affinity of the interaction, and the equivalence in both directions. Geometry parameters that affect the velocity. Therefore, the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio kd/ka is equal to the dissociation constant KD (Nature 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59: 439-473).
  • Immune cells includes cells of the immune system involved in protecting the body against both infectious diseases and foreign substances.
  • Immune cells can include, for example, neutrophils, eosinophils, basophils, lymphocytes, such as B cells and T cells, and monocytes.
  • T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, ⁇ T cells, regulatory T cells, suppressor T cells, and natural killer cells.
  • multifunctional fusion polypeptide means a non-naturally occurring binding molecule designed to target two or more antigens.
  • Multifunctional fusion polypeptides as described herein are typically genetically engineered fusion proteins designed to bring two different desired biological functions into a single binding molecule.
  • a multifunctional fusion polypeptide can be a multifunctional binding molecule.
  • FiBody refers to a bispecific fusion polypeptide or a multifunctional fusion protein obtained by replacing part or all of the constant region of a bispecific antibody with a ligand and its receptor-specific affinity.
  • the "YBody” technology mentioned in the present invention was developed by Wuhan Youzhiyou Company in 2012. This technology is based on the "Knob-into-Holes” technology, and one of the heterodimers formed is a normal heavy chain , and the other is the N-terminal linking scFv of the Fc functional region, forming an asymmetric bispecific antibody.
  • the term “about” or “approximately” means a difference of 30, 25, 20, 25, 10, 9, 8, 7, 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% quantification, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length. In certain embodiments, when the term “about” or “approximately” precedes a numerical value, it means that the stated value is plus or minus a range of 15%, 10%, 5% or 1%.
  • the words “comprising”, “including” and “comprising” will be understood to mean the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
  • Consisting of means including and limited to what is followed by the phrase “consisting of.” Thus, the phrase “consisting of” means that the listed element is required or required and no other elements may be present.
  • Consisting essentially of means that any elements listed following the phrase are included, and are limited to those that contribute to or do not interfere with the activity or effect of the listed elements as detailed in the present invention other elements. Thus, the phrase “consisting essentially of” means that the listed elements are required or required, but that other elements are optional and may depend on whether they affect the activity or function of the listed elements exist or not.
  • references throughout this disclosure to "one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “another embodiment,” or “a further embodiment” ” or a combination thereof indicates that a particular feature, structure, or characteristic described in relation to the described embodiment is included in at least one embodiment of the present invention.
  • appearances of the aforementioned terms in various places throughout this specification are not necessarily all referring to the same embodiment.
  • the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • the present invention provides novel bispecific fusion polypeptides comprising a ligand (or fragment thereof) and its receptor (or fragment thereof), said ligand (or fragment thereof) and its receptor (or fragment thereof), respectively Independently replace CH1 and CL of the Fab on one side of the antibody, specifically, the bispecific fusion polypeptide comprises a first antigen-binding portion, and the first antigen-binding portion comprises: a first polypeptide, the first polypeptide is derived from N-terminus to C-terminus comprising the first heavy chain variable domain VH1 of the first antibody operably linked to the first conjugated fragment;
  • a second polypeptide comprising, from the N-terminus to the C-terminus, the first light chain variable domain VL1 of the first antibody operably linked to the second conjugated fragment
  • the first conjugate fragment is a receptor
  • the second conjugated fragment is a ligand
  • the first conjugate fragment is a ligand
  • the second conjugated fragment is a receptor
  • the bispecific fusion polypeptide further comprises a second antigen-binding portion that is different from the first antigen-binding portion.
  • Alternative polypeptide fusion modes for the second antigen-binding moiety include selected from:
  • CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or fragment thereof) and its receptor (or fragment thereof), i.e.
  • the second antigen-binding portion comprises: a third polypeptide comprising, from the N-terminus to the C-terminus, the second heavy chain variable domain VH2 of the second antibody operably linked to a third Conjugated fragments, and
  • a fourth polypeptide comprising, from the N-terminus to the C-terminus, the second light chain variable domain VL2 of the second antibody operably linked to the fourth conjugated fragment;
  • the third conjugate fragment is a receptor and the fourth conjugate fragment is a ligand; or the third conjugate fragment is a ligand and the fourth conjugate fragment is a receptor; and
  • the third conjugate fragment and/or the fourth conjugate fragment and the first conjugate fragment and/or the second conjugate fragment are selected from different receptors and ligands.
  • the Fab on the other side of the antibody retains the original CH1 and CL, that is,
  • the second antigen binding portion comprises: a third polypeptide comprising, from the N-terminus to the C-terminus, the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain constant region CH1, and
  • a fourth polypeptide comprising, from the N-terminus to the C-terminus, the second light chain variable domain VL2 of the second antibody operably linked to the antibody light chain constant region CL.
  • the present invention utilizes the specific binding force of the ligand and its receptor itself to creatively and operably link it with the antigen binding region (antibody variable region), and the linking includes linking with one of the antigen binding regions, The other antigen-binding region is still connected to CH1 and CL; or both antigen-binding regions are connected to ligand receptors, but the two antigen-binding regions are connected to ligand receptors of different types, so as to avoid mismatching of different antigen-binding regions.
  • the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide comprising an antibody Fab comprising two different antigen-binding moieties, a first antigen-binding moiety and a second antigen-binding moiety part, characterized in that CH1 and CL on one side of the Fab are independently substituted by ligands and their receptors, and CH1 and CL on the other side of the Fab are not substituted, and the receptors both recognize and bind
  • the active site of the ligand also includes a functionally active site that produces a response; the light chain of the first antigen-binding moiety does not mispair with the heavy chain of the second antigen-binding moiety.
  • CH1 and CL on the other side of the Fab are independently replaced by a second ligand and its receptor, the first ligand and its receptor, and the second ligand and its receptor different.
  • the multifunctional fusion protein can not only exert dual target specificity, but also exert the biological activity of ligand receptor conduction.
  • the ligand and its receptors are IL15 and IL15R ⁇ , and in addition to the dual-target targeting effect of the multifunctional fusion polypeptide, IL15R ⁇ can also present IL-15 to IL-2/15R ⁇ dimer forms a ternary complex, activates JAK and STAT model pathways, promotes the proliferation and activation of target cells, and increases the secretion level of IFN- ⁇ and TNF- ⁇ ; JAK/STAT, Ras/MAPK-enhances proliferation signals ; Up-regulation of Bcl-2, Bcl-XL (anti-apoptotic protein), down-regulation of Bim, Puma (pro-apoptotic protein) -- attenuating apoptosis signal.
  • Receptor is a substance on the cell membrane or in the cell that can recognize and bind to biologically active molecules, and the biologically active substances that can bind to the receptor are collectively referred to as "ligand”.
  • receptors According to the location of receptors in cells, they are divided into two categories: cell surface receptors and intracellular receptors.
  • the receptor itself contains at least two active sites: one is the active site that recognizes and binds to the ligand; the other is the functional active site responsible for generating the response, which can only be combined with the ligand to form a binary complex and allosteric Only then can a response response be generated, thereby initiating a series of biochemical reactions, which ultimately lead to the biological effects of target cells.
  • Receptors are generally glycoproteins, and the binding between wild-type receptors and ligands is not mediated by covalent bonds, but mainly by ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. When the receptor binds to the ligand, it has the characteristics of saturation, high affinity and specificity.
  • Receptors and ligands that cooperate with each other have relatively specific binding affinities, and optionally biological effects.
  • the receptor contains only an active site that recognizes and binds a ligand, and does not contain a functionally active site that generates a response (eg, a function that activates the biological effects of downstream signaling pathways).
  • the receptor and/or ligand is a natural receptor-ligand structure, and the receptor includes both an active site that recognizes the binding ligand and a functional active site responsible for generating a response, capable of exerting
  • the bispecific fusion protein is a multifunctional fusion protein, which not only has bispecificity, but also can play ligand receptor function.
  • the receptors and/or ligands are modified on the basis of the natural sequence, the modifications include but are not limited to: truncation, insertion and/or mutation; the purpose of these modifications includes but is not limited to : increase or decrease the binding force of ligand and receptor; enhance, decrease or eliminate the biological function of ligand receptor; increase, decrease or eliminate glycosylation sites in receptor and or ligand protein; decrease or eliminate Ligand toxicity.
  • the amino acid sequences of the receptors and/or ligands each independently consist of 10-1000 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands are each independently consists of 20-800 amino acids; in some embodiments, the amino acid sequences of the receptor and/or ligand each independently consist of 30-600 amino acids; in some embodiments, the receptor and/or The amino acid sequences of the ligands each independently consist of 40-400 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands each independently consist of 50-300 amino acids; in some embodiments , the amino acid sequences of the receptors and/or ligands each independently consist of 55-260 amino acids.
  • the amino acid sequence of the receptor and/or ligand can also be independently selected from 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 amino acids.
  • the molecular weights of the receptors and/or ligands are each independently selected from 1KD to 100KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 2KD to 80KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 3KD to 70KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 60KD; In some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 50KD; in some embodiments, the molecular weights of the receptors and/or ligands are each independently selected from 4KD to 40KD; In some embodiments, the receptor and/or ligand molecular weights are each independently selected from 5KD to 30KD.
  • the molecular weight of the receptor and/or ligand can be independently selected from 1KD, 2KD, 3KD, 4KD, 4.5KD, 5KD, 6KD, 7KD, 8KD, 9KD, 10KD, 11KD, 15KD, 18KD, 20KD, 25KD, 30KD , 35KD, 40KD, 45KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD.
  • the binding mode of the receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) can be covalent binding, non-covalent interaction or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonding , hydrophobic, ionic, and van der Waals bonds.
  • the affinity between the inserted or replaced conjugated fragments when the affinity between the inserted or replaced conjugated fragments is lower than expected (eg, the two variable regions in the antigen-binding moiety cannot be brought together so that they can specifically recognize the antigen, or Failure to prevent heavy chain mismatches between 2 or more heavy chain constant regions, or failure to prevent mismatches between antigen binding moieties to achieve a combination of specific VL-VH moieties), can be achieved by targeting the antibody to the ligand and/or Or receptors are engineered to increase affinity.
  • At least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments, the non-natural interchain bond is formed between a first mutated residue contained in the receptor and a second mutated residue contained in the ligand; in some embodiments, the first and the second mutated residue At least one of the groups is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
  • At least one native glycosylation site is absent in the receptor and/or ligand.
  • the receptors and ligands are selected from interleukins and their receptors.
  • the inventors have carried out a study on the stereoscopic conformation of a large number of interleukins and their receptors, and found that a large number of interleukins or IFN-like molecules can be divided into 4 types of stereoscopic conformations: class A-toju type, class B-bowtie type , C-baseball hand type, D type-clamp type, as shown in Table 3:
  • the ligand and its receptor are selected from class A interleukins and their receptors, eg, IL15/IL15R, IL2/IL2R, IL4/IL-4R ⁇ +R ⁇ , IL-6/IL-6R , IL-11/IL-11R, IL-13/IL-13R1, IL-20/IL20R ⁇ +IL20R ⁇ , IL24/IL20R ⁇ +IL20R ⁇ .
  • class A interleukins and their receptors eg, IL15/IL15R, IL2/IL2R, IL4/IL-4R ⁇ +R ⁇ , IL-6/IL-6R , IL-11/IL-11R, IL-13/IL-13R1, IL-20/IL20R ⁇ +IL20R ⁇ , IL24/IL20R ⁇ +IL20R ⁇ .
  • the ligand and its receptor are selected from class D interleukins and their receptors, eg, IL7/IL7R, IL21/IL21R, IL23A/IL12B.
  • the interleukin and its receptor have the following amino acid sequences:
  • the receptor-ligand combination is selected from IL15 and IL15R ⁇ , and "IL15R ⁇ ” and “IL15RA” are interchangeable in the present invention.
  • the IL15 and IL15R ⁇ comprise at least one non-natural interchain bond, in some embodiments, the non-natural interchain bond is a disulfide bond, and the IL15 and/or the IL15R ⁇ comprise at least one non-natural interchain bond.
  • One amino acid is mutated to cysteine, in some embodiments, the mutation is located at the contact interface of IL-15 and IL15R ⁇ , in some embodiments, the cysteine mutation of IL15 is selected from E90C, the The cysteine mutation of IL15R ⁇ was selected from P67C.
  • the amino acid positions described in IL15 are referenced (SEQ ID NO. 26), and the amino acid positions described in IL15R ⁇ are referenced (SEQ ID NO. 27).
  • At least one native glycosylation site is absent in the IL15 or IL15R ⁇ , in some embodiments the glycosylation site is an N-glycosylation site, in some embodiments , the IL15 comprises at least one of the following amino acid mutations: N71Q, N79Q or N112Q; in some embodiments, the glycosylation site is an O glycosylation site, and in some embodiments, the IL15R ⁇ at least comprises One of the following amino acid mutations: T2A, T81A and/or T86A.
  • the receptor-ligand combination is selected from IL2 and IL2R ⁇ .
  • the IL2 and IL2R ⁇ comprise at least one non-natural interchain bond
  • the non-natural interchain bond is a disulfide bond
  • the IL2 and/or the IL2R ⁇ comprise at least one non-natural interchain bond.
  • One amino acid is mutated to cysteine
  • the S at position 75 of the IL2 is mutated to a C
  • the N-terminus of the IL2R ⁇ is extended by two or three amino acids.
  • the extended amino acid at position 2 is cysteine
  • the extended amino acid at position 1 is a non-polar fatty acid amino acid, an aromatic amino acid, or a non-polar R group. Any of a charged amino acid, a positively charged R group, or a negatively charged R group.
  • the extended amino acid at position 2 is cysteine
  • the extended amino acids at positions 1 and 3 are non-polar fatty acid amino acids, aromatic amino acids , any one of an amino acid with an uncharged R group, an amino acid with a positive charge in the R group, or an amino acid with a negative charge in the R group.
  • the amino acid position of IL2 is referenced (SEQ ID NO. 21), and the amino acid position of IL2R ⁇ is referenced to (SEQ ID NO. 22 or SEQ ID NO. 23).
  • the insertion or replacement positions of the cooperating receptor (or fragment thereof) and ligand (or fragment thereof) can be located, for example:
  • the receptor or fragment thereof is inserted or replaced in the CL region, and the ligand or fragment thereof is inserted or replaced in the CH1 region; or
  • the receptor or fragment thereof is inserted or replaced in the CH1 region, and the ligand or fragment thereof is inserted or replaced in the CL region.
  • the bispecific fusion polypeptide provided by the present invention comprises a first antigen-binding portion and a second antigen-binding portion, and has two antigen specificities.
  • the first antigen-binding portion and the second antigen-binding portion are different, and can be the first antigen.
  • the binding moiety and the second antigen-binding moiety bind to different antigens, or the first antigen-binding moiety and the second antigen-binding moiety bind to different epitopes of the same antigen.
  • the target for the bispecific fusion protein is a tumor.
  • the targets bound by the first antigen-binding moiety and the second antigen-binding moiety are both expressed on tumor cells; in some embodiments, the targets bound by the first antigen-binding moiety are on tumor cells, and the second antigen-binding moiety binds Part of the binding target is on an immune cell; in some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety bind to an immune cell.
  • T-cell redirected killing is an ideal mechanism of action in many therapeutic areas.
  • Various bispecific antibody formats have been implicated in T cell redirection in preclinical and clinical trials (May C et al (2012) Biochem Pharmacol, 84(9)): 1105-1112, pp; Frankel SR, and Baeuerle PA, (2013) CURR OPIN Chemical Biology, Vol. 17(3):385-92, pp.).
  • All T-cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen-binding sites, one of which binds to a surface antigen on the target cell and the other to a T-cell surface antigen .
  • T cell surface antigens the epsilon subunit of human CD3 derived from the TCR protein complex is most frequently targeted as a target for redirected T cell killing.
  • Tumor-associated antigens that can be targeted include, but are not limited to: alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733 , BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a- e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147,
  • T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69, and CD90.
  • Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage.
  • the targets bound by the first antigen-binding moiety and the second antigen-binding moiety are both immune checkpoints or their ligands, and the immune checkpoint molecules include but are not limited to: TIGIT, PD-1, TIM- 3.
  • the target bound by the first antigen-binding portion is PD-1, and the target bound by the second antigen-binding portion is PD-L1; in some embodiments, the target bound by the first antigen-binding portion is PD-1, the target bound by the second antigen binding moiety is TIGIT; in some embodiments, the target bound by the first antigen binding moiety is PD-1, and the target bound by the second antigen binding moiety is GTLA4; In embodiments, the target bound by the first antigen-binding portion is PD-1, and the target bound by the second antigen-binding portion is LAG3; in some embodiments, the target bound by the first antigen-binding portion is PD-1, The target bound by the second antigen binding moiety is TIM-3; in some embodiments, the target bound by the first antigen binding moiety is PD-1, and the target bound by the second antigen binding moiety is CD47; in some embodiments In some embodiments, the target bound by the first antigen-binding portion is PD-1, the
  • the first antigen binding moiety targets a tumor-associated antigen and the second antigen binding moiety targets an immune checkpoint.
  • the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets PD-1; in some embodiments, the first antigen-binding moiety targets VEGF and the second antigen-binding moiety targets PD-1 L1; in some embodiments, the first antigen binding moiety targets Claudin18.2 and the second antigen binding moiety targets PD-L1; in some embodiments, the first antigen binding moiety targets HER2 and the second antigen binding moiety targets HER2 Targets CTLA-4; in some embodiments, the first antigen-binding moiety targets CD20 and the second antigen-binding moiety targets CD47; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets HER2 Targets CD47.
  • the first antigen binding moiety and the second antigen binding moiety simultaneously target tumor heterogeneity.
  • exemplary common targets for tumors include, but are not limited to, HGF and VEGF, IGF-1R and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and FcyRIIIa, CD20 and FcyRIIIa, Her2 and FcyRIIIa.
  • the bispecific fusion polypeptides of the invention are capable of binding VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF
  • exemplary common targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNF ⁇ , IL-6 and TNF ⁇ , IL-6 and IL-1, IgE and IL-13, IL-1 and IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and FcyRIIb, and CD79 and FcyRIIb.
  • Exemplary targets for the treatment of inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and ⁇ V ⁇ 3; TNF and NGF; TNF and IL- TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A;
  • Targets involved in rheumatoid arthritis include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1 ⁇ ; TNF and MIF; TNF and IL-17 ; and TNF and IL-15.
  • Targets for the treatment of systemic lupus erythematosus include but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6S T, INHA, INHBA, KLF6, DPP4, FCER2, , R2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA
  • MS multiple sclerosis
  • MS including but not limited to: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200 , IFN ⁇ , GM-CSF, FGF, C5, CD52 and CCR2.
  • Targets for the treatment of sepsis include but are not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NF ⁇ B1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NF ⁇ B1, PTAFR, TLR2, TLR4, GPR44 , HMOX1, midkine, IRAK1, NF ⁇ B2, SERPINA1, SERPINE1, and TREM1.
  • bispecific fusion proteins of the invention antibodies against any combination of these antigens can be prepared; that is, each of these antigens can optionally and independently be included or excluded by the multispecific antibodies according to the invention .
  • the first antigen-binding portion and the second antigen-binding portion target different epitopes of the same antigen.
  • At least one of the two antigen-binding fragments may also include a secretion signal sequence.
  • a secretion signal sequence refers to a sequence that induces secretion of an expressed protein or peptide by linking to a coding sequence located outside the cell membrane or at the N-terminus outside the cell, and the signal sequence may be a peptide sequence consisting of about 18-30 amino acids. . All proteins that can be transported outside the cell membrane have distinct signal sequences that are cleaved by signal peptidases on the cell membrane. In general, for foreign proteins that are not naturally expressed by the host cell, a secretion signal sequence that enables secretion of the protein into the periplasm or into the culture medium, or a modified sequence may be employed.
  • it comprises a heavy chain constant region Fc that is a heterodimeric (heterodimeric Fc fusion protein).
  • the Fc includes but is not limited to the following combinations:
  • the introduced mutation of the Fc constant region is based on KiH technology (Knob-into-Holes), that is, an amino acid mutation is introduced into one heavy chain of the Fc constant region, and the introduced amino acid volume is larger than the original amino acid residue volume, A protruding "knob"-like structure (Knob) is formed, and another mutation is introduced in the other chain region of the Fc constant region, and the introduced amino acid volume is smaller than the original amino acid residue volume, forming a depression, a "hole”-like structure ( Hole), so that the convex heavy chain is more inclined to pair with the concave heavy chain, thereby avoiding the mispairing of the heavy chain.
  • KiH technology KiH technology
  • the introduction of mutations into the Fc constant region is based on electrostatic interactions, such as ART-lg technology, developed by Chugai, a Roche subsidiary, that specifically alters the charge of the Fc constant region domain to promote heterologous recombination.
  • the pairing of the chains is equivalent to the KiH technology of the charge version, which is described in the patent application WO2006106905, which is incorporated into the present invention in its entirety.
  • the introduction of mutations into the Fc constant region is based on SEED technology
  • SEED heterodimerization is another steric mutation-based design strategy that utilizes IgG and IgACH3 domains (also known as AG SEED CH3 Complementarity to alternating sequences derived from GASEED CH3).
  • IgG and IgA CH3 derivatives generate complementary sequences and thus preclude the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technology is described in patent application WO2007110205, which is incorporated herein in its entirety.
  • the introduction of mutation into the Fc constant region is based on the change of the isoelectric point, which is convenient for improving the rate of heterodimer formation and maintaining the stability of the Fc region.
  • This technology is described in WO2014145806, which is incorporated into this patent in its entirety. .
  • the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.
  • the Fc constant region is associated as a heterodimer based on any combination of the above techniques, eg, in some embodiments, the Fc constant region is mutated based on a combination of KIH and electrostatic interactions .
  • the XmAb bispecific platform approach can improve the thermostability of bispecific antibodies by combining electrostatic interactions, CH3 domain conformation and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutations of native IgG1 to S364K and K370S heterodimers to form hydrogen bonds between the two, followed by L368D/K370S substitutions to drive salt bridge interactions to promote heterodimers.
  • all or part of the CH2, CH3 or CH4 region is inserted or replaced with a receptor and its ligand.
  • the inserted or replaced regions are independently located in the CH2, CH3, or CH4 regions, or anywhere between adjacent regions (eg, CH1-CH2 junction, CH2-CH3 junction, CH3- CH4 junction);
  • any two of the above-mentioned constant regions eg, any of the CL-CH1, CH2-CH2, CH3-CH3, or CH4-CH4 regions
  • the two cooperating affixes of the replacement regions are inserted or replaced.
  • the value of x can be selected from 1 ⁇ 9, eg 2, 3, 4, 5, 6, 7, 8.
  • the N-terminus and/or C-terminus of the conjugated fragment is linked to the antigen-binding fragment via a linker peptide.
  • the number of amino acids of the linking peptide is 1-30; , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.
  • the amino acids of the linking peptide are nonsense polypeptides that do not have additional functions other than linking (eg, protein localization, enzyme cleavage site, etc.).
  • the linker peptide is a flexible linker peptide
  • the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.
  • the amino acid sequence of the linking peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.
  • the connecting peptide is usually flexible, which can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
  • the linker peptide is a rigid linker peptide; ie, a relatively inflexible peptide linker.
  • Rigid linker peptides do not require a complete lack of flexibility, but are less flexible than flexible linker peptides such as glycine-rich peptide linkers. Due to their relative lack of flexibility, rigid linker peptides reduce the movement of the two protein domains (in the present case stabilizer protein and thermostable reverse transcriptase) linked together by the rigid linker peptide.
  • a linker peptide that provides an ordered chain eg, an alpha helix structure
  • arginine, leucine, glutamic acid, glutamine and methionine all show relatively high propensity for helical linker structures.
  • non-helical linkers containing many proline residues can also exhibit significant rigidity.
  • rigid linking peptides include polylysine and poly-DL-alanine polylysine.
  • rigid linker peptides are described in the linker database described by George et al., Protein Engineering, 15, pp. 871-79 (2003).
  • the rigid linker peptide is also a non-cleavable linker peptide, ie a non-cleavable rigid linker peptide.
  • the present invention also relates to isolated nucleic acids encoding bispecific fusion polypeptides or multifunctional fusion proteins as described above.
  • isolated nucleic acid refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in either single- or double-stranded form.
  • isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, the polypeptides also include native polynucleotides, sugars, or base-altered analogs.
  • the polynucleotide is a light chain polynucleotide.
  • the isolated nucleic acid includes the nucleotide sequence encoding the amino acid sequence of the protein complex, as well as the nucleotide sequence complementary thereto.
  • the complementary sequences include fully complementary sequences and substantially complementary sequences, which refers to sequences that hybridize under stringent conditions known in the art to a nucleotide sequence encoding an amino acid sequence of a protein complex.
  • nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such alterations include additions, deletions, or non-conservative or conservative substitutions.
  • a polynucleotide encoding an amino acid sequence of a protein complex can be construed to include a nucleotide sequence that is substantially identical to the isolated nucleic acid. Said substantial identity aligns the nucleotide sequence with another random sequence in a manner that maximizes their correspondence, which can show greater than 80 when the aligned sequences are analyzed using algorithms common in the art % homology, greater than 90% homology, or greater than 95% homology.
  • the present invention also relates to vectors containing nucleic acids as described above.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40.
  • the vector may contain a selectable marker (e.g., a tag that facilitates enrichment, such as a his tag; or a tag that facilitates detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector then contain regulatory elements such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (e.g. transcription termination signals, or polyadenylation signals and multimerization) necessary to affect expression in a given target cell U sequence, etc.).
  • the vectors can be cloning vectors and expression vectors. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved. Prokaryotic expression vectors are commonly used PET series, pGEX series, and eukaryotic expression vectors are commonly used pcDNA3. pEGFP-N1, pSV2, etc.
  • the vector can be a composition, for example, a mixture of multiple plasmids, and different plasmids carry a part of the antibody or its fragment.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • a variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as E. coli or Bacillus stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insects Cells (such as Lepidopteran cells including Spodoptera frugiperda cells) or mammalian cells (such as Chinese Hamster Ovary (CHO) cells, NSO cells, Small Hamster Kidney (BHK) cells, monkey kidney cells, Hela cells, human hepatocellular carcinoma cells or 293 cells, etc.).
  • prokaryotic cells such as E. coli or Bacillus stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insects Cells such as Lepidopteran cells including Spodoptera frugiperda cells
  • mammalian cells such as Chinese Hamster Ovary (CHO) cells, NSO cells, Small Hamster Kid
  • bispecific fusion polypeptides or multifunctional fusion proteins of the invention can be prepared using any method known in the art.
  • Bispecific fusion polypeptides or multifunctional fusion proteins expressed in host cells are collected.
  • bispecific antibodies Early methods of constructing bispecific antibodies included chemical cross-linking or hybridoma or tetravalent tumor methods (eg, Staerz UD et al. Nature, 314:628-31, 1985; Milstein C et al. Nature, 305:537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160: 1686-1701, 1984).
  • the chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. For example the chemical conjugation of two different monoclonal antibodies, or for example the chemical conjugation of two antibody fragments such as two Fab fragments.
  • the hybridoma method is the production of bispecific monoclonal antibodies by means of cell hybridomas or ternary hybridomas that are fused by established hybridomas, or established hybridomas and derived from mice. obtained by fusion of lymphocytes. While these techniques are used to make BiAbs, various production problems make such complexes difficult to use, such as generation of mixed populations containing different combinations of antigen binding sites, difficulties in protein expression, need to purify the BiAb of interest, low yield, production High cost.
  • Recent methods utilize genetically engineered constructs that are capable of producing a homogeneous product of a single BiAb without extensive purification to remove unwanted by-products.
  • Such constructs include tandem scFvs, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as Ch1/Ck domains or DNLTM (Chames & Baty, Curr. Opin. Drug. Discov. Devel., 12:276-83, 2009; Chames & Baty, mAbs, 1:539-47).
  • Related purification techniques are well known.
  • Antibodies can also be produced using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs produced by individual lymphocytes selected for production of specific antibodies, for example by Babcook J et al, Proc. Natl. Acad. Sci. USA. 93: 7843-7848, 1996; WO 92/02551; WO 2004/051268 and methods described in WO 2004/106377.
  • Antigenic polypeptides used to generate, e.g., for immunizing hosts or for panning antibodies such as for phage display (or yeast or bacterial cell surface expression) can be derived from genetically engineered hosts comprising expression systems by methods well known in the art Cells are prepared, or they can be recovered from natural biological sources.
  • nucleic acid encoding one or both polypeptide chains of a bispecific antibody can be introduced into cultured host cells by a variety of known methods (eg, transformation, transfection, electroporation, bombardment with nucleic acid-coated microparticles, etc.).
  • the nucleic acid encoding the bispecific antibody may be inserted into a vector suitable for expression in the host cell prior to introduction into the host cell.
  • such vectors may contain sequence elements that enable expression of the inserted nucleic acid at the RNA and protein levels.
  • the bispecific antibodies of the invention can be used to detect any or all of these antigens by conventional immunological assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or histoimmunohistochemistry (eg in biological samples such as serum or plasma).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • histoimmunohistochemistry eg in biological samples such as serum or plasma.
  • the present invention provides a method for detecting an antigen in a biological sample, the method comprising: contacting the biological sample with the bispecific antibody or antibody part antigen of the present invention that can specifically recognize the antigen, and detecting the antigen-binding agent An antibody (or antibody portion), or a non-binding antibody (or antibody portion), thereby detecting the antigen in the biological sample.
  • Suitable detectable substances include various enzymes, repair groups, fluorescent substances, luminescent substances and radioactive substances.
  • suitable enzymes include, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase;
  • suitable repair group complexes include streptavidin/biotin and Avidin/biotin;
  • suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, rosin B, dichlorotriazinylamine fluorescein, Dan Sulfonyl chloride or phycoerythrin;
  • luminescent substances include 3-aminophthaloyl cyclic hydrazine;
  • suitable radioactive substances include I 125 , I 131 , 35 S or 3 H.
  • the bispecific fusion polypeptide or multifunctional fusion protein of the present invention or the nucleic acid encoding the same can be applied to the preparation of pharmaceutical compositions or sterile compositions, for example, by combining the bispecific fusion polypeptide or multifunctional fusion protein with a pharmaceutically acceptable Carriers, excipients or stabilizers are mixed.
  • the pharmaceutical composition may include one or a combination (eg, two or more different) functional fragments thereof of the antibodies of the invention.
  • a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen.
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.
  • the bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition may be in a form that is conjugated to a second activator (functional molecule).
  • the second activator can be a random functional molecule capable of preventing or treating the target disease, and can include compounds, peptides, polypeptides, nucleic acids, carbohydrates, lipids, or inorganic particles.
  • the bispecific fusion polypeptide or multifunctional fusion protein may itself have therapeutic activity; however, it may function to target the second activator to a specific disease area.
  • the disease area may be those organs, tissues, or cells in which bispecific antibodies that specifically bind to the antigen are aggregated and distributed.
  • the drug targeted to the diseased area is present in high concentrations so that the effect of the drug is increased compared to the amount injected. Therefore, the pharmaceutical composition can be used to treat drug-resistant tumors and can reduce side effects and adverse drug reactions due to non-specific drug distribution.
  • Activators comprising bispecific fusion polypeptides or multifunctional fusion proteins in pharmaceutical compositions can be contained in microcapsules, or in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles) and nanocapsules), or contained in macroemulsions, the microcapsules can be prepared by techniques such as coacervation or interfacial polymerization, examples being hydroxymethylcellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules.
  • the present invention also relates to the application of the above-mentioned bispecific fusion polypeptide or multifunctional fusion protein in the preparation of medicaments for treating diseases.
  • the disease may be, for example, cancer, immune disorders, metabolic diseases, and microbial infections.
  • cancer refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases.
  • the microorganism in a microbial infection can be an exogenous pathogen or a population of cells bearing an exogenous pathogen, such as a virus.
  • the present invention is applicable to exogenous pathogens such as bacteria, fungi, viruses, mycoplasmas and parasites.
  • Pathogens that can be treated with the present invention can be any infectious organism known in the art that is pathogenic in animals, including organisms such as Gram-negative or Gram-positive cocci or bacilli, bacteria, DNA viruses, and RNA viruses , including but not limited to DNA viruses such as papillomavirus, parvovirus, adenovirus, herpes virus, and vaccinia virus, and DNA viruses such as arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus , Paramyxovirus, Reovirus, Retrovirus and Rhabdovirus RNA virus.
  • organisms such as Gram-negative or Gram-positive cocci or bacilli, bacteria, DNA viruses, and RNA viruses , including but not limited to DNA viruses such as papillomavirus, parvovirus, adenovirus, herpes virus, and vaccinia virus, and DNA viruses such as arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus
  • antibiotic-resistant bacteria such as antibiotic-resistant Streptococcus species and Staphlococcus species, or those that are susceptible to antibiotics but cause recurrent infections treated with antibiotics that eventually develop resistant organisms bacteria.
  • antibiotics can be treated with the ligand-immunogen conjugates of the invention in combination with lower doses of antibiotics than would normally be administered to patients to avoid the development of these antibiotic-resistant bacterial strains.
  • the present invention also applies to any fungi, mycoplasma species, parasites or other infectious organisms that cause disease in animals.
  • the present invention can be used to treat parasitic infections, including but not limited to infections caused by the following parasites: Taenia solium, Schistosoma, Ascaris, Amoeba and Plasmodium, Trypanosoma, Leishmania Protozoa (Leishmania) and Toxoplasma (Toxoplasma) species.
  • Parasites of particular interest are those that express folate receptors and bind folate; however, there are numerous references in the literature for ligands that exhibit high affinity for infectious organisms.
  • penicillins and cephalosporins which are known for their antibiotic activity and which bind specifically to bacterial cell wall precursors, can likewise be used as ligands for the preparation of ligand-immunogen conjugates for use in accordance with the present invention.
  • the ligand-immunogen conjugates of the present invention can also be directed against a population of cells bearing endogenous pathogens, wherein the pathogen-specific antigen is preferentially expressed on the surface of cells bearing the pathogen, and serves as a specific antigen for the antigen.
  • Receptors for sexually bound ligands can be directed against a population of cells bearing endogenous pathogens, wherein the pathogen-specific antigen is preferentially expressed on the surface of cells bearing the pathogen, and serves as a specific antigen for the antigen.
  • the present invention also relates to a method of preventing and/or treating and administering a therapeutically effective amount of a pharmaceutical composition to prevent and/or treat the diseases as described above.
  • the methods of the present invention can be used in human clinical medicine and veterinary applications.
  • the host animal that carries the population of pathogenic organisms and is treated with the ligand-immunogen conjugate may be a human, or in the case of veterinary applications, a laboratory, agricultural, domestic or wild animal.
  • the present invention may be applicable to host animals including, but not limited to: humans; laboratory animals, such as rodents (eg, mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; domesticated animals, such as dogs, cats, and rabbits ; agricultural animals such as cattle, horses, pigs, sheep, goats; and captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.
  • rodents eg, mice, rats, hamsters, etc.
  • rabbits, monkeys, chimpanzees chimpanzees
  • domesticated animals such as dogs, cats, and rabbits
  • agricultural animals such as cattle, horses, pigs, sheep, goats
  • captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.
  • compositions can be injected into entities including rats, mice, domestic animals, and/or humans by various routes. All injection methods are contemplated, eg, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or topical injections are possible. The composition can be injected by other methods known in the art.
  • a “therapeutically effective amount” as used herein refers to an amount sufficient to treat a disease based on a reasonable benefit-to-loss ratio.
  • a therapeutically effective amount can vary depending on the patient for a variety of reasons, such as the type of disease, severity, onset, age of the entity, weight, rate of excretion, susceptibility to reaction, health status, and/or complications; and/or drug activity, injection route, injection cycle and number of injections, and/or drug combination; can also be appropriately selected by those of ordinary skill in the art according to the purpose of treatment.
  • the injection amount can be divided randomly into multiples such that the amount is about 0.001-100 mg/kg adult body weight.
  • Bispecific fusion polypeptides or multifunctional fusion proteins of the invention or nucleic acids or polynucleotides encoding antibodies of the invention can also be administered, for example, in combination with standard cancer treatments (eg, surgery, radiation, and chemotherapy).
  • standard cancer treatments eg, surgery, radiation, and chemotherapy
  • antitumor therapy using the compositions of the invention and/or effector cells equipped with these compositions is used in combination with chemotherapy.
  • Non-limiting examples of antibody combination treatments of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and combinations thereof.
  • the FiBody is a recombinantly obtained bispecific antibody that utilizes the specific affinity between the ligand and its receptor to replace CL and CH1 on one side of the bispecific antibody, which can avoid or reduce the bispecific antibody Mismatch between light and heavy chains.
  • interleukins and their receptors are used as examples to construct FiBody, and they are divided into four categories according to the three-dimensional conformation of interleukins and their receptors, as shown in Figure 5:
  • Bispecific antibodies were constructed based on the above four types of interleukins and their receptors.
  • the VH targeting the primary antibody is selected to be linked to the receptor protein through Linker, and then linked to the Fc of the antibody through Hinge; the VL targeting the primary antibody is linked to the ligand protein through Linker to reduce or avoid light chain and heavy
  • the chain is mismatched; the other end is the complete Fab structure targeting the second antibody (anti-TIGIT antibody), and the Fc constituting the first antibody and the Fc constituting the second antibody have conventional KiH remodeling to reduce or avoid heavy chain errors. match. or
  • the VH targeting the first antibody is selected to be linked to the ligand protein through Linker, and then linked to the Fc of the antibody through Hinge; the VL targeting the second antibody is linked to the receptor protein (IL15) through Linker to reduce or avoid light
  • the chain is mismatched with the heavy chain; the other end is the complete Fab structure targeting the first antibody, and the Fc constituting the first antibody and the Fc constituting the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatch.
  • the bispecific antibody is modified with disulfide bonds, as shown in Figure 8.
  • Ligand receptor disulfide bond transformation Select the VH targeting the secondary antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through Linker, and then connect to the Fc of the antibody through Hinge; the VL targeting the secondary antibody is linked through Linker It is linked to the ligand protein (IL15); the other end is the complete Fab structure targeting the first antibody.
  • the Fc of the first antibody and the Fc of the second antibody have conventional KiH transformation to avoid heavy chain mismatch.
  • the receptor and ligand proteins are mutated to form intermolecular disulfide bonds and further improve the stability of the molecule, for example:
  • Disulfide bond engineering positions include, but are not limited to, the following mutation sites:
  • Fab-IL15/IL15RA_Fc specifically selects the VH targeting the second antibody (anti-TIGIT) to connect to the receptor protein (IL15RA) through Linker, and then connects to the Fc of the antibody through Hinge; the VL targeting the second antibody Linked to the ligand protein (IL15) through Linker; the other end is the complete Fab structure targeting the first antibody, the Fc of the first antibody and the Fc of the second antibody have conventional KiH transformation to avoid heavy chain mismatch .
  • the receptor and ligand proteins are mutated to form intermolecular disulfide bonds and further improve the stability of the molecule; further, the glycosylation sites on the receptor and ligand proteins are modified to eliminate the Molecular heterogeneity, such as:
  • IL15/IL15RA and its constituent bispecific antibodies in order to avoid the interaction of IL15/IL15RA and its constituent bispecific antibodies with the IL2/15R ⁇ / ⁇ C complex, causing unwanted nonspecific binding, we investigated IL15/IL15RA and its constituent bispecifics. Antibodies were engineered to reduce or completely lose the affinity of IL15/IL15RA for the IL2/15R ⁇ / ⁇ C complex.
  • the VH targeting the second antibody is connected to the receptor protein (IL15RA) through Linker, and then connected to the Fc of the antibody through Hinge; VL is connected to the ligand protein (IL15) through Linker; the other end is the complete Fab structure targeting the first antibody.
  • the Fc of the first antibody and the Fc of the second antibody have conventional KiH transformation to avoid heavy chain errors. match.
  • the purpose of mutating the ligand protein is to reduce or inactivate the biological function of the receptor and ligand complex, such as:
  • the VH of the second antibody is specifically selected to be connected to the VL of the second antibody through Linker to form an scFv structure, and then connected to the Fc of the antibody through Hinge; the other end is the targeting
  • the complete Fab structure of the primary antibody (this structure is similar to Wuhan Youzhiyou Y-Body, named as YBody), the Fc of the primary antibody and the Fc of the secondary antibody have conventional KiH transformation to avoid heavy chain errors. match. For example:
  • VH targeting the second antibody is linked to the VL of the second antibody through Linker to form an scFv structure, and then the linker is connected with the complete Fc targeting the first antibody.
  • the C-terminal connection of ; forms a symmetrical structure.
  • the VH targeting the second antibody is specifically selected to connect to the CL domain, and then connected to the Fc of the antibody through Hinge, and the VL targeting the second antibody (anti-TIGIT) is connected to
  • the CH1 domain forms the light chain; the other end is the complete Fab structure targeting the first antibody, and the Fc constituting the first antibody and the Fc constituting the second antibody have conventional KiH modifications to avoid heavy chain mismatch.
  • VH targeting the first antibody is linked to the receptor protein (IL15RA) through Linker, and then linked to the Fc of the antibody through Hinge; targeting the second antibody (anti-TIGIT antibody) VL is connected to the ligand protein (IL15) through Linker; the other end is the VH targeting the second antibody is connected to CH1 through a conventional sequence, and then connected to the Fc of the antibody through Hinge, and the VL targeting the first antibody is connected through a conventional sequence.
  • the two Fcs have conventional KiH reengineering to avoid heavy chain mismatches. For example:
  • Molecule number R1042 PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL ( Figure 10 left) first polypeptide @PD-L1_VH_IL15RA_Fc-Knob (SEQ ID NO.1) second polypeptide @TIGIT_VL_IL15(SEQ ID NO.6) third polypeptide @TIGIT_VH_CH1_Fc-Hole(SEQ ID NO.3) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.8)
  • the VH targeting the second antibody is linked to the receptor protein (IL15RA) through Linker, and then linked to the Fc of the antibody through Hinge; the VH targeting the first antibody (anti-PD-L1 antibody)
  • the VL is connected to the ligand protein (IL15) through Linker; the other end is the VH targeting the first antibody is connected to CH1 through a conventional sequence, and then connected to the Fc of the antibody through Hinge, and the VL targeting the second antibody is connected through a conventional sequence.
  • the two Fcs have conventional KiH reengineering to avoid heavy chain mismatches.
  • Molecule number R1043 PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-LI-VL first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO: 5) second polypeptide @PD-L1_VL_IL15 (SEQ ID NO: 2) third polypeptide @PD-L1_VH_CH1_Fc-Hole(SEQ ID NO.7) Fourth polypeptide @TIGIT_VL_CL(SEQ ID NO.4)
  • VH targeting the secondary antibody to connect to the receptor protein (IL21R) through Linker, and then connect to the Fc of the antibody through Hinge
  • the VL targeting the primary antibody is linked through Linker Connected to the ligand protein (IL21); the other end is the VL targeting the second antibody (anti-PD-L1 antibody) connected to CL, and the VH of the targeting structure first antibody (anti-TIGIT antibody) is connected to CH1, It is then connected to the Fc of the antibody through Hinge, and the Fc at both ends has conventional KiH transformation.
  • a bispecific antibody with disulfide bond engineering (disulfide bond engineered IL2/IL2R ⁇ complex): the heavy chain of an antibody targeting tumor cells or immune cell surface antigens (eg TIGIT antibody)
  • the variable region (VH) is linked to the IL2R ⁇ mutant through Linker, and then linked to the Fc of hIgG1 antibody through Hinge (hinge region), and the light chain variable region (VL) of the antibody targeting the same antigen (TIGIT antibody) is linked through Linker.
  • Linked to IL2 mutants antibodies targeting immune cells or tumor cell surface antigens (eg PD-L1 antibodies) have VHs directly linked to the constant region of hIgG1 antibodies (hlgG1), antibodies targeting the same antigen (PD-L1 antibodies)
  • the VL of IL-2 is directly linked to human immunoglobulin light chain kappa antibody ( ⁇ -IgLC) to prepare a targeted disulfide bond to transform IL2/IL2R ⁇ complex.
  • the inventors designed two disulfide bond-engineered IL2/IL2R ⁇ complexes, namely disulfide bond-engineered IL2/IL2R ⁇ complex 1 and disulfide bond-engineered IL2/IL2R ⁇ complex 2; and IL2/IL2R ⁇ complex 3 is the compound of the existing report, and the compound 4 is the IL2/IL2R ⁇ complex without disulfide bond modification;
  • the specific composition and amino acid sequence of the compound 1, 2, 3, and 4 are as follows:
  • sequence structures of disulfide-engineered IL2/IL2R ⁇ complex 1 are: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant, PD-L1 VH-hIgG1 and PD-L1 VL- ⁇ -IgLC
  • the amino acid sequence of TIGIT VH-IL2R ⁇ mutant-Fc is as shown in SEQ ID NO.77
  • the amino acid sequence of TIGIT VL-IL2 mutant is as shown in SEQ ID NO.78
  • the amino acid sequence of PD-L1 VH-hIgG1 As shown in SEQ ID NO.79
  • the amino acid sequence of PD-L1 VL- ⁇ -IgLC is shown in SEQ ID NO.80.
  • the sequence structure of disulfide bond-engineered IL2/IL2R ⁇ complex 2 is: TIGIT VH-IL2 mutant-Fc, TIGIT VL-IL2R ⁇ mutant, PD-L1 VH-hIgG1 and PD-L1 VL- ⁇ -IgLC; among them, TIGIT
  • the amino acid sequence of VH-IL2 mutant-Fc is shown in SEQ ID NO. 81
  • the amino acid sequence of TIGIT VL-IL2R ⁇ mutant is shown in SEQ ID NO. 82
  • the amino acid sequence of PD-L1 VH-hIgG1 is shown in SEQ ID NO. 79
  • the amino acid sequence of PD-L1 VL- ⁇ -IgLC is shown in SEQ ID NO.80.
  • the sequence structure of IL2/IL2R ⁇ complex 3 is: TIGIT VH-IL2R ⁇ (L42C)-Fc, TIGIT VL-IL2(F42C), PD-L1VH-hIgG1 and PD-L1 VL- ⁇ -IgLC; among them, TIGITVH-IL2R ⁇ (
  • the amino acid sequence of L42C)-Fc is shown in SEQ ID NO.83
  • the amino acid sequence of TIGITVL-IL2(F42C) is shown in SEQ ID NO.84
  • the amino acid sequence of PD-L1 VH-hIgG1 is shown in SEQ ID NO.79 shown
  • the amino acid sequence of PD-L1 VL- ⁇ -IgLC is shown in SEQ ID NO.
  • the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. During the intracellular period, the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130 rpm, and cells were counted before transfection. 2E6 cells were inoculated into 1 L shake flasks, and the culture system was about 300 ml.
  • the transiently transfected cell expression solution was centrifuged at 9000 rpm/20 min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter. Purification was performed using ProA affinity chromatography. The process is as follows, using AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV of equilibration buffer (10mM PBS), load the sample onto the chromatography column, and allow the target protein to adsorb on the chromatography column. Other impurities penetrate and separate.
  • equilibration buffer 10mM PBS
  • neutralization buffer 1M Tris, pH 8.0
  • Embodiment 10 FiBody physical and chemical detection
  • HPLC-SEC detection results of sample R0951 are shown in Figure 11
  • HPLC-SEC detection results of sample R1042 are shown in Figure 12
  • HPLC-SEC detection results of sample R0809 are shown in Figure 13
  • HPLC-SEC detection results of sample R1110 are shown in Figure 13.
  • the detection results of SEC are shown in Figure 14.
  • bispecific antibodies with mispaired forms were also expressed and had levels similar to normal molecules, but with significantly lower purity.
  • HPLC-SEC detection results of R1262 are shown in Figure 15, the results show that: the free light chain is successfully removed, and the disulfide bond modified IL2/IL2R ⁇ complex 1 prepared by the present invention has high purity.
  • the binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay.
  • R0950, R0951, R0952, R0954, R0955, R0960, R1123/R1119/R1120/R1124, R1042/R1043, R0810 are shown in Figures 16-19; unexpectedly, R0950 (@TIGIT at the Fab end) has low binding activity R0951 ⁇ R0960 (@TIGIT at IL15/IL15R end); R0951 ⁇ R0960 binding activity is similar to that of positive control R0226 (Tigit monoclonal antibody, OMP-313R12, WO2016191643); Class A and Class D interleukins and their receptors are replaced After CH1 and CL, the binding force of the targeting region was not affected.
  • Disulfide bond engineering optimized samples R1081, R1085 and glycosylation samples modified molecules were comparable to the tigit-end affinities of the molecules before modification.
  • R1042, R1043 and R1124 are mismatched test molecules, and their TIGIT binding activity is significantly reduced; R0810 is a ScFv structure molecule, and its binding activity is also weaker than that of the control molecule R0226.
  • R1262 The results of R1262 are shown in Figure 20. The results show that the affinity of the disulfide-modified IL2/IL2R ⁇ complex 1 (R1262) is comparable to that of the unmodified IL2/IL2R ⁇ complex (R1115), indicating that the disulfide-modified IL2/IL2R ⁇ complex will not Affects the affinity of the targeting region.
  • the binding activity of double antibody molecules (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay.
  • R0802 is (PD-L1 monoclonal antibody, 176F9, VH sequence is shown in SEQ ID NO: 36, VL sequence is shown in SEQ ID NO: 37), R0514 is (PD-L1 monoclonal antibody, Avelumab), R0919 is ( PD-L1 monoclonal antibody, VH is shown in SEQ ID NO: 75, VL sequence is shown in SEQ ID NO: 76), R0968 is (PD-L1 monoclonal antibody, VH is shown in SEQ ID NO: 71) , the VL sequence is shown in SEQ ID NO: 72).
  • R1262 The results of R1262 are shown in Figure 26.
  • the results show that the affinity of the disulfide-modified IL2/IL2R ⁇ complex 1 (R1262) is comparable to that of the non-disulfide-modified IL2/IL2R ⁇ complex 4 double antibody (R1115). , indicating that disulfide bond modification does not affect the affinity of the targeting region.
  • the binding activity of the double antibody molecule (TIGIT end) blocking ligand to CHO-TIGIT cells was detected by FCM assay.
  • the detection results of R1262 are shown in Figure 27.
  • the results show that: the disulfide bond-modified IL2/IL2R ⁇ complex 1 (R1262) is compared with the non-disulfide bond-modified IL2/IL2R ⁇ complex 4 (R1115).
  • the blocking effect of the binding force to the region is comparable.
  • Antibody dilution use FACS buffer to dilute all molecules to an initial concentration of 400nM, a volume of 180 ⁇ l, 3-fold serial dilution (60+120), 10 concentrations; cell counting and plating: R0255-2 (CHO-mTigit)/293T-IL15R -28 cells were centrifuged at 250g for 5min, the supernatant was discarded, the cell density was adjusted to 2E+06 with FACS buffer, and 100 ⁇ L/tube were evenly distributed into 96-well V-plates; the above diluted antibody was added to the cells, 100 ⁇ L/ Well, incubate at 2-8 degrees for 0.5h; take out the 96-well plate, centrifuge at 250g for 5min, carefully remove the supernatant, add 200 ⁇ L/well of FACS buffer, centrifuge again at 250g for 5min, carefully remove the supernatant; prepare PE fluorescent secondary antibody with FACS buffer (1:500 dilution), add 100 ⁇ L/well to the corresponding 96-well plate,
  • R1042, R1043 and R0951 have comparable activities, indicating that no mismatch is produced, and even the wrong Fv can be expressed; wherein R0655 is (IL15/IL15RFc fusion protein, see SEQ ID NO: 38)
  • the samples (R1072, R1081, R1082, R0954, R1084-R1086) optimized for disulfide bond modification in the examples of the present invention were detected by SDS-PAGE electrophoresis.
  • the results are shown in Figure 30.
  • the R1072 molecule without disulfide bond modification has a band between the molecular weights of 25KD and 35KD, indicating that there is a free light chain;
  • the ligand receptor disulfide bond modified molecules are R0954, R1085, and R1086, of which R0954 , R1086 electrophoresis results show that there are still non-covalent light chains (bands between 25KD and 35KD), and no non-covalent light chains in R1085 (no bands between 25KD and 35KD), indicating that the disulfide bond of R1085 was successfully transformed. .
  • the light and heavy chain disulfide bond modified molecules are R1081, R1082 and R1084.
  • the electrophoresis results show that there is no non-covalent light chain (no band between 25KD and 35KD), indicating that the disulfide bond modification of R1081, R1082 and R1084 is successful.
  • Example 9 the disulfide-bonded IL2/IL2R ⁇ complexes 1 and 2 and IL2/IL2R ⁇ complex 3 constructed in Example 8) and R1115 in Example 2.
  • R1115 is an unmodified molecule with disulfide bonds, and there is a band between the molecular weights of 25KD and 35KD, indicating the existence of free light chains; the disulfide bond modified IL2/IL2R ⁇ complexes 1 and 2 have a molecular weight of There is no band between 25KD and 35KD, which is the same as that of IL2/IL2R ⁇ complex 3, indicating that the free light chain was successfully removed and the disulfide bond was successfully transformed.
  • Example 14 Influence of the types of amino acids at positions 1 and 3 of the N-terminal extension of the amino acid sequence of IL2R ⁇ mutants on the formation of disulfide bonds introduced after IL2/IL2R ⁇ complex engineering
  • the N-terminus of the amino acid sequence of the IL2R ⁇ mutant is extended by three amino acids;
  • the effects of the types of amino acids at position 3 and position 3 on the formation of disulfide bonds introduced after the transformation of IL2/IL2R ⁇ complexes were carried out as follows:
  • the amino acid at position 3 to be extended is an aromatic amino acid (take phenylalanine as an example), an amino acid with an uncharged R group (take serine as an example), an amino acid with a positive charge in the R group (take lysine as an example) or Any one of the negatively charged amino acids in the R base (take aspartic acid as an example), construct and prepare disulfide bonds to transform IL2/IL2R ⁇ complexes 5, 6, 7, and 8 (referred to as R1493, R1494, R1495, R1496), together with disulfide bond engineering IL2/IL2R ⁇ complex 1 (the 3rd amino acid of the N-terminal extension of the amino acid sequence of IL2R ⁇ mutant in IL2/IL2R ⁇ complex 1 is glycine (non-polar fatty acid amino acid)) as an experiment group, the IL2/IL2R ⁇ complex 4 without disulfide bond modification was used as the control group.
  • the disulfide-engineered IL2/IL2R ⁇ complexes 5, 6, 7, and 8 were prepared in the same process as the disulfide-engineered IL2/IL2R ⁇ complex 1 in Example 8.
  • sequence structures of disulfide bond-engineered IL2/IL2R ⁇ complexes 5, 6, 7, and 8 are: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant (SEQ ID NO: 78), PDL1 VH-hIgG1 ( SEQ ID NO: 79) and PDL1 VL- ⁇ -IgLC (SEQ ID NO: 80); wherein, the amino acid sequences of TIGITVH-IL2R ⁇ mutant-Fc of disulfide bond engineered IL2/IL2R ⁇ complexes 5, 6, 7, 8 As shown in sequence SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 (wherein, the three amino acids extended from the N-terminus of the amino acid sequence of the IL2R ⁇ mutant are shown in bold, Underlined is the extended 3rd amino acid);
  • the obtained disulfide bond-modified IL2/IL2R ⁇ complexes 5, 6, 7, 8 and complex 1 were detected by SDS-PAGE electrophoresis, and the disulfide bond-unmodified IL2/IL2R ⁇ complex 4 was used as the control group to explore the interleukin
  • the effect of the type of amino acid at position 3 extending from the N-terminal of the amino acid sequence of the 2 receptor ⁇ mutant on the formation of disulfide bonds introduced after the transformation of the IL2/IL2R ⁇ complex is shown in Figure 32.
  • the disulfide bond-modified IL2/IL2R ⁇ complexes 5, 6, 7, and 8 all successfully removed the free light chain, and the disulfide bond was successfully modified; it showed that the extended third amino acid was a non-polar fatty acid amino acid, aromatic Family amino acids, uncharged R group amino acids, R group positively charged amino acids or R group negatively charged amino acids will not affect the formation of disulfide bonds introduced after IL2/IL2R ⁇ complex transformation.
  • the first and third amino acids of the extension are selected as non-polar fatty acid amino acids (take glycine as an example), aromatic amino acids (take phenylalanine as an example), and amino acids with uncharged R groups (take serine as an example) , any combination in the positively charged amino acid of R group (take lysine as an example) or the amino acid with negative charge of R group (take aspartic acid as an example), construct and prepare disulfide bond to transform IL2/IL2R ⁇ complex 9, 10, 11, 12, 13 (referred to as R1662, R1663, R1664, R1665, R1666 in sequence), together with the disulfide bond-modified IL2/IL2R ⁇ complex 1 as the experimental group, with the disulfide bond unmodified IL2/IL2R ⁇ complex Item 4 was the control group.
  • the disulfide-engineered IL2/IL2R ⁇ complexes 9, 10, 11, 12, and 13 were prepared in the same manner as the disulfide-engineered IL2/IL2R ⁇ complex 1 in Example 8.
  • sequence structures of disulfide bond-engineered IL2/IL2R ⁇ complexes 9, 10, 11, 12, and 13 are: TIGIT VH-IL2R ⁇ mutant-Fc, TIGIT VL-IL2 mutant (SEQ ID NO: 78), PDL1 VH- hIgG1 (SEQ ID NO: 79) and PDL1 VL- ⁇ -IgLC (SEQ ID NO: 80); wherein, TIGIT VH-IL2R ⁇ mutants of disulfide-engineered IL2/IL2R ⁇ complexes 9, 10, 11, 12, 13 -The amino acid sequence of Fc is shown in sequence SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 (wherein, interleukin 2 receptor alpha mutant The three amino acids extended from the N-terminus of the amino acid sequence are shown in bold, and the extended amino acids 1 and 3 are underlined);
  • the obtained disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, 13 and complex 1 were detected by SDS-PAGE electrophoresis, and the disulfide bond-unmodified IL2/IL2R ⁇ complex 4 was used as a control group to explore leukocytes.
  • the effect of the combination of amino acids at positions 1 and 3 of the N-terminal extension of the amino acid sequence of IL-2 receptor ⁇ mutants on the formation of disulfide bonds introduced after the transformation of the IL2/IL2R ⁇ complex is shown in Figure 33.
  • the disulfide bond-modified IL2/IL2R ⁇ complexes 9, 10, 11, 12, and 13 all successfully removed the free light chain, and the disulfide bond was successfully modified; indicating that the extended first position Any combination of the amino acid at position 3 being a non-polar fatty acid amino acid, an aromatic amino acid, an amino acid with an uncharged R group, a positively charged amino acid in the R group, or a negatively charged amino acid in the R group will not affect the IL2/IL2R ⁇ complex. Formation of disulfide bonds introduced after transformation.
  • SEQ ID NO: 37 @PD-L1:VL (SEQ ID NO: 37)

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Abstract

涉及生物医药领域，具体而言，涉及一种双特异性融合多肽。该双特异性融合多肽包括抗原结合部分，其包含第一抗原结合部分，所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段，和第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，所述第一缀合片段和所述第二缀合物片段能够特异性结合；其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。

Description

双特异性多功能融合多肽
相关申请的交叉引用
本公开要求于2021年4月22日提交中国专利局的申请号为“202110436970.6”名称为“抗体或其功能片段”、2021年7月30日提交中国专利局的申请号为“202110871320.4”名称为“抗体或其功能片段”、2021年9月24日提交中国专利局的申请号为“202111121937.0”名称为“双特异性多功能融合多肽”、2022年3月10日提交中国专利局的申请号为“202210240917.3”名称为“双特异性多功能融合多肽”的共4件中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本发明涉及生物医药领域，具体而言，涉及一种包含配体和受体的双特异性融合多肽和/多功能融合多肽。
背景技术
为了拓宽蛋白类药物的临床疗效，全球生物技术科研工作者一直在努力开发不同结构、类型的生物大分子药物。双特异性抗体是目前临床上最热门的新型生物大分子药物结构。双特异性抗体(Bispecific Antibodies，BsAb)是指可以同时结合两个不同抗原或一个抗原不同表位的抗体，可通过特有的作用方式，发挥单抗无法实现的生物学功能。
随着重组蛋白表达和基因工程技术的进步，双特异性抗体形式越来越多样化，截止目前，已有超过20种的双特异性抗体形式开发成技术平台。
在IgG型双抗的组装过程中，两条天然重链和两条天然轻链可随机产生10种可能的组合，其中只有一种是目标双抗产物。10种不同双抗产物的生化特性类似，从中分离出目标双抗的难度极大，导致目标双抗产率低、纯度低，增加成本影响疗效。
双抗技术平台的核心价值在于解决重链和重链错配、轻链和重链错配的问题，解决重链与重链错配的技术平台主要有：Knob-into-Holes(KiH)、ART-Ig、链交换工程结构域(SEED)技术、XmAb。
解决重链与轻链错配的技术平台主要有：
1.共同轻链技术：由Genentech于2006年开发，从针对多种抗原的噬菌体展示筛选中发现的抗体通常共享相同的VL结构域，这反映了噬菌体文库中轻链库的大小非常有限。公共轻链策略的使用简化了工业生产中的抗体工程和纯化过程。优势：解决了LC/HC和HC/HC错配的问题。不足：需要在噬菌体展示筛选中发现抗体共享的VL，难度较大，专利申请WO2008027236。
2.CrossMab技术：2007年由罗氏研发，通过交换Fab片段的重链和轻链结构域序列来解决轻链问题，通常有三种方式实现：交换VH和VL，交换CH1和CL或交换VH和VL以及CH1和CL，优势：同时结合KiH结构可以解决轻重链错配的问题，保留了原始抗原的亲和力，不足：表达量比较低，专利申请WO2009080251。
解决重链与重链错配的技术已相对成熟，解决轻链与重链错配的技术仍有改进空间，有鉴于此，特提出本发明。
发明内容
本发明旨在一定程度上解决相关技术中的技术问题之一。
发明人提出了一种新的双特异性抗体开发思路，利用配体和受体的特异性亲合力，替换抗体或其功能片段中的CH1和CL，从而避免或减少重链和轻链发生错配；进一步地，所述替换可同时或独立地选自CH2、CH3以及任选地CH4，从而促进重链异二聚体地形成。另一方面，本发明提出地双特异性抗体是一种多功能融合蛋白，所述多功能融合蛋白不仅能发挥双靶点特异性，且能发挥配受体传导地生物学活性。
在一个方面，本发明提供一种双特异性融合多肽，其包含第一抗原结合部分，所述第一抗原结合部分包含：
第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段，和
第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
所述第一缀合片段和所述第二缀合物片段能够特异性结合；
其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。
在一些实施方式中，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
所述第二抗原结合部分包括：
第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
其中，所述第三缀合片段和所述第四缀合物片段能够特异性结合；所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体。
在一些实施方式中，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
所述第二抗原结合部分包括：
第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
在一些实施方式中，所述受体仅包含识别并结合配体的活性部位，不包含产生应答反应的功能活性部位。
在一些实施方式中，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；在一些实施方式中，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间；在一些实施方式中，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基；在一些实施方式中，所述非天然链间键为二硫键。
在一些实施方式中，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
在一些实施方式中，所述受体及其配体选自白细胞介素及其受体。
在一些实施方式中，所述白细胞介素及其受体立体构像为托举型，选自IL15/IL15R、IL2/IL2R、IL4/IL-4Rα+Rγ、IL-6/IL-6R、IL-11/IL-11R、IL-13/IL-13R1、IL-20/IL20Rα+IL20Rβ和/或IL24/IL20Rα+IL20Rβ。
在一些实施方式中，所述白细胞介素及其受体立体构像为钳型，选自IL7/IL7R、IL21/IL21R、IL23A/IL12B。在一些实施方式中，所述配体和受体选自IL15和IL15Rα。
在一些实施方式中，所述IL15第90位的E突变为C，且所述IL15Rα第67位的P突变位C。
在一些实施方式中，所述第一重链可变结构域VH1与第一轻链可变结构域VL1之间存在非天然二硫键，优选地，第一重链可变结构域VH1与第一轻链可变结构域VL1包含以下任一突变组合：
组合 VH VL
1 37C 95C
2 44C 100C
3 44C 101C
4 44C 105C
5 45C 87C
6 45C 98C
7 100C 50C
8 100bC 49C
9 98C 46C
10 101C 46C
11 105C 43C
12 106C 57C
13 108C 43C
在一些实施方式中，所述IL15第61位的D突变为N，第64位的E突变为Q，和/或第65位的N突变位D。
在一些实施方式中，所述IL15至少一个N糖基化位点不存在，优选地，所述N糖基化位点选自N71、N79和/或N112；优选地，所述IL15包含以下氨基酸突变：N71Q、N79Q和/或N112Q。
在一些实施方式中，所述IL15Rα至少一个O糖基化位点不存在，优选地，所述O糖基化位点选自T2、T81和/或T86；优选地，所述IL15Rα包含以下氨基酸突变：T2A、T81A和/或T86A。
在一些实施方式中，所述配体和受体选自IL2和IL2Rα。
在一些实施方式中，所述IL2第75位的S突变为C，且所述IL2Rα的N端延伸两个或三个氨基酸。
优选地，所述IL2Rα的N端延伸两个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种。
优选地，所述IL2Rα的N端延伸三个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位和第3位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种。
在一些实施方式中，所述双特异性融合多肽包含抗体Fc恒定区；在一些实施方式中，所述抗体Fc恒定区是异源二聚体；在一些实施方式中，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体；在一些实施方式中，所述抗体Fc恒定区包含CH2、CH3以及任选的CH4，所述CH2、CH3和/或任选的CH4被替换成所述受体及其配体。
在一些实施方式中，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；在一些实施方式中，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；在一些实施方式中，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；在一些实施方式中，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞。在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够衔接T细胞和肿瘤抗原；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够衔接NK细胞和肿瘤抗原；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够协同抑制信号通路；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够形成蛋白复合物。
本发明还涉及分离的核酸，其编码如上所述的双特异性融合多肽。
本发明还涉及含有如上所述核酸的载体。
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。
本发明还涉及制备双特异性融合多肽的方法，包括：
用如上所述的载体转化宿主细胞；
培养所转化的宿主细胞；和
收集宿主细胞中表达的双特异性融合多肽。
本发明还涉及药物组合物，其包含如上所述的双特异性融合多肽，和药学上可接受的载体，赋形剂，或稳定剂。
本发明还涉及如上所述的双特异性融合多肽在制备用于治疗疾病的药物中的应用。
附图说明
图1为4种经典的双抗平台：图1A为KiH异源二聚Fc改造技术；图1B为CrossMab双特异性抗体技术；图1C为武汉友芝友YBody双抗技术(非对称型scFv双抗)；图1D为对称型scFv双抗；
图2为本发明提供的一种新型双特性抗体FiBody，由具有特异性亲和力的配受体替换一侧Fab的CH1、CL；
图3为示例性的展示FiBody的4中可行方案：图3-1为改造的配受体，配受体之间具有非天然存在的链间键；图3-2为两侧Fab的CH1、CL均被受体、配体取代，两侧选自不同的配受体；图3-3为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH3段也被配受体替换；图3-4为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH2也被配受体替换；其他的可行性改造方式还有很多；
图4为示例性的当本发明的双特异性抗体用于治疗肿瘤时，双特异性抗体的抗原结合部分的靶向结合包括示例性的3种类型：图4-A第一抗原结合部分靶向T细胞，第二抗原结合部分靶向肿瘤细胞；图4-B第一抗原结合部分和第二抗原结合部分均靶向肿瘤细胞；图4-C第一抗原结合部分与第二抗原结合部分均靶向T细胞；图4-D示例性的体现本发明双特性抗体可选的为三功能融合蛋白，除发挥不同的抗原结合，还能激活配受体通路，激发配受体生物学活性；
图5为白细胞介素及其受体的立体构像图，可以分为四类：A类为托举型，B类为蝴蝶结型，C类为棒球手型，D类为钳型；
图6为四类立体构像的白介素及其受体的举例，A类托举型为IL2/IL2R，B类蝴蝶结型为IL22/IL22R，C类蝴蝶结型为IL18/IL18R，D类钳型为IL21/IL21R；
图7为本发明实施例中基于IL15(配体)与IL15RA(受体)FiBody设计，第二抗原结合区VH与IL15RA相连，第二抗原结合区VL与IL15相连；
图8为二硫键改造优化结构示意图；
图9为IL15/IL15RA与IL2/15Rβ/γC复合物相互作用立体结构示意图；
图10为本发明实施例中错配分子R1042/R1124结构示意图；
图11为本发明实施例中样品R0951的HPLC-SEC检测结果；
图12为本发明实施例中样品R1042的HPLC-SEC检测结果；
图13为本发明实施例中样品R0809的HPLC-SEC检测结果；
图14为本发明实施例中样品R1110的HPLC-SEC检测结果；
图15为本发明实施例中样品R1262的HPLC-SEC检测结果。
图16为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；
图17为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1123/R1119/R1120/R1124)；
图18为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1042/R1043)；
图19为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R0810)；
图20为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1262)；其中，hIgG1为亚型对照抗体。
图21为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；
图22为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1115-R1120、R1123-R1124)；
图23为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R0950、R1042、R1043)；
图24为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1081-R1086)；
图25为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1072、R1109-R1111)；
图26为本发明实施例中FCM法检测双抗PD-L1端与CHO-PD-L1细胞结合活性(R1262)；
图27为本发明实施例中R1262对配体与靶向区结合力的阻断检测结果；
图28为本发明实施例中样品受配体复合物(IL15/IL15R)的结合活性(R0950、R0951、R0952、R0954、R0955、R0960)；
图29为本发明实施例中样品受配体复合物(IL15/IL15R)的结合活性(R1042、R1043)；
图30为本发明实施例中二硫键改造优化样品凝胶电泳检测结果(R1072、R1081、R1082、R0954、R1084-R1086)；
图31为本发明实施例9制备得到的FiBody样品(实施例8对应构建的二硫键改造IL2/IL2Rα复合物1和2、IL2/IL2Rα复合物3)和实施例2的R1115的凝胶电泳检测结果；其中，复合物1和2为二硫键改造IL2/IL2Rα复合物，复合物3为现有报道的IL2/IL2Rα复合物，复合物4为二硫键未改造IL2/IL2Rα复合物(样品R1115)。
图32为本发明实施例14中制备的纯化后的二硫键改造IL2/IL2Rα复合物5、6、7、8以及复合物1的凝胶电泳检测结果，二硫键未改造IL2/IL2Rα复合物4作为对照。
图33为本发明实施例14中制备的纯化后的二硫键改造IL2/IL2Rα复合物9、10、11、12、13以及复合物1的凝胶电泳检测结果，二硫键未改造IL2/IL2Rα复合物4作为对照。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。在本发明中引用的所有文献，包括公开出版物、专利和专利申请，都通过引用的方式全文并入本文。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
术语解释：
术语“抗原结合部分”或“抗原结合结构域”意指对抗原决定簇赋予其结合特异性的抗原结合分子的部分。在一些实施方案中，所述“抗原结合部分”为抗体功能片段。
术语“氨基酸”意指由DNA和RNA编码的20个天然存在的氨基酸之一。
非极性脂肪酸氨基酸的单字母缩写分别为甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、异亮氨酸(I)、甲硫氨酸(M)；芳香族氨基酸的单字母缩写分别为苯丙氨酸(F)、色氨酸(W)、酪氨酸(Y)；R基不带电荷的氨基酸的单字母缩写分别为是丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、脯氨酸(P)、天冬氨酸(N)、谷氨酰胺(Q)；R基带正电荷的氨基酸的单字母缩写分别为赖氨酸(K)、精氨酸(R)、组氨酸(H)；R基带负电荷的氨基酸的单字母缩写分别为天冬氨酸(D)、谷氨酸(E)。
术语“野生型或WT”意指在自然界发现的氨基酸序列或核苷酸序列，包含等位变异。WT蛋白质具有未经过有意修饰的氨基酸序列或核苷酸序列。
术语“抗体”涵盖任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体、双特异性(双价)抗体或双特异性融合多肽。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一个 可变区(“HCVR”)以及第一、第二和第三恒定区(分别为CH1、CH2、CH3)组成，而每条轻链由一个可变区(“LCVR”)以及一个恒定区(CL)组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。
抗体呈“Y”型，主干由两条重链的第二(CH2)、第三(CH3)以及任选地第四恒定区(CH4)组成，其通过二硫键结合。“Y”型结构的每条臂包含其中一条重链的可变区(VH)和第一恒定区(CH1)，其与一条轻链的可变区(VL)和恒定区(CL)结合。轻链和重链的可变区负责抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)(轻(L)链的CDR包含LCDR1、LCDR2、LCDR3，重(H)链的CDR包含HCDR1、HCDR2、HCDR3。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。HCVR和LCVR各包含4个FR，并且CDR和FR自氨基端至羧基端依以下顺序排列：FRI、CDR1、FR2、CDR2、FR3、CDR3、FR4。
重链和轻链的恒定区不参与抗原结合，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异形体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
高变区通常包含来自轻链可变区中约氨基酸残基24-34(LCDR1；“L”表示轻链)、50-56(LCDR2)和89-97(LCDR3)以及重链可变区中约31-35B(HCDR1；“H”表示重链)、50-65(HCDR2)和95-102(HCDR3)的氨基酸残基；Kabat等人，《免疫学相关蛋白质的序列(SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST)》，第5版.马里兰州贝塞斯达美国国家卫生研究院公共卫生服务部(Public Health Service，National Institutes of Health，Bethesda，Md.)(1991)和/或那些形成高变环的残基(例如轻链可变区中的残基26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)以及重链可变区中的26-32(HCDR1)、53-55(HCDR2)和96-101(HCDR3)；Chothia和Lesk(1987)《分子生物学杂志(J.Mol.Biol.)》196：901-917。
在一些实施方式中，所述抗体为双特异抗体(BiAb)。术语“双特异性”在本文中是指两种不同抗原，或甚至当这两者是相同抗原时，它们每一个都具有针对不同表位的结合特异性。所述表位可以源自不同抗原或相同抗原。术语“双特异性融合多肽”和“双特异性抗体”在本文中是指所有制得的具有全长抗体或带抗原结合位点的片段的产物。所述抗体可以是人抗体，非人抗体(如小鼠来源抗体)，人源化抗体，或嵌合抗体(如人-小鼠嵌合抗体或不同亚型抗体的嵌合)。在一些情况下，抗体的变体是在本发明所提供的抗体序列上发生保守修饰或保守置换或取代所得到的。“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。所属领域技术人员将能够使用熟知的技术确定如本文所阐明的抗原结合分子的合适变体。对于核苷酸和氨基酸序列，术语“同一性”表明当具有适当的插入或缺失的情况下最佳比对和比较时两个核酸或两个氨基酸序列之间的同一性程度。
术语“Fab”为免疫球蛋白中不含或含一小部分残余Fc片段的Fab片段，例如，Fab片断包括重链和轻链的可变区、以及所有或部分的第一恒定域。出于简单，后文中的术语“Fab”也可指诸如F(ab)2这样的片段。
术语“Fc”或“Fc区”或“Fc结构域”意指包含抗体的恒定区，在一些情况下排除第一恒定区免疫球蛋白结构域(例如CH1)或其一部分的全部或一部分，并且在一些情况下进一步排除铰链的全部或一部分的多肽。因此，Fc可指IgA、IgD和IgG的最后两个恒定区免疫球蛋白结构域(例如CH2和CH3)，IgE和IgM的最后三个恒定区免疫球蛋白结构域，以及任选地这些结构域的柔性铰链N端的全部或一部分。对于IgA和IgM，Fc可以包含J链。对于IgG来说，Fc结构域包含免疫球蛋白结构域CH2和CH3(Cγ2和Cγ3)以及位于CH1(Cγ1)与CH2(Cγ2)之间的较低铰链区。尽管Fc区的边界可以变化，但是人类IgG重链Fc区通常被定义为包括其羧基端的残基E216、C226或A231，其中编号根据如Kabat中的EU索引。在一些实施方案中，如下文更全面地描述，对Fc区进行氨基酸修饰，例如所述Fc为异源二聚体。
本文的“修饰”是指多肽序列中的氨基酸取代、插入和/或缺失或与蛋白质化学连接的部分的改变。本文的“氨基酸修饰”是指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起见，除非另外指出，否则氨基酸修饰始终是由DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20个氨基酸。
“表位”在本文中意指与特定抗原结合结构域，例如抗体分子的可变区(称为互补位)相互作用的决定子。表位是例如氨基酸或糖侧链的分子的分组，并且通常具有特定的结构特征以及特定的电荷特征。单个分子可具有超过一个表位。表位可以包含直接参与结合的氨基酸残基(也称为表位的免疫显性组分)和不直接参与结合的其它氨基酸残基，例如被特异性抗原结合肽有效阻断的氨基酸残基；换句话说，氨基酸残基在特异性抗原结合肽的覆盖面积内。表位可以是构形的也可以是线性的。构形表位是由来自线性多肽链的不同 区段的氨基酸空间并置而产生。线性表位是由多肽链中的相邻氨基酸残基产生的表位。构形和非构形表位的区别可以在于在变性溶剂存在下，与前者而非后者的结合丧失。表位通常包括独特空间构象中的至少3个，并且更通常至少5个或8-10个氨基酸。识别相同表位的抗原结合分子可以在简单的免疫分析中验证，显示一种抗原结合分子阻断另一种抗原结合分子与靶抗原结合的能力。如下所概述，本发明不仅包括本文中所列举的抗原结合分子和抗原结合结构域，还包括与所列举的抗原结合分子或抗原结合结构域结合的表位竞争结合的抗原结合分子和抗原结合结构域。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指有指向的、能被相应物质竞争阻断的某种配基在体外或体内与特异结构位点相互作用的生物结合过程。如抗原和抗体或受体和配体之间的结合。
特异性结合的强度或亲和力可以根据相互作用的解离常数(KD)表示，其中较小的KD表示较大的亲和力，并且较大的KD表示较低的亲和力。例如KD为至少约10 -4M、至少约10 -5M、至少约10 -6M、至少约10 -7M、至少约10 -8M、至少约10 -9M、替代地至少约10 -10M、至少约10 -11M、至少约10 -12M、或更大的抗原结合力来展现。结合特性可以通过所属领域众所周知的方法，例如生物层干涉测量法和基于表面等离振子共振的方法来确定。一种这样的方法需要测量抗原结合位点/抗原或受体/配体复合物缔合和解离的速率，其中速率取决于复合物搭配物的浓度、相互作用的亲和力以及在两个方向上同等地影响速率的几何参数。因此，可以确定缔合速率(ka)和解离速率(kd)，并且kd/ka的比例等于解离常数KD(《自然(Nature)》361：186-187(1993)和Davies等人(1990)《生物化学年鉴(Annual RevBiochem)》59：439-473)。
术语“免疫细胞”包括参与保护机体抵抗传染性疾病和外来物质二者的免疫系统的细胞。免疫细胞可以包括例如嗜中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，淋巴细胞，如B细胞和T细胞，和单核细胞。T细胞可以包括例如，CD4+、CD8+、T辅助细胞、细胞毒性T细胞、γδT细胞、调节性T细胞、抑制性T细胞和天然杀伤细胞。
术语“多功能融合多肽”意指设计来靶向两个或更多个抗原的非天然存在的结合分子。本文所述的“多功能融合多肽”通常是遗传工程化的融合蛋白，其经设计以将两个不同的所需的生物学功能带入单个结合分子。例如，多功能融合多肽可以是多功能结合分子。
术语“FiBody”，是利用配体及其受体特异性亲和力取代双特异性抗体部分或全部恒定区，从而得到的双特异性融合多肽或多功能融合蛋白。
本发明中提到的“YBody”技术由武汉友芝友公司于2012年开发，该技术是在“Knob-into-Holes”技术的基础上，形成异源二聚体的其中一条为正常重链，另外一条为Fc功能区的N端链接scFv，形成了不对称的双特异性抗体。
术语“约”或“大约”是指与参照定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度相差30、25、20、25、10、9、8、7、6、5、4、3、2或1％的定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度。在特定实施方式中，当术语“约”或“大约”位于数值之前时，表示所述值加上或减去15％、10％、5％或1％的范围。
除非上下文另有规定，词语“包含”、“包括”和“含有”将被理解为表示包括所述的步骤或要素或一组步骤或要素，但不排除任何其他步骤或要素或一组步骤或要素。“由......组成”所表示的是包括并且限于短语“由......组成”所接的内容。因此，短语“由......组成”表示所列出的要素是需要的或必需的，并且没有其他要素可存在。“基本由......组成”所表示的是包括列于此短语之后的任意要素，并且限于有助于或不妨碍所列的要素的如在本发明中详述的活性或作用的其他要素。因此，短语“基本由......组成”表示所列出的要素是需要的或必需的，但其他要素是可选地并可取决于其是否影响所列出的要素的活性或作用而存在或不存在。
在本发明全文中提及的“一个实施方式”、“实施方式”、“特定实施方式”、“相关实施方式”、“某种实施方式”、“另外的实施方式”或“进一步的实施方式”或其组合表示所描述的与所述实施方式相关的特定特征、结构或特性包含于本发明的至少一个实施方式中。因此，在本说明书全文各处出现前述用语未必都指同一实施方式。此外，所述特定特征、结构或特性可在一个或多个实施方式中以任意适宜方式组合。
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。
在说明书和权利要求中的术语“第一”、“第二”用于区分相似元素，而不一定用于描述顺序或时间次序。应当理解，如此使用的术语在合适环境下是可互换的，并且本文描述的本发明的实施方案能够以与本文描述或举例说明不同的其他顺序操作。
双特异性融合多肽
本发明提供了新的双特异性融合多肽，其包含配体(或其片段)及其受体(或其片段)，所述配体(或其片段)及其受体(或其片段)分别独立地替换抗体一侧Fab的CH1和CL，具体地，所述双特异性融合多肽包含第一抗原结合部分，所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包 含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段；
第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。
所述双特异性融合多肽还包含第二抗原结合部分，所述第二抗原结合部分与第一抗原结合部分不同。对于第二抗原结合部分可选的多肽融合方式包括选自：
1.抗体另一侧Fab的CH1和CL被另一种配体(或其片段)及其受体(或其片段)替换，即
所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体。
2.抗体另一侧Fab保留原来的CH1和CL，即，
所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
本发明利用配体及其受体本身特有的特异性结合力，将其创造性地与抗原结合区(抗体可变区)可操作性地连接，所述连接包括与其中之一抗原结合区连接，另一抗原结合区仍与CH1和CL连接；或者两种抗原结合区都与配受体连接，只是两种抗原结合区连接不同种类地配受体，从而避免不同抗原结合区发生错配。
在一些实施方式中，本发明提供的双特异性融合多肽是一种多功能融合多肽，其包含抗体Fab，所述Fab包含两种不同的抗原结合部分，第一抗原结合部分和第二抗原结合部分，其特征在于，所述Fab一侧的CH1和CL独立地被配体及其受体所取代，所述Fab的另一侧CH1和CL未被取代，所述受体既包含识别并结合配体的活性部位，也包含产生应答反应的功能活性部位；所述第一抗原结合部分的轻链不会与所述第二抗原结合部分的重链错配。在一些实施方式中，所述Fab的另一侧CH1和CL独立地被第二配体及其受体取代，所述第一配体及其受体与所述第二配体及其受体不同。
所述多功能融合蛋白不仅能发挥双靶点特异性，且能发挥配受体传导地生物学活性。例如，在某个特定的实施方式中，所述配体及其受体为IL15和IL15Rα，所述多功能融合多肽除具有双靶点靶向作用外，IL15Rα还能将IL-15递呈给IL-2/15Rβγ二聚体形成三元复合物，激活JAK和STAT型号通路，促进靶细胞增殖与活化、IFN-γ、TNF-α分泌水平提升；JAK/STAT，Ras/MAPK-增强增殖信号；Bcl-2、Bcl-XL(抗凋亡蛋白)的上调、Bim、Puma(促凋亡蛋白)的下调--减弱凋亡信号。
i.配体和受体
“受体(receptor)”是细胞膜上或细胞内能识别生物活性分子并与之结合的物质，能与受体结合的生物活性物质统称为“配体(ligand)”。
根据受体在细胞中的位置，将其分为细胞表面受体和细胞内受体两大类。受体本身至少含有两个活性部位：一个是识别并结合配体的活性部位；另一个是负责产生应答反应的功能活性部位，这一部位只有在与配体结合形成二元复合物并变构后才能产生应答反应，由此启动一系列的生化反应，最终导致靶细胞产生生物效应。
受体一般为糖蛋白，野生型受体与配体之间的结合不通过共价键介导，主要靠离子键、氢键、范德华力和疏水作用而相互结合。受体在与配体结合时，具有饱和性、高亲和性、专一性等特性。
互相配合的受体和配体具有相对特异结合的亲和力，以及任选的生物学效应。在一些实施方式中，所述受体仅包含识别并结合配体的活性部位，不包含产生应答反应的功能活性部位(例如激活下游信号通路的生物学效应的功能)。在一些实施方式中，所述受体和/或配体为天然的受配体结构，所述受体既包含识别结合配体的活性部位，又包含负责产生应答反应的功能活性部位，能够发挥相应的生物学功能，所述双特异性融合蛋白是一种多功能融合蛋白，不仅具有双特异性，而且能发挥配受体功能。
在一些实施方式中，所述受体和/或配体在天然序列的基础上做了修饰，所述修饰包括但不限于：截短、插入和/或突变；这些修饰的目的包括但不限于：增加或降低配体和受体的结合力；增强、降低或消除配体受体的生物学功能；增加、减少或消除受体和或配体蛋白中的糖基化位点；降低或消除受配体毒性。
在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由10～1000个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由20～800个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由30～600个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由40～400个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由50～300个氨基酸组成；在一些实施方式中，所述受体和/或配体的氨基酸序列各自独立地由55～260个氨基酸组成。例如，受体和/或配体的氨基酸序列也可以独立地选自20、30、40、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900个氨基酸。
在一些实施方式中，所述受体和/或配体分子量各自独立地选自1KD～100KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自2KD～80KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自3KD～70KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～60KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～50KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自4KD～40KD；在一些实施方式中，所述受体和/或配体分子量各自独立地选自5KD～30KD。例如，受体和/或配体的分子量可以独立地选自1KD、2KD、3KD、4KD、4.5KD、5KD、6KD、7KD、8KD、9KD、10KD、11KD、15KD、18KD、20KD、25KD、30KD、35KD、40KD、45KD、50KD、60KD、70KD、80KD、90KD、100KD。
受体(或其片段)和其相应的配体(或其片段)的结合方式可以是共价结合、非共价相互作用或其组合；非共价键的例子包括，但不限于，氢键、疏水键、离子键、和范德华键。在一些实施方式中，当被插入或替换的缀合片段之间的亲和力低于预期时(例如不能拉近抗原结合部分中的两个可变区以使其获得特异性识别抗原的功能，或者不能防止2个或多个重链恒定区之间的重链错配，或者不能防止抗原结合部分之间错配以实现特定VL-VH部分的组合)，可以通过对抗体所述配体和/或受体进行改造以增加亲和力。在一些实施方式中，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；在一些实施方式中，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间；在一些实施方式中，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基；在一些实施方式中，所述非天然链间键为二硫键。
在一些实施方式中，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
在一些实施方式中，所述受体和配体选自白细胞介素及其受体。
发明人对大量的白细胞介素及其受体进行了立体构像研究，发现大量的白细胞介素或IFN类分子立体构像可以分为4类：A类-托举型、B类-蝴蝶结型、C-棒球手型、D类-钳型，如表3所示：
表3
Figure PCTCN2022088198-appb-000001
Figure PCTCN2022088198-appb-000002
在一些实施例中，所述配体及其受体选自A类白细胞介素及其受体，例如IL15/IL15R、IL2/IL2R、IL4/IL-4Rα+Rγ、IL-6/IL-6R、IL-11/IL-11R、IL-13/IL-13R1、IL-20/IL20Rα+IL20Rβ、IL24/IL20Rα+IL20Rβ。
在一些实施例中，所述配体及其受体选自D类白细胞介素及其受体，例如IL7/IL7R、IL21/IL21R、IL23A/IL12B。
在一些实施方式中，所述白细胞介素及其受体具有如下表氨基酸序列：
Figure PCTCN2022088198-appb-000003
在一些实施方式中，所述受体-配体的组合选自IL15与IL15Rα，在本发明“IL15Rα”和“IL15RA”可以互换。
在一些实施方式中，所述IL15和IL15Rα包含至少一个非天然的链间键，在一些实施方式中，所述非天然链间键为二硫键，所述IL15和/或所述IL15Rα至少包含一个氨基酸突变为半胱氨酸，在一些实施方式中，所述突变位于IL-15和IL15Rα的接触界面上，在一些实施方式中，所述IL15的半胱氨酸突变选自E90C，所述IL15Rα的半胱氨酸突变选自P67C。IL15所述氨基酸位置参照(SEQ ID NO.26)，IL15Rα所述氨基酸 位置参照(SEQ ID NO.27)。
在一些实施方式中，至少一个天然糖基化位点在所述IL15或IL15Rα中不存在，在一些实施方式中，所述糖基化位点为N糖基化位点，在一些实施方式中，所述IL15至少包含以下之一氨基酸突变：N71Q，N79Q或N112Q；在一些实施方式中，所述糖基化位点为O糖基化位点，在一些实施方式中，所述IL15Rα至少包含以下之一氨基酸突变：T2A、T81A和/或T86A。
在一些实施方式中，所述受体-配体的组合选自IL2与IL2Rα。
在一些实施方式中，所述IL2和IL2Rα包含至少一个非天然的链间键，在一些实施方式中，所述非天然链间键为二硫键，所述IL2和/或所述IL2Rα至少包含一个氨基酸突变为半胱氨酸，在一些实施方式中，所述IL2第75位的S突变为C，且所述IL2Rα的N端延伸两个或三个氨基酸。
在一些实施方式中，所述IL2Rα的N端延伸两个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种。
在一些实施方式中，所述IL2Rα的N端延伸三个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位和第3位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种。IL2所述氨基酸位置参照(SEQ ID NO.21)，IL2Rα所述氨基酸位置参照(SEQ ID NO.22或SEQ ID NO.23)。
互相配合的受体(或其片段)以及配体(或其片段)的插入或替换位置可以位于，例如：
受体或其片段插入或替换CL区，配体或其片段插入或替换CH1区；或
受体或其片段插入或替换CH1区，配体或其片段插入或替换CL区。
ii.抗原结合部分
本发明提供的双特异性融合多肽，包含第一抗原结合部分和第二抗原结合部分，具有两种抗原特异性，第一抗原结合部分与第二抗原结合部分是不同的，可以是第一抗原结合部分与第二抗原结合部分结合不同的抗原，也可以是第一抗原结合部分与第二抗原结合部分结合相同抗原的不同表位。
在一些实施方式中，所述双特异性融合蛋白针对的靶标是肿瘤。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在肿瘤细胞表达；在一些实施方式中，第一抗原结合部分结合的靶点在肿瘤细胞，第二抗原结合部分结合的靶点在免疫细胞；在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在免疫细胞。
T细胞重定向杀伤是许多治疗领域中理想的作用机制。在临床前和临床试验中，各种双特异性抗体形式参与T细胞重定向(May C等人(2012)Biochem Pharmacol，84(9))：1105年至1112年，第；弗兰克尔SR，和Baeuerle PA，(2013)CURR OPIN化学生物学，第17卷(3)：385-92，页)。所有T细胞重新靶向的双特异性抗体或其片段已被工程化以具有至少两个抗原结合位点，其中一个位点与靶细胞上的表面抗原结合另一个位点与T细胞表面抗原结合。在T细胞表面抗原中，源自TCR蛋白质复合物的人CD3的ε亚基最常被靶向作为重定向T细胞杀伤的靶标。
可被靶向的肿瘤相关联抗原包括但不限于：α-胎蛋白(AFP)、α-辅肌动蛋白-4、A3、对A33抗体有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、碳酸酐酶IX、CASP-8/m、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA-4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p(CSAp)、CEA(CEACAM5)、CEACAM6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、成纤维细胞生长因子(FGF)、Flt-1、Flt-3、叶酸盐受体、G250抗原、Claudin18.2、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜BCMA促性腺激素(HCG)和其亚基、HER2/neu、HMGB-1、缺氧诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、PAM4抗原、胰腺癌粘蛋白、PD-1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、P1GF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素、存活素-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、Thomson-Friedenreich抗原、肿瘤坏死抗原、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bcl-2、bcl-6、Kras、致癌基因标记物以及致癌基因产物(参见，例如Sensi等人，Clin Cancer Res2006，12：5023-32；Pamiani 等人，JImmunol2007，178：1975-79；Novellino等人Cancer Immunol Immunother2005，54：187-207)。
虽然对于效应T细胞具有特异性的抗体或其它结合分子优选地结合至CD3抗原，但是在效应T细胞上表达的其它抗原是已知的并且可由T-细胞重定向复合物靶向。示例性T-细胞抗原包括但不限于，CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69和CD90。
免疫检查点是免疫系统中的抑制途径，其对维持自身耐受性和调节外周组织中生理性免疫应答的持续时间和幅度以使附带组织损伤最小化至关重要。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点均为免疫检查点或其配体，所述免疫检查点分子包括但不限于：TIGIT、PD-1、TIM-3、LAG3、GTLA4、BTLA、BTN1A1、VISTA、LAIR、CD96、PVRIG、LILRA3、LILRA4、LILRB1、LILRB2、LILRB3、LLRB4、NKG-2A、CD47、CD200R1、CD300、Dectin-1、ICOS、NKp30、CD28、CD28H、CRTAM、DNAM-1、4-1-BB、BAFF、CD27、CD30、CD40、DR3、GITR、HVEM、LIGHT、OX40、TACI、2B4、CD2、CD48、CD229、SLAM、SLAMF5、GRAAC、TIM1、TIM4、CD7、DPPIV。
在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为PD-L1；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIGIT；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为LAG3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIM-3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为CD47；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原结合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为TIGIT。
在一些实施方式中，第一抗原结合部分靶向肿瘤相关抗原，第二抗原结合部分靶向免疫检查点。在一些实施方式中，第一抗原结合部分靶向HER2，第二抗原结合部分靶向PD-1；在一些实施方式中，第一抗原结合部分靶向VEGF，第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向Claudin18.2，第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向HER2，第二抗原结合部分靶向CTLA-4；在一些实施方式中，第一抗原结合部分靶向CD20，第二抗原结合部分靶向CD47；在一些实施方式中，第一抗原结合部分靶向HER2，第二抗原结合部分靶向CD47。
在一些实施方式中，第一抗原结合部分和第二抗原结合部分同时靶向肿瘤异质性。用于肿瘤的示例性共同靶标包括但不限于HGF和VEGF，IGF-1R和VEGF，Her2和VEGF，CD19和CD3，CD20和CD3，Her2和CD3，CD19和FcγRIIIa，CD20和FcγRIIIa，Her2和FcγRIIIa。本发明的双特异性融合多肽能够结合VEGF和磷脂酰丝氨酸；VEGF和ErbB3；VEGF和PLGF；VEGF和ROBO4；VEGF和BSG2；VEGF和CDCP1；VEGF和ANPEP；VEGF和c-MET；HER-2和ERB3；HER-2和BSG2；HER-2和CDCP1；HER-2和ANPEP；EGFR和CD64；EGFR和BSG2；EGFR和CDCP1；EGFR和ANPEP；IGF1R和PDGFR；IGF1R和VEGF；IGF1R和CD20；CD20和CD74；CD20和CD30；CD20和DR4；CD20和VEGFR2；CD20和CD52；CD20和CD4；HGF和c-MET；HGF和NRP1；HGF和磷脂酰丝氨酸；ErbB3和IGF1R；ErbB3和IGF1，2；c-Met和Her-2；c-Met和NRP1；c-Met和IGF1R；IGF1，2和PDGFR；IGF1，2和CD20；IGF1，2和IGF1R；IGF2和EGFR；IGF2和HER2；IGF2和CD20；IGF2和VEGF；IGF2和IGF1R；IGF1和IGF2；PDGFRa和VEGFR2；PDGFRa和PLGF；PDGFRa和VEGF；PDGFRa和c-Met；PDGFRa和EGFR；PDGFRb和VEGFR2；PDGFRb和c-Met；PDGFRb和EGFR；RON和c-Met；RON和MTSP1；RON和MSP；RON和CDCP1；VGFR1和PLGF；VGFR1和RON；VGFR1和EGFR；VEGFR2和PLGF；VEGFR2和NRP1；VEGFR2和RON；VEGFR2和DLL4；VEGFR2和EGFR；VEGFR2和ROBO4；VEGFR2和CD55；LPA和S1P；EPHB2和RON；CTLA4和VEGF；CD3和EPCAM；CD40和IL6；CD40和IGF；CD40和CD56；CD40和CD70；CD40和VEGFR1；CD40和DR5；CD40和DR4；CD40和APRIL；CD40和BCMA；CD40和RANKL；CD28和MAPG；CD80和CD40；CD80和CD30；CD80和CD33；CD80和CD74；CD80和CD2；CD80和CD3；CD80和CD19；CD80和CD4；CD80和CD52；CD80和VEGF；CD80和DR5；CD80和VEGFR2；CD22和CD20；CD22和CD80；CD22和CD40；CD22和CD23；CD22和CD33；CD22和CD74；CD22和CD19；CD22和DR5；CD22和DR4；CD22和VEGF；CD22和CD52；CD30和CD20；CD30和CD22；CD30和CD23；CD30和CD40；CD30和VEGF；CD30和CD74；CD30和CD19；CD30和DR5；CD30和DR4；CD30和VEGFR2；CD30和CD52；CD30和CD4；CD138和RANKL；CD33和FTL3；CD33和VEGF；CD33和VEGFR2；CD33和CD44；CD33和DR4；CD33和DR5；DR4和CD137；DR4和IGF1，2；DR4和IGF1R；DR4和DR5；DR5和CD40；DR5和CD137；DR5和CD20；DR5和EGFR；DR5和IGF1，2；DR5和IGFR，DR5和HER-2，以及EGFR和DLL4。其他靶标组合包括EGF/erb-2/erb-3家族的一个或多个成员。
此外，用于自身免疫病症和炎性病症的示例性共同靶标包括但不限于IL-1和TNFα，IL-6和TNFα，IL-6和IL-1，IgE和IL-13，IL-1和IL-13，IL-4和IL-13，IL-5和IL-13，IL-9和IL-13，CD19和FcγRIIb，以及CD79和FcγRIIb。
用于治疗炎性疾病的示例性靶点包括但不限于：TNF和IL-17A；TNF和RANKL；TNF和VEGF；TNF和SOST；TNF和DKK；TNF和αVβ3；TNF和NGF；TNF和IL-23p19；TNF和IL-6；TNF和SOST；TNF和IL-6R；TNF和CD-20；IgE和IL-13；IL-13和IL23p19；IgE和IL-4；IgE和IL-9；IgE和IL-9；IgE和IL-13；IL-13和IL-9；IL-13和IL-4；IL-13和IL-9；IL-13和IL-9；IL-13和IL-4；IL-13和IL-23p19；IL-13和IL-9；IL-6R和VEGF；IL-6R和IL-17A；IL-6R和RANKL；IL-17A和IL-1β；IL-1β和RANKL；IL-1β和VEGF；RANKL和CD-20；IL-1α和IL-1β；IL-1α和IL-1β。
参与类风湿性关节炎(RA)的靶点包括但不限于：TNF和IL-18；TNF和IL-12；TNF和IL-23；TNF和IL-1β；TNF和MIF；TNF和IL-17；和TNF和IL-15。
治疗系统性红斑狼疮(SLE)的靶点包括但不限于：CD20，CD22，CD19，CD28，CD4，CD24，CD37，CD38，CD40，CD69，CD72，CD74，CD79A，CD79B，CD80，CD81，CD83，CD86，IL-4，IL-6，IL10，IL2，IL4，IL11，TNFRSF5，TNFRSF6，TNFRSF8，C5，TNFRSF7，TNFSF5，TNFSF6，TNFSF7，BLR1，HDAC4，HDAC5，HDAC7A，HDAC9，ICOSL，IGBP1，MS4A1，RGSI，SLA2，IFNB1，AICDA，BLNK，GALNAC4S-6S T，INHA，INHBA，KLF6，DPP4，FCER2，，R2，ILIR2，ITGA2，ITGA3，MS4A1，ST6GALI，CDIC，CHSTIO，HLA-A，HLA-DRA，NT5E，CTLA4，B7.1，B7.2，BlyS，BAFF，IFN-α和TNF-α。
用于治疗多发性硬化症(MS)的靶点，包括但不限于：IL-12，TWEAK，IL-23，CXCL13，CD40，CD40L，IL-18，VEGF，VLA-4，TNF，CD45RB，CD200，IFNγ，GM-CSF，FGF，C5，CD52和CCR2。
用于治疗脓毒症的靶点包括但不限于：TNF，IL-1，MIF，IL-6，IL-8，IL-18，IL-12，IL-10，IL-23，FasL，LPS，Toll-样受体，TLR-4，组织因子，MIP-2，ADORA2A，IL-1B，CASP1，CASP4，NFκB1，PROC，TNFRSFIA，CSF3，CCR3，ILIRN，MIF，NFκB1，PTAFR，TLR2，TLR4，GPR44，HMOX1，中期因子，IRAK1，NFκB2，SERPINA1，SERPINE1，和TREM1。
为了形成本发明的双特异性融合蛋白，可以制备针对这些抗原的任意组合的抗体；即，这些抗原中的每一个可以任选地和独立地被根据本发明的多特异性抗体包括或不包括。
在一些实施方式中，第一抗原结合部分和第二抗原结合部分靶向同一抗原的不同表位。
在一些实施方式中，至少一个两个抗原结合片段还可以包括分泌信号序列。
分泌信号序列是指，通过连接至编码序列位于细胞膜外侧或细胞外侧的N端而诱导所表达的蛋白或肽的分泌的序列，所述信号序列可以是由约18-30个氨基酸组成的肽序列。所有能转运到细胞膜外侧的蛋白有不同的信号序列，所述信号序列被细胞膜上的信号肽酶切割。通常，对于并非宿主细胞天然表达的外来蛋白而言，可以采用能将该蛋白分泌到细胞周质或培养基中的分泌信号序列，或采用修饰的序列。
iii.异二聚体Fc融合蛋白
在一些实施方式中，其包含重链恒定区Fc，所述Fc恒定区是异源二聚体(异二聚体Fc融合蛋白)。
所述Fc包括但不限于如下组合：
CH2；
CH2+CH3；
CH2+CH3+CH4；
所述Fc恒定区引入突变以避免重链错配。
在一些实施方式中，所述Fc恒定区引入突变为基于KiH技术(Knob-into-Holes)，即在其中Fc恒定区一条重链中引入氨基酸突变，引入的氨基酸体积大于最初氨基酸残基体积，形成一个突起的类似“杵”的型结构(Knob)，在Fc恒定区另一条链区引入另一突变，引入的氨基酸体积小于最初氨基酸残基体积，形成一个凹陷，类似“臼”的结构(Hole)，从而凸型重链更倾向和凹型重链配对，从而避免重链发生错配。该技术由基因泰克研发，记载于专利申请WO1996027011中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于静电相互作用，例如ART-lg技术，该技术由罗氏子公司Chugai开发，特异性的改变Fc恒定区结构域的电荷，促进异源重链的配对，相当于电荷版的KiH技术，该技术记载于专利申请WO2006106905中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于SEED技术，SEED异二聚化是另一种基于空间突变的设计策略，该策略利用了从IgG和IgACH3域(也称为AG SEED CH3和GA SEED CH3)衍生的交替序列的互补性。IgG和IgA CH3衍生物产生互补序列，因此在组装两个互补的重链异源二聚体的同时，排除了缺乏互补性的同源二聚体的组装。该技术记载于专利申请WO2007110205中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于等电点改变，便于提高异源二聚体形成率以及保持Fc区域稳定性的改造，该技术记载于WO2014145806，该专利全文引入本专利。
在一些实施方式中，所述Fc恒定区基于亲水相互作用或增加的柔性而缔合成为异源二聚体。
在一些实施方式中，所述Fc恒定区基于以上技术的任意组合缔合成为异源二聚体，例如，在一些实施方式中，所述Fc恒定区基于KIH和静电相互作用的组合进行了突变。例如，XmAb双特异性平台方法可以通过结合静电相互作用，CH3域构象和氢键提高双特异性抗体的热稳定性。具体的，该策略将天然IgG1的Fc侧链突变交换为S364K和K370S异二聚体，以在两者之间形成氢键，然后进行L368D/K370S取代驱动盐桥相互作用以促进异二聚体的形成，专利申请WO2014145907，该专利全文引入本专利。
在一些实施方式中，所述CH2、CH3或CH4区域的全长或部分被插入或替换成受体及其配体。
在一些实施方式中，被插入或替换的区域独立地位于CH2、CH3或CH4区，或任意像个相邻的区之间的位置(如CH1-CH2交界处、CH2-CH3交界处、CH3-CH4交界处)；
在一些实施方式中，当上述任意两个恒定区(例如CL-CH1、CH2-CH2、CH3-CH3或CH4-CH4区之任一项)被插入或替换时，替换区域两个互相配合的缀合片段之间的亲和力，K D＜1×10 -3(M)，例如如x×10 -4(M)、x×10 -5(M)、x×10 -6(M)、x×10 -7(M)、x×10 -8(M)、x×10 -9(M)、x×10 -10(M)、x×10 -11(M)；x的值可选自1～9，例如2、3、4、5、6、7、8。
在一些实施方式中，所述缀合片段的N端和/或C端通过连接肽与所述抗原结合片段连接。
在一些实施方式中，所述连接肽的氨基酸数目为1～30个；可以是1，2，3，4，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个；优选5～20个。
在一些实施方式中，所述连接肽的氨基酸是不具有除连接以外的额外功能(例如蛋白定位、酶切位点等)的无意义多肽。
在一些实施方式中，所述连接肽为柔性连接肽；
在一些实施方式中，所述连接肽的氨基酸序列选自Gly、Ser、Pro、Ala以及Glu中的一种或多种。
在一些实施方式中，所述连接肽的氨基酸序列选自(GGGGS)n、(GGGS)n、(GGS)n、(GS)n或(G)n，其中n选自1，2，3，4，5或6。
连接肽通常是柔性的，可以减少融合蛋白与目的蛋白之间的空间位阻，从而更有利于蛋白正确折叠。
在另外的实施方案中，连接肽是刚性接头肽；即相对非柔性肽接头。刚性连接肽不要求完全缺乏柔性，而是比柔性接头肽如富甘氨酸肽接头柔性少。由于其相对缺乏柔性，刚性连接肽降低通过刚性连接肽连接在一起的两个蛋白结构域(在当前情况下是稳定剂蛋白和热稳定逆转录酶)的运动。提供有序链(例如α螺旋结构)的连接肽可提供刚性接头肽。例如，精氨酸、亮氨酸、谷氨酸、谷氨酰胺和甲硫氨酸都显示出相对高的螺旋接头结构倾向。然而，包含许多脯氨酸残基的非螺旋接头也可表现显著的刚性。刚性连接肽的实例包括聚赖氨酸和聚-DL-丙氨酸聚赖氨酸。刚性肽接头的进一步描述由Wriggers等，Biopolymers，80，第736-46页(2005)提供。此外，刚性接头肽在由George等，Protein Engineering，15，第871-79页(2003)描述的接头数据库中描述。优选地，刚性连接肽也是非可切割接头肽，即非可切割刚性连接肽。
分离的核酸
本发明还涉及分离的核酸，其编码如上所述的双特异性融合多肽或多功能融合蛋白。
术语“分离的核酸”在本文中是指以单链或双链形式存在的脱氧核糖核酸或核糖核酸聚合物。所述分离的核酸包括RNA基因组序列，DNA(gDNA和cDNA)或从DNA转录的RNA序列，而且，除非特别指明，所述多肽还包括天然多核苷酸、糖、或碱基改变的类似物。根据本发明一个方面，所述多核苷酸是轻链多核苷酸。
所述分离的核酸包括编码蛋白复合物氨基酸序列的核苷酸序列，也包括与其互补的核苷酸序列。所述互补序列包括完全互补的序列和基本上互补的序列，这是指能在本领域已知的严谨条件下与编码蛋白复合物氨基酸序列的核苷酸序列杂交的序列。
而且，编码蛋白复合物氨基酸序列的核苷酸序列可以被改变或突变。所述改变包括添加、缺失、或非保守取代或保守取代。编码蛋白复合物氨基酸序列的多核苷酸可以被解释为，包括相对于该分离的核酸有实质性同一性的核苷酸序列。所述实质性同一性将该核苷酸序列与另外的随机序列以使得它们最大对应的方式进行比对，当用本领域常见的算法分析所比对的序列时，所述序列可显示大于80％的同源性，大于90％的同源性，或大于95％的同源性。
载体
本发明还涉及含有如上所述核酸的载体。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包 括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。所述载体可以包含选择标记(例如便于富集的标签，例如his tag；或便于被检测的标签，例如GFP)，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。所述载体可以是克隆载体与表达载体。在表达或是制备抗体或片段时，常涉及原核表达载体和真核表达载体，原核表达载体常用PET系列、pGEX系列，真核表达载体常用pcDNA3.1、pcDNA3.4、pcDNA4、pEGFP-N1、pEGFP-N1、pSV2等。
在本发明中，载体可以为组合物，例如为多种质粒的混合物，不同质粒负载抗体或其片段的一部分。
宿主细胞
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。
可以使用的多种培养的宿主细胞包括，例如，原核细胞、真核细胞、细菌细胞(如大肠杆菌或嗜热脂肪芽胞杆菌(Bacilisstearothermophilus))、真菌细胞(如酿酒酵母或毕赤酵母)、昆虫细胞(如包括草地夜蛾细胞在内的鳞翅目昆虫细胞)或哺乳动物细胞(如中国仓鼠卵巢(CHO)细胞、NS0细胞、小仓鼠肾(BHK)细胞、猴肾细胞、Hela细胞、人肝细胞癌细胞或293细胞等等)。
制备双特异性融合多肽或多功能融合蛋白的方法
可采用本领域任何已知的方法制备本发明的双特异性融合多肽或多功能融合蛋白。
例如：用如上所述的载体转化宿主细胞；
培养所转化的宿主细胞；和
收集宿主细胞中表达的双特异性融合多肽或多功能融合蛋白。
特别的，可采用如下方法。
早期构建双特异性抗体的方法有化学交联法或杂合杂交瘤或四价体瘤法(例如，Staerz UD等，Nature，314：628-31，1985；Milstein C等，Nature，305：537-540，1983；Karpovsky B等，J.Exp.Med.，160：1686-1701，1984)。化学偶联法是将2个不同的单克隆抗体用化学偶联的方式连接在一起，制备出双特异性单克隆抗体。例如两种不同单克隆抗体的化学结合，或例如两个抗体片段如两个Fab片段的化学结合。杂合杂交瘤法是通过细胞杂交法或者三元杂交瘤的方式产生双特异性单克隆抗体，这些细胞杂交瘤或者三元杂交瘤是通过建成的杂交瘤融合，或者建立的杂交瘤和从小鼠得到的淋巴细胞融合而得到的。虽然这些技术用于制造BiAb，但各种产生问题使得此类复合物难以使用，诸如产生含有抗原结合位点的不同组合的混合群体、蛋白质表现方面的困难、需要纯化目标BiAb、低产率、生产费用高等。
最近的方法利用经过基因工程改造的构建体，其能够产生单一BiAb的均质产物而无需彻底纯化以去除不需要的副产物。此类构建体包括串联scFv、二抗体、串联二抗体、双可变结构域抗体和使用诸如Ch1/Ck结构域或DNLTM的基元的异源二聚(Chames&Baty，Curr.Opin.Drug.Discov.Devel.，12：276-83，2009；Chames&Baty，mAbs，1：539-47)。相关纯化技术是公知的。
还可以使用单淋巴细胞抗体方法通过克隆和表达由选择用于产生特异性抗体的单个淋巴细胞产生的免疫球蛋白可变区cDNA来产生抗体，例如由Babcook J等人，Proc.Natl.Acad.Sci.USA.93：7843-7848，1996；WO 92/02551；WO 2004/051268和WO 2004/106377所述的方法。
用于产生例如用于免疫宿主或用于淘选诸如用于噬菌体展示(或酵母细胞或细菌细胞表面表达)的抗体的抗原多肽可以通过本领域熟知的方法从包含表达系统的遗传工程改造的宿主细胞制备，或者它们可以是从天然生物来源回收。例如，可将编码双特异性抗体的一条或两条多肽链的核酸通过多种已知的方法(如转化、转染、电穿孔、用核酸包被的微粒轰击等)引入培养的宿主细胞。在一些实施方案中，编码双特异性抗体的核酸在被引入宿主细胞前可先插入至适于在宿主细胞中表达的载体中。典型的所述载体可包含使插入的核酸能够在RNA和蛋白质水平上表达的序列元件。
本发明的双特异性抗体，或其部分可通过常规的免疫学分析方法，例如酶联免疫吸附试验(ELISA)，放射免疫分析(RIA)或组织免疫组织化学用于检测任一或所有这些抗原(例如在生物样品，如血清或血浆中)。本发明提供检测生物样品中的抗原的方法，该方法包括：使所述生物样品与本发明的可特异识别所述抗原的双特异性抗体，或抗体部分抗原相接触，并检测与抗原结合的抗体(或抗体部分)，或非结合抗体(或抗体部分)，由此检测所述生物样品中的所述抗原。所述抗体用可检测的物质进行直接或间接的标记，以便于检测结合或非结合抗体。合适的可检测物质包括多种酶，修复基团，荧光物质，发光物质和放射性物质。合适的酶的例子包括，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，乙酰胆碱酯酶；合适的修复基团复合物的例子包括链霉抗生物素蛋白/生物素和抗生物素蛋白/生物素；合适的荧光物质的例子包括7-羟基香豆素，荧光素，荧光素异硫氰酸盐，硷性蕊香红B，二氯三嗪基胺荧光素，丹磺酰氯或藻红蛋白；发光物质的例子包括3-氨基邻苯二甲酰环肼；合适的放射性物质的例子包括I 125、I 131、 35S或 3H。
药物组合物
本发明的双特异性融合多肽或多功能融合蛋白或编码其的核酸可以应用于制备药物组合物或无菌组 合物，例如，将双特异性融合多肽或多功能融合蛋白与药学上可接受的载体、赋形剂或稳定剂混合。药物组合物可包括一种或组合的(如两种或更多不同的)本发明的抗体其功能片段。例如，本发明的药物组合物可包含与靶抗原上的不同表位结合的具有互补活性的抗体或抗体片段(或免疫缀合物)的组合。治疗和诊断剂的制剂可通过以例如冻干粉末、浆液、水性溶液或悬浮液的形式与药学可接受的载体、赋形剂或稳定剂混合来制备。
药物组合物中的双特异性融合多肽或多功能融合蛋白可以是与第二种激活剂(功能分子)结合的形式。所述第二种激活剂可以是能预防或治疗目标疾病的随机功能分子，可包括化合物，肽，多肽，核酸，碳水化合物，脂质，或无机粒子。在所述药物组合物中，双特异性融合多肽或多功能融合蛋白可以本身具有治疗活性；但它可以发挥将所述第二种激活剂靶向特异性疾病区的功能。所述疾病区可以是与抗原特异性结合的双特异性抗体所聚集和分布的那些器官，组织，或细胞。靶向所述疾病区的药物以高浓度存在，使得药物效应相比注射的量增加。因此，药物组合物可以用于治疗耐药性肿瘤，并可以减少因非特异性药物分布所致的副作用和不利的药物反应。
药物组合物中包含双特异性融合多肽或多功能融合蛋白的激活剂可以容纳在微胶囊中，或容纳在胶体性质的药物运送系统(如脂质体，白蛋白小球体，微乳剂，纳米颗粒及纳米胶囊)中，或者容纳在大乳剂(macroemulsions)中，所述微胶囊可以通过诸如凝聚(coacervation)技术或界面聚合作用来制备，例子分别有羟甲基纤维素或明胶微胶囊和聚-(异丁烯酸甲酯)微胶囊。
医药用途与治疗方法
本发明还涉及如上所述的双特异性融合多肽或多功能融合蛋白在制备用于治疗疾病的药物中的应用。
根据本发明一个方面，所述疾病可以是例如，癌症、免疫性病症、代谢性疾病以及微生物感染。
术语“癌症”是指以体内异常细胞的不受控生长为特征的一大类疾病。“癌症”包括良性和恶性癌症以及休眠肿瘤或微转移。
在一些实施方式中，微生物感染中微生物可以是外源病原体或带有外源病原体例如病毒的细胞群体。本发明适用于诸如细菌、真菌、病毒、支原体和寄生虫的外源病原体。可以用本发明治疗的病原体可以是任何本领域熟知的在动物体内致病的感染性生物，包括诸如以下的生物：革兰氏阴性或革兰氏阳性球菌或杆菌的细菌、DNA病毒和RNA病毒，包括但不限于诸如乳头瘤病毒、细小病毒、腺病毒、疱疹病毒和痘苗病毒的DNA病毒、以及诸如沙粒病毒、冠形病毒、鼻病毒、呼吸道合胞病毒、流感病毒、细小核糖核酸病毒、副粘病毒、呼肠孤病毒、逆转录病毒和弹状病毒的RNA病毒。特别感兴趣的是抗生素抗性细菌，例如抗生素抗性链球菌(Streptococcus species)和葡萄球菌(Staphlococcus species)，或者是对抗生素敏感但引起用抗生素治疗的复发性感染、以致最终产生抗性生物的细菌。这类生物可以用本发明的配体-免疫原缀合物与低于正常给予患者的剂量的抗生素联合治疗，以避免产生这些抗生素抗性细菌菌株。本发明也适用于任何真菌、支原体种、寄生虫或在动物中致病的其它感染性生物。可以用本发明方法治疗的真菌的实例包括生长为霉或酵母样的真菌，包括例如引起诸如以下疾病的真菌：癣、组织胞浆菌病、芽生菌病、曲霉病、隐球菌病、孢子丝菌病、球孢子菌病、类球孢子菌病和念珠菌病。本发明可以用来治疗寄生虫感染，包括但不限于由以下寄生虫引起的感染：体绦虫、血吸虫、组织蛔虫、变形虫和疟原虫属(Plasmodium)、锥虫属(Trypanosoma)、利什曼原虫属(Leishmania)和弓形体属(Toxoplasma)种。特别感兴趣的寄生虫是表达叶酸受体并结合叶酸的寄生虫；然而，在文献中关于对感染性生物表现出高亲和性的配体有大量的参考文献。例如，已知其抗生素活性并且与细菌细胞壁前体特异性结合的青霉素和头孢菌素同样可以用作制备按照本发明使用的配体-免疫原缀合物的配体。本发明的配体-免疫原缀合物也可以针对带有内源病原体的细胞群体，其中所述病原体特异性抗原优先在带有所述病原体的细胞表面表达，并且用作与所述抗原特异性结合的配体的受体。
本发明还涉及一种预防和/或治疗和施用治疗有效量的药物组合物以预防和/或治疗如上所述疾病的方法。
本发明的方法可以用于人类临床医学和兽医学应用。因此，带有致病生物群体并且用配体-免疫原缀合物治疗的宿主动物可以是人类，或者在兽医学应用的情况下，可以是实验室动物、农用动物、驯养动物或野生动物。本发明可以适用于包括但不限于以下的宿主动物：人类；实验室动物，诸如啮齿动物(例如小鼠、大鼠、仓鼠等)、兔、猴、黑猩猩；驯养动物，例如狗、猫和兔；农用动物，例如牛、马、猪、绵羊、山羊；和关养的野生动物，例如熊、熊猫、狮、虎、豹、大象、斑马、长颈鹿、大猩猩、海豚和鲸。
药物组合物可通过多种途径注射至实体中，所述实体包括大鼠、小鼠、家养动物、和/或人类。所有注射方法都可以预期，例如，口服，直肠，静脉，鼻，腹部，皮下，或局部注射都是有可能的。组合物可以用本领域已知的其它方法来注射。
“治疗有效量”在本文中是指，根据合理的益损比来看，能治疗疾病的足够量。治疗有效量可以因患者引起的多种原因而有不同，所述原因例如，疾病类型、严重程度、发作、实体的年龄、体重、排泄速度、反应易感性、健康状态、和/或并发症；和/或药物活性、注射途径、注射周期和注射次数、和/或药物组合； 也可以由本领域普通技术人员根据治疗目的进行适当选择。例如，注射量可以随机分为多次，使得该量为约0.001-100mg/kg成人体重。
本发明的双特异性融合多肽或多功能融合蛋白或编码本发明抗体的核酸或多核苷酸还可与例如标准癌症治疗(例如，手术、放射和化学疗法)组合施用。例如，使用本发明的组合物和/或装备了这些组合物的效应细胞的抗肿瘤疗法与化学疗法联合使用。本发明抗体组合治疗的非限制性实例包括手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1、FiBody设计
在一些实施例中，FiBody是利用配体及其受体之间特异性亲和力，取代双特异性抗体一侧的CL和CH1，重组获得的双特异性抗体，其能够避免或减少双特异性抗体轻链与重链发生错配。
本实施例以白细胞介素及其受体为例构建FiBody，根据白细胞介素及其受体的立体构像将其分为四类，见图5：
Figure PCTCN2022088198-appb-000004
基于以上4类白细胞介素及其受体分别构建双特异性抗体。
实施例2、基于白细胞介素及其受体FiBody的构建
选取靶向第一抗体的VH通过Linker连接在受体蛋白上，再通过Hinge与抗体的Fc连接；靶向第一抗体的VL通过Linker连接在配体蛋白上，以降低或避免轻链与重链发生错配；另一端为靶向第二抗体(抗TIGIT抗体)的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链发生错配。或
选取靶向第一抗体的VH通过Linker连接在配体蛋白上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在受体蛋白(IL15)上，以降低或避免轻链与重链发生错配；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链错配。
Figure PCTCN2022088198-appb-000005
一些具体的实施示例如下表：
Figure PCTCN2022088198-appb-000006
Figure PCTCN2022088198-appb-000007
Figure PCTCN2022088198-appb-000008
Figure PCTCN2022088198-appb-000009
Figure PCTCN2022088198-appb-000010
实施例3具有二硫键改造的双特异性抗体
3.1为了进一步改善双特异性抗体稳定性并且延长双特异性抗体的半衰期，对双特异性抗体进行二硫键改造，见图8。
配受体二硫键改造：选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对受体、配体蛋白进行突变，目的形成分子间二硫键，进一步提高分子的稳定性，具体例如：
Figure PCTCN2022088198-appb-000011
Figure PCTCN2022088198-appb-000012
3.2在VH、VL之间设计二硫键改造，形成dsFv，帮助非共价设计的受配体重、轻链之间形成共价二硫键链接。二硫键改造位置包括但不限于以下突变位点：
组合 VH VL
1 37C 95C
2 44C 100C
3 44C 101C
4 44C 105C
5 45C 87C
6 45C 98C
7 100C 50C
8 100bC 49C
9 98C 46C
10 101C 46C
11 105C 43C
12 106C 57C
13 108C 43C
具体例如：
Figure PCTCN2022088198-appb-000013
Figure PCTCN2022088198-appb-000014
实施例4、消除糖基化的突变改造
Fab-IL15/IL15RA_Fc的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对受体、配体蛋白进行突变，目的形成分子间二硫键，进一步提高分子的稳定性；进一步的，对受体、配体蛋白上的糖基化位点进行改造，目的是消除分子的异质性，具体例如：
Figure PCTCN2022088198-appb-000015
Figure PCTCN2022088198-appb-000016
Figure PCTCN2022088198-appb-000017
实施例5、具有降低IL15/IL15RA与IL2/15Rβ/γC复合物亲和力改造的IL15/IL15RA及其组成的双特异性抗体
在某些应用中，为了避免IL15/IL15RA及其组成的双特异性抗体与IL2/15Rβ/γC复合物相互作用，引起不需要的非特异性结合，我们对IL15/IL15RA及其组成的双特异性抗体进行改造，以降低或完全丧失IL15/IL15RA与IL2/15Rβ/γC复合物亲和力。通过检查IL15/IL15RA与IL2/15Rβ/γC复合物作用界面晶体结构，以及使用MolecularOperating Environment(MOE；Chemical Computing Group，Montreal，Quebec，加拿大)软件建模，我们预测在IL15/IL15RA界面处可以进行氨酸突变改造以便降低或完全丧失IL15/IL15RA与IL2/15Rβ/γC复合物亲和力，如图9中描绘的。
描述Fab-IL15/IL15RA_Fc的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对配体蛋白进行突变，目的是降低或失活受、配体复合物的生物学功能，具体例如：
Figure PCTCN2022088198-appb-000018
实施例6、构建基于scFv、CrossMab结构的双特异性抗体作为实验对照
正如前文所描述，scFv和CrossMab都是常用的双特异性抗体构建技术手段，在这里作为设计对照，跟我们的分子进行对比：
基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Hinge与抗体的Fc连接；另一端为靶向第一抗体的完整Fab结构，(此结构与武汉友芝友Y-Body相似，命名为YBody)，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如：
Figure PCTCN2022088198-appb-000019
描述基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Linker与完整的靶向第一抗体的Fc的C端连接；组成一个对称的结构。具体例如：
Figure PCTCN2022088198-appb-000020
Figure PCTCN2022088198-appb-000021
基于CrossMab结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH连接至CL结构域，再通过Hinge与抗体的Fc连接，靶向第二抗体(抗TIGIT)的VL连接至CH1结构域，形成轻链；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如：
Figure PCTCN2022088198-appb-000022
实施例7、抗体重-轻链错配测试
轻链错配是双抗平台面临的一个难点问题。为了验证本平台防错配性能，我们专门设计了受体、配体分布在抗体两边的Fab，故意设计错配的重、轻链结构，并进行表达验证。
描述Fab-IL15/IL15RA_Fc错配的构建方法：
R1042：具体选取靶向第一抗体(抗PD-L1抗体)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第二抗体(抗TIGIT抗体)的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第二抗体的VH通过常规序列连接在CH1，再通过Hinge与抗体的Fc连接，靶向第一抗体的VL通过常规序列连接在CL，两个Fc具有常规的KiH改造，以避免重链错配。具体例如：
分子编号 R1042：PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL(图10左)
第一多肽 @PD-L1_VH_IL15RA_Fc-Knob(SEQ ID NO.1)
第二多肽 @TIGIT_VL_IL15(SEQ ID NO.6)
第三多肽 @TIGIT_VH_CH1_Fc-Hole(SEQ ID NO.3)
第四多肽 @PD-L1_VL_CL(SEQ ID NO.8)
R1043：具体选取靶向第二抗体(抗TIGIT抗体)的VH通过Linker连接在受体蛋白(IL15RA)上，再通过Hinge与抗体的Fc连接；靶向第一抗体(抗PD-L1抗体)的VL通过Linker连接在配体蛋白(IL15)上；另一端为靶向第一抗体的VH通过常规序列连接在CH1，再通过Hinge与抗体的Fc连接，靶向第二抗体的VL通过常规序列连接在CL，两个Fc具有常规的KiH改造，以避免重链错配。
分子编号 R1043：PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-LI-VL
第一多肽 @TIGIT_VH_IL15RA_Fc-Knob(SEQ ID NO：5)
第二多肽 @PD-L1_VL_IL15(SEQ ID NO：2)
第三多肽 @PD-L1_VH_CH1_Fc-Hole(SEQ ID NO.7)
第四多肽 @TIGIT_VL_CL(SEQ ID NO.4)
描述Fab-IL21/IL21R_Fc错配的构建方法：
选取靶向第二抗体(抗PD-L1抗体)的VH通过Linker连接在受体蛋白(IL21R)上，再通过Hinge与抗体的Fc连接；靶向第一抗体(抗TIGIT抗体)的VL通过Linker连接在配体蛋白(IL21)上；另一端为靶向第二抗体(抗PD-L1抗体)的VL连接在CL上，靶向结构第一抗体(抗TIGIT抗体)的VH连 接在CH1上，再通过Hinge与抗体的Fc连接，两端的Fc具有常规的KiH改造。具体例如：
Figure PCTCN2022088198-appb-000023
实施例8、具有二硫键改造的双特异性抗体
在IL2、IL2Rα之间设计二硫键改造(将野生型白细胞介素2的氨基酸序列(SEQ ID NO.21)中第75位的氨基酸突变为半胱氨酸；并将白细胞介素2受体α的氨基酸序列(SEQ ID NO.22或SEQ ID NO.23)的N端延伸两个或三个氨基酸，其中，延伸的第2位氨基酸为半胱氨酸)，帮助非共价连接的IL2和IL2Rα之间形成共价二硫键连接。
示例性的阐述一种具有二硫键改造的双特异性抗体(二硫键改造IL2/IL2Rα复合物)的构建方法：靶向肿瘤细胞或免疫细胞表面抗原的抗体(例如TIGIT抗体)的重链可变区(VH)通过Linker连接在IL2Rα突变体上，再通过Hinge(铰链区)与hIgG1抗体的Fc连接，靶向相同抗原的抗体(TIGIT抗体)的轻链可变区(VL)通过Linker与IL2突变体连接；靶向免疫细胞或肿瘤细胞表面抗原的抗体(例如PD-L1抗体)的VH直接与hIgG1抗体的恒定区(hIgG1)连接，靶向相同抗原的抗体(PD-L1抗体)的VL直接与人免疫球蛋白轻链kappa抗体(κ-IgLC)连接，制备具有靶向性的二硫键改造IL2/IL2Rα复合物。
根据以上构建方法，发明人设计了2种二硫键改造IL2/IL2Rα复合物，即二硫键改造IL2/IL2Rα复合物1和二硫键改造IL2/IL2Rα复合物2；而IL2/IL2Rα复合物3为现有报道的复合物，复合物4为二硫键未改造IL2/IL2Rα复合物；复合物1、2、3、4的具体组成和氨基酸序列如下：
二硫键改造IL2/IL2Rα复合物1(样品R1262)的序列结构为：TIGIT VH-IL2Rα突变体-Fc、TIGIT VL-IL2突变体、PD-L1 VH-hIgG1和PD-L1 VL-κ-IgLC；其中，TIGIT VH-IL2Rα突变体-Fc的氨基酸序列如SEQ ID NO.77所示，TIGIT VL-IL2突变体的氨基酸序列如SEQ ID NO.78所示，PD-L1 VH-hIgG1的氨基酸序列如SEQ ID NO.79所示，PD-L1 VL-κ-IgLC的氨基酸序列如SEQ ID NO.80所示。
Figure PCTCN2022088198-appb-000024
Figure PCTCN2022088198-appb-000025
二硫键改造IL2/IL2Rα复合物2的序列结构为：TIGIT VH-IL2突变体-Fc、TIGIT VL-IL2Rα突变体、PD-L1 VH-hIgG1和PD-L1 VL-κ-IgLC；其中，TIGIT VH-IL2突变体-Fc的氨基酸序列如SEQ ID NO.81所示，TIGIT VL-IL2Rα突变体的氨基酸序列如SEQ ID NO.82所示，PD-L1 VH-hIgG1的氨基酸序列如SEQ ID NO.79所示，PD-L1 VL-κ-IgLC的氨基酸序列如SEQ ID NO.80所示。
Figure PCTCN2022088198-appb-000026
IL2/IL2Rα复合物3的序列结构为：TIGIT VH-IL2Rα(L42C)-Fc、TIGIT VL-IL2(F42C)、PD-L1VH-hIgG1和PD-L1 VL-κ-IgLC；其中，TIGITVH-IL2Rα(L42C)-Fc的氨基酸序列如SEQ ID NO.83所示，TIGITVL-IL2(F42C)的氨基酸序列如SEQ ID NO.84所示，PD-L1 VH-hIgG1的氨基酸序列如SEQ ID NO.79所示，PD-L1 VL-κ-IgLC的氨基酸序列如SEQ ID NO.80所示；IL2Rα(L42C)代表：将IL2Rα的氨基酸序列中第42位亮氨酸突变为半胱氨酸，IL2(F42C)代表：将IL2的氨基酸序列中第42位苯丙氨酸突变为半胱氨酸。
Figure PCTCN2022088198-appb-000027
实施例9、FiBody样品的制备
蛋白瞬转表达：
将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到目的蛋白。
Expi293在37℃、8％二氧化碳、130rpm条件培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300ml。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀，标记为A管；将1.5mg转染试剂PEI加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养。直到D7-D10后收样。
蛋白纯化：
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下，使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。
实施例10、FiBody理化检测
样品经过一步纯化后通过HPLC-SEC进行检测(分析柱TOSOH，TSKgel G2000)纯度，各个样品的表达量及纯度结果见下表。
Figure PCTCN2022088198-appb-000028
样品R0951的HPLC-SEC进行检测结果如图11所示，样品R1042的HPLC-SEC进行检测结果如图12所示，样品R0809的HPLC-SEC进行检测结果如图13所示，样品R1110的HPLC-SEC进行检测结果如图14所示。
结果显示，相比于非对称scFv(Y-Body，R0809)、对称ScFv(R0810)、CrossMab(R0959)结构的双特异性抗体，FiBody平台制备双特异性抗体(包括各种改造优化抗体)具有更高的表达量和/或更高的纯度。
意外的是具有错误配对形式的双特异性抗体(样品R1042、R1043、R1124)也能表达并具有类似正常分子的表达量，但是具有明显低的纯度。
R1262的HPLC-SEC检测结果如图15所示，结果说明：成功去除了游离轻链，且本发明制备得到的二硫键改造IL2/IL2Rα复合物1的纯度高。
实施例11、FiBody抗原亲和力检测
TIGIT端结合活性分析
通过FCM实验方法检测双抗分子(TIGIT端)与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL，3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL，2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h，再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1∶500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。
R0950、R0951、R0952、R0954、R0955、R0960，R1123/R1119/R1120/R1124，R1042/R1043，R0810的结果如图16-图19所示；意外的是R0950(@TIGIT在Fab端)结合活性低于R0951～R0960(@TIGIT在IL15/IL15R端)；R0951～R0960结合活性与阳性对照R0226(Tigit单克隆抗体，OMP-313R12，WO2016191643)相近；A类和D类白细胞介素及其受体替换CH1和CL后，靶向区结合力并未受到影响，与阳性对照(R0226\R0774(VH如序列如SEQ ID NO：73所示，VL序列如SEQ ID NO：74所示)，Tigit单克隆抗体)表现出了相当的亲和力；C类分子的替换CH1和CL后靶向区受到影响，靶向结合力明显低于阳性对照(R0226\R0774，Tigit单克隆抗体)。
二硫键改造优化样品R1081、R1085及糖基化样品改造分子与改造之前分子相比tigit端亲和力结果相当。
R1042、R1043及R1124为错配测试分子，其TIGIT结合活性显著降低；R0810是ScFv结构分子，结合活性也弱于对照分子R0226。
R1262的结果如图20所示，结果显示：二硫键改造IL2/IL2Rα复合物1(R1262)与二硫键未改造IL2/IL2Rα复合物(R1115)的亲和力相当，说明二硫键改造不会影响靶向区的亲和力。
PD-L1端结合活性分析
通过FCM实验方法检测双抗分子(PD-L1端)与CHO-PD-L1细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL，3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL，2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h，再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1∶500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。
R0950、R0951、R0952、R0954、R0955、R0960，R1072、R1115-R1120、R1123-R1124，R0950、R1042、R1043，R1072、R1081-R1086，R1072、R1109-R1111的结果如图21-图25所示，意外的是@PD-L1放在IL15端的活性要好于放在Fab端；R1042、R1043及R1124是对应错配分子，活性明显很弱，其他FiBody均显示了与阳性抗体(PD-L1单克隆抗体)相当的亲和力。其中R0802为(PD-L1单抗，176F9，VH序列如SEQ ID NO：36所示，VL序列如SEQ ID NO：37所示)，R0514为(PD-L1单抗，Avelumab)，R0919为(PD-L1单抗，VH如序列如SEQ ID NO：75所示，VL序列如SEQ ID NO：76所示)，R0968为(PD-L1单抗，VH如序列如SEQ ID NO：71所示，VL序列如SEQ ID NO：72所示)。
R1262的结果如图26所示，结果显示：二硫键改造IL2/IL2Rα复合物1(R1262)与二硫键未改造IL2/IL2Rα复合物4的双抗(R1115)相比，两者亲和力相当，说明二硫键改造不会影响靶向区的亲和力。
TIGIT端结合阻断分析
通过FCM实验方法检测双抗分子(TIGIT端)阻断配体与CHO-TIGIT细胞结合活性。配制3％BSA缓冲液：称取4.5g BSA到150mL 1×PBS中，混匀后放置冰上备用；抗体稀释：将受试抗体(R1262)、阳性对照抗体(R1115)用3％BSA稀释成初始浓度为800nM，亚型对照抗体(hIgG1抗体)用3％BSA稀释成初始浓度为20μg/mL，体积300μL。3倍梯度稀释(100μL+200μL)共10个浓度；细胞计数并铺板：将R0254-3细胞计数后，按100μL，2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h，再加入50μL配体，2-8度孵育0.5h；350×g离心5min后，去掉上清，按200μL/孔3％BSA；350×g离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1∶500×稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350×g离心5min后，去掉上清，按100μL/孔加入1×PBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程，上机检测。
R1262检测结果如图27所示，结果显示：二硫键改造IL2/IL2Rα复合物1(R1262)与二硫键未改造IL2/IL2Rα复合物4(R1115)相比，两者对配体与靶向区结合力的阻断效果相当。
实施例12、FiBody受体配体复合物(IL15/IL15R)的结合活性
抗体稀释：用FACS buffer将所有分子稀释成初始浓度400nM，体积180μl，3倍梯度稀释(60+120)，10个浓度；细胞计数并铺板：将R0255-2(CHO-mTigit)/293T-IL15R-28细胞离心250g 5min后弃去上清，用FACS buffer调整细胞密度为2E+06，按100μL/管均分到96孔V型板中；将上述稀释好的抗体加入到细胞中，100μL/孔，2-8度孵育0.5h；取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200μL/孔，再次250g离心5min，小心去上清；用FACS buffer配制PE荧光二抗(1∶500稀释)，按100μL/孔加入对于的96孔板中，重悬细胞，2-8度孵育30min；取出96孔板，250g离心5min，小心去上清后，加入FACS buffer 200μL/孔，再次250g离心5min，小心去上清；用1xPBS 100μL/孔重悬，FACS检测。
结果如图28、图29所示；R0952(IL15、IL15RA换位)、R0960(减活)分子具有很低的IL15受体复合物结合活性；R0955(去糖基化)IL15活性下降；R0953、R0954共价连接后活性与R0951类似，说明对结构影响很小。
错配分子R1042、R1043与R0951活性相当，说明没有产生错配，即使是错误的Fv也能表达出来；其中R0655为(IL15/IL15RFc融合蛋白，见SEQ ID NO：38)
实施例13、二硫键改造分子电泳检测
对本发明实施例中二硫键改造优化样品(R1072、R1081、R1082、R0954、R1084-R1086)进行SDS-PAGE电泳检测。
结果如图30所示，未进行二硫键改造的R1072分子在分子量25KD～35KD之间有条带，说明存在游离轻链；配受体二硫键改造分子为R0954、R1085、R1086，其中R0954、R1086电泳结果显示，仍有非共价轻链存在(25KD～35KD之间有条带)，R1085无非共价轻链存在(25KD～35KD之间无条带)，说明R1085二硫键改造成功。
轻重链二硫键改造分子为R1081、R1082、R1084，电泳结果显示无非共价轻链存在(25KD～35KD之间无条带)，说明R1081、R1082、R1084二硫键改造成功。
对实施例9制备得到的FiBody样品(实施例8对应构建的二硫键改造IL2/IL2Rα复合物1和2、IL2/IL2Rα复合物3)和实施例2的R1115进行SDS-PAGE电泳检测。
检测结果如图31所示，其中，R1115为二硫键未改造分子，在分子量25KD～35KD之间有条带，说明存在游离轻链；二硫键改造IL2/IL2Rα复合物1和2在分子量25KD～35KD之间无条带，与IL2/IL2Rα复合物3相同，说明成功去除了游离轻链，二硫键改造成功。
实施例14、IL2Rα突变体的氨基酸序列的N端延伸的第1位和第3位氨基酸的种类对IL2/IL2Rα复合物改造后引入的二硫键形成的影响
当第二抗原结合部分通过接头间接地与IL2Rα突变体的N端相连时，IL2Rα突变体的氨基酸序列的N端延伸三个氨基酸；为了进一步明确IL2Rα突变体的氨基酸序列的N端延伸的第1位和第3位氨基酸的种类对IL2/IL2Rα复合物改造后引入的二硫键形成的影响，进行如下实验：
1、延伸的第3位氨基酸的种类对IL2/IL2Rα复合物改造后引入的二硫键形成的影响
选择延伸的第3位氨基酸为芳香族氨基酸(以苯丙氨酸为例)、R基不带电荷的氨基酸(以丝氨酸为例)、R基带正电荷的氨基酸(以赖氨酸为例)或R基带负电荷的氨基酸(以天冬氨酸为例)中的任一种，构建并制备二硫键改造IL2/IL2Rα复合物5、6、7、8(简称依次为R1493、R1494、R1495、R1496)，与二硫键改造IL2/IL2Rα复合物1(IL2/IL2Rα复合物1中IL2Rα突变体的氨基酸序列的N端延伸的第3位氨基酸为甘氨酸(非极性脂肪酸氨基酸))一起作为实验组，以二硫键未改造IL2/IL2Rα复合物4为对照组。
二硫键改造IL2/IL2Rα复合物5、6、7、8与实施例8中制备二硫键改造IL2/IL2Rα复合物1的过程相同。
二硫键改造IL2/IL2Rα复合物5、6、7、8的序列结构均为：TIGIT VH-IL2Rα突变体-Fc、TIGIT VL-IL2突变体(SEQ ID NO：78)、PDL1 VH-hIgG1(SEQ ID NO：79)和PDL1 VL-κ-IgLC(SEQ ID NO：80)；其中，二硫键改造IL2/IL2Rα复合物5、6、7、8的TIGITVH-IL2Rα突变体-Fc的氨基酸序列如依次SEQ ID NO：85、SEQ ID NO：86、SEQ ID NO：87、SEQ ID NO：88所示(其中，IL2Rα突变体的氨基酸序列的N端延伸的三个氨基酸以粗体示出，带有下划线的为延伸的第3位氨基酸)；
Figure PCTCN2022088198-appb-000029
Figure PCTCN2022088198-appb-000030
将获得的二硫键改造IL2/IL2Rα复合物5、6、7、8以及复合物1进行SDS-PAGE电泳检测，二硫键未改造IL2/IL2Rα复合物4为对照组，以探究白细胞介素2受体α突变体的氨基酸序列的N端延伸的第3位氨基酸的种类对IL2/IL2Rα复合物改造后引入的二硫键形成的影响，实验结果如图32所示，可以看出，与对照组相比，二硫键改造IL2/IL2Rα复合物5、6、7、8均成功去除了游离轻链，二硫键改造成功；表明延伸的第3位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸，均不会影响IL2/IL2Rα复合物改造后引入的二硫键的形成。
2、延伸的第1位和第3位氨基酸组合的种类对IL2/IL2Rα复合物改造后的二硫键形成的影响
选择延伸的第1位和第3位氨基酸为非极性脂肪酸氨基酸(以甘氨酸为例)、芳香族氨基酸(以苯丙氨酸为例)、R基不带电荷的氨基酸(以丝氨酸为例)、R基带正电荷的氨基酸(以赖氨酸为例)或R基带负电荷的氨基酸(以天冬氨酸为例)中的任一种组合，构建并制备二硫键改造IL2/IL2Rα复合物9、10、11、12、13(简称依次为R1662、R1663、R1664、R1665、R1666)，与二硫键改造IL2/IL2Rα复合物1一起作为实验组，以二硫键未改造IL2/IL2Rα复合物4为对照组。
二硫键改造IL2/IL2Rα复合物9、10、11、12、13与实施例8中制备二硫键改造IL2/IL2Rα复合物1的过程相同。
二硫键改造IL2/IL2Rα复合物9、10、11、12、13的序列结构均为：TIGIT VH-IL2Rα突变体-Fc、TIGIT VL-IL2突变体(SEQ ID NO：78)、PDL1 VH-hIgG1(SEQ ID NO：79)和PDL1 VL-κ-IgLC(SEQ ID NO：80)；其中，二硫键改造IL2/IL2Rα复合物9、10、11、12、13的TIGIT VH-IL2Rα突变体-Fc的氨基酸序 列如依次SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：93所示(其中，白细胞介素2受体α突变体的氨基酸序列的N端延伸的三个氨基酸以粗体示出，带有下划线的为延伸的第1位和第3位氨基酸)；
Figure PCTCN2022088198-appb-000031
Figure PCTCN2022088198-appb-000032
将获得的二硫键改造IL2/IL2Rα复合物9、10、11、12、13以及复合物1进行SDS-PAGE电泳检测，二硫键未改造IL2/IL2Rα复合物4为对照组，以探究白细胞介素2受体α突变体的氨基酸序列的N端延伸的第1位和第3位氨基酸组合的种类对IL2/IL2Rα复合物改造后引入的二硫键形成的影响，实验结果如图33所示，可以看出，与对照组相比，二硫键改造IL2/IL2Rα复合物9、10、11、12、13均成功去除了游离轻链，二硫键改造成功；表明延伸的第1位和第3位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸的任意组合，均不会影响IL2/IL2Rα复合物改造后引入的二硫键的形成。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列36：@PD-L1：VH(SEQ ID NO：36)
Figure PCTCN2022088198-appb-000033
序列37：@PD-L1：VL(SEQ ID NO：37)
Figure PCTCN2022088198-appb-000034
R0655氨基酸序列：(SEQ ID NO：38)
Figure PCTCN2022088198-appb-000035
R0968PD-L1 VH氨基酸序列：(SEQ ID NO：71)
Figure PCTCN2022088198-appb-000036
R0968PD-L1 VL氨基酸序列：(SEQ ID NO：72)
Figure PCTCN2022088198-appb-000037
R0774TIGIT VH氨基酸序列：(SEQ ID NO：73)
Figure PCTCN2022088198-appb-000038
R0774TIGIT VL氨基酸序列：(SEQ ID NO：74)
Figure PCTCN2022088198-appb-000039
R0919PD-L1 VH氨基酸序列：(SEQ ID NO：75)
Figure PCTCN2022088198-appb-000040
R0919PD-L1 VL氨基酸序列：(SEQ ID NO：76)
Figure PCTCN2022088198-appb-000041

Claims (30)

  1. 一种双特异性融合多肽，其包含第一抗原结合部分，所述第一抗原结合部分包含：
    第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段，和
    第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
    所述第一缀合片段和所述第二缀合物片段能够特异性结合；
    其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。
  2. 根据权利要求1所述的双特异性融合多肽，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
    所述第二抗原结合部分包括：
    第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
    第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
    其中，所述第三缀合片段和所述第四缀合物片段能够特异性结合；所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
    所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体。
  3. 根据权利要求1所述的双特异性融合多肽，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
    所述第二抗原结合部分包括：
    第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
    第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
  4. 如上任一项所述的双特异性融合多肽，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力。
  5. 根据权利要求4所述的双特异性融合多肽，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间。
  6. 根据权利要求4所述的双特异性融合多肽，所述非天然链间键形成于第一重链可变结构域VH1包含的第一突变残基和第一轻链可变结构域VL1包含的第二突变残基之间。
  7. 根据权利要求5或6所述的双特异性融合多肽，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基。
  8. 根据权利要求7所述的双特异性融合多肽，所述非天然链间键为二硫键。
  9. 如上任一项所述的双特异性融合多肽，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
  10. 如上任一项所述的双特异性融合多肽，所述受体及其配体选白细胞介素及其受体。
  11. 根据权利要求10所述的双特异性融合多肽，所述白细胞介素及其受体立体构像为托举型，选自IL15/IL15R、IL2/IL2R、IL4/IL-4Rα+Rγ、IL-6/IL-6R、IL-11/IL-11R、IL-13/IL-13R1、IL-20/IL20Rα+IL20Rβ和/或IL24/IL20Rα+IL20Rβ。
  12. 根据权利要求10所述的双特异性融合多肽，所述白细胞介素及其受体立体构像为钳型，选自IL7/IL7R、IL21/IL21R、IL23A/IL12B。
  13. 根据权利要求11所述的双特异性融合多肽，所述配体和受体选自IL15和IL15Rα。
  14. 根据权利要求13所述的双特异性融合多肽，所述IL15第90位的E突变为C，且所述IL15Rα第67位的P突变位C。
  15. 根据权利要求13所述的双特异性融合多肽，所述第一重链可变结构域VH1与第一轻链可变结构域VL1之间存在非天然二硫键，优选地，第一重链可变结构域VH1与第一轻链可变结构域VL1包含以下任一突变组合：
    组合 VH VL 1 37C 95C 2 44C 100C 3 44C 101C
    4 44C 105C 5 45C 87C 6 45C 98C 7 100C 50C 8 100bC 49C 9 98C 46C 10 101C 46C 11 105C 43C 12 106C 57C 13 108C 43C
    。
  16. 根据权利要求13～15任一项所述的双特异性融合多肽，所述IL15第61位的D突变为N，第64位的E突变为Q，和/或第65位的N突变位D。
  17. 根据权利要求13～16任一项任一项所述的双特异性融合多肽，所述IL15至少一个N糖基化位点不存在；
    优选地，所述N糖基化位点选自N71、N79和/或N112；优选地，所述IL15包含以下氨基酸突变：N71Q、N79Q和/或N112Q。
  18. 根据权利要求13～17任一项任一项所述的双特异性融合多肽，所述IL15Rα至少一个O糖基化位点不存在；
    优选地，所述O糖基化位点选自T2、T81和/或T86；优选地，所述IL15Rα包含以下氨基酸突变：T2A、T81A和/或T86A。
  19. 根据权利要求11所述的双特异性融合多肽，所述配体和受体选自IL2和IL2Rα。
  20. 根据权利要求19所述的双特异性融合多肽，所述IL2第75位的S突变为C，且所述IL2Rα的N端延伸两个或三个氨基酸；
    所述IL2Rα的N端延伸两个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种；
    所述IL2Rα的N端延伸三个氨基酸时，延伸的第2位氨基酸为半胱氨酸，延伸的第1位和第3位氨基酸为非极性脂肪酸氨基酸、芳香族氨基酸、R基不带电荷的氨基酸、R基带正电荷的氨基酸或R基带负电荷的氨基酸中的任一种。
  21. 如上任一项所述的双特异性融合多肽，其包含抗体Fc恒定区。
  22. 根据权利要求21所述的双特异性融合多肽，所述抗体Fc恒定区是异源二聚体。
  23. 根据权利要求21或22所述的双特异性融合多肽，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体。
  24. 根据权利要求21或22任一项所述的双特异性融合多肽，所述抗体Fc恒定区包含CH2、CH3以及任选的CH4，所述CH2、CH3和/或任选的CH4被替换成所述受体及其配体。
  25. 如上任一项所述的双特异性融合多肽，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；
    优选地，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；
    优选地，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；
    优选地，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞。
  26. 分离的核酸，其编码权利要求1～25任一项所述的双特异性融合多肽。
  27. 含有权利要求26所述核酸的载体。
  28. 含有权利要求26所述核酸或者权利要求27所述载体的宿主细胞。
  29. 药物组合物，其包含权利要求1～25任一项所述的双特异性融合多肽，和药学上可接受的载体，赋形剂，或稳定剂。
  30. 权利要求1～25任一项所述的双特异性融合多肽在制备用于治疗疾病的药物中的应用。
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