WO2022100652A1 - 一种同时靶向cd3和cd137的融合蛋白及其制备方法和用途 - Google Patents

一种同时靶向cd3和cd137的融合蛋白及其制备方法和用途 Download PDF

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WO2022100652A1
WO2022100652A1 PCT/CN2021/130015 CN2021130015W WO2022100652A1 WO 2022100652 A1 WO2022100652 A1 WO 2022100652A1 CN 2021130015 W CN2021130015 W CN 2021130015W WO 2022100652 A1 WO2022100652 A1 WO 2022100652A1
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antibody
antigen
peptide linker
binding fragment
fusion protein
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PCT/CN2021/130015
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English (en)
French (fr)
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高新
钱尼良
潘秀颉
徐桂利
刘玉杰
李宏杰
杨翠马
徐勤枝
窦晓倩
刘蕴慧
郁振军
白贵军
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高新
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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
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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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
    • 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
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application generally relates to the field of antibodies. More specifically, the present application relates to a fusion protein targeting CD3 and CD137 at the same time and its preparation method and use.
  • the BITE molecule exhibits potent anti-tumor capabilities, its production remains low, and the bispecific antibody sometimes triggers severe side effects, including liver damage and an excessive immune response, in which white blood cells secrete large amounts of toxic cytokines Signal. Such cytokine "storms" can lead to fever and, in severe cases, organ damage.
  • a new fusion protein is provided herein, specifically a new multispecific antibody, which can simultaneously target CD3 and CD137 on activated T cells, and is compatible with CD3.
  • the affinity of both CD137 and CD137 antigens is low (KD is 10-1000nM), but the affinity of the molecule for binding to T cells is not lower than that of traditional CD3 bispecific antibodies. Therefore, while increasing effective T cell recruitment, the multispecific antibody also has the potential to reduce T cell hyperactivation caused by high-affinity molecules targeting CD3, reducing the risk of potential adverse reactions; in addition, this novel Multispecific antibodies have higher molecular weights and can prevent excessive renal clearance and thus increase their in vivo half-life.
  • the application provides a fusion protein comprising:
  • first peptide linker is used to link the heavy chain of the anti-CD137 antibody or antigen-binding fragment thereof and the heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof
  • second peptide linker is used to link the the light chain of an anti-CD137 antibody or antigen-binding fragment thereof and the light chain of said anti-CD3 antibody or antigen-binding fragment thereof
  • the fusion protein further comprises:
  • the heavy chain of the first antibody or antigen-binding fragment thereof is linked to the heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof through the third peptide linker
  • the light chain of the first antibody or antigen-binding fragment thereof is linked to the light chain of the anti-CD3 antibody or antigen-binding fragment thereof through the fourth peptide linker
  • the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker are each independently selected from the group consisting of sequences set forth in Seq ID NO. 1-2 (Seq ID NO. 1-2).
  • Seq ID NO. 1-2 Seq ID NO. 1-2
  • 1 Xaa Pro Pro Cys Pro Ala Pro Glu
  • Seq ID NO.2 Glu Pro Ala Pro Cys Pro Pro Pro Xaa, wherein Xaa can be any amino acid except Cys or absent) any one of the peptide linkers, where X represents any amino acid except Cys, or a deletion.
  • each of the first to fourth peptide linkers is a hinge region of a native antibody, wherein the hinge region is mutated to delete only one cysteine.
  • any of the first to fourth peptide linkers are independently selected from a C229 deletion mutated IgGl hinge region or a C229 deletion mutated IgGl hinge region with the hinge region D224-S242 inverted.
  • the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or single chain antibody molecules such as sc-Fv.
  • the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUCl, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met and GPC3.
  • the first antigen, CD137 molecule and CD3 molecule are independently derived from mammals, preferably non-human primates or humans.
  • the affinity constant of the first antibody or antigen-binding fragment thereof for binding to the first antigen is the affinity constant of the anti-CD137 antibody or antigen-binding fragment thereof for a CD137 molecule or an anti-CD3 antibody or antigen thereof 10-1000 times the affinity constant of the binding fragment to the CD3 molecule.
  • the application provides a fusion protein, which sequentially comprises from the N-terminus to the C-terminus:
  • disulfide bond can be formed between the first peptide linker and the second peptide linker, and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker, each independently is selected from the following: a peptide linker comprising any one of the sequences shown in Seq ID NO. 1-2, wherein X represents any amino acid except Cys, or deletion.
  • each of the first to fourth peptide linkers is a hinge region of a native antibody, wherein the hinge region is mutated to delete only one cysteine.
  • any of the first to fourth peptide linkers are independently selected from a C229 deletion mutated IgGl hinge region or a C229 deletion mutated IgGl hinge region with the hinge region D224-S242 inverted.
  • the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUCl, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met and GPC3.
  • the first antigen, the CD137 molecule and the CD3 molecule are independently derived from a mammal, preferably a non-human primate or a human.
  • the affinity constant of the Fab fragment for binding to the first antigen is the affinity constant of the anti-CD137 antibody or antigen-binding fragment thereof and the CD137 molecule or the affinity constant of the anti-CD3 antibody or antigen-binding fragment thereof and the CD3 molecule 10-1000 times the affinity constant.
  • the application provides a nucleic acid encoding the fusion protein of the first aspect or the second aspect.
  • the application provides an expression vector comprising the nucleic acid of the third aspect.
  • the present application provides a host cell comprising the nucleic acid of the third aspect or the expression vector of the fourth aspect.
  • the host cell is a mammalian cell.
  • Mammalian cells can include, but are not limited to, CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, and PER.C6 cells.
  • the application provides a method for preparing the fusion protein of the first aspect or the second aspect, comprising:
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the fifth aspect host cells, and a pharmaceutically acceptable carrier.
  • the present application provides the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect. Use in medicines for the treatment, amelioration or prevention of tumors, autoimmune diseases or infectious diseases.
  • the present application provides a method for treating, ameliorating or preventing tumors, autoimmune diseases or infectious diseases, comprising administering to an individual in need the fusion protein of the first aspect or the second aspect, the third The nucleic acid of the aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect.
  • the application provides the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect for use in therapy , the use of improving or preventing tumors, autoimmune diseases or infectious diseases.
  • Figure 1 shows the schematic structure of the fusion protein constructed in this application, wherein A shows the schematic structure of the trispecific antibody T1 simultaneously targeting CD3 ⁇ CD137 ⁇ CD19, B shows the trispecific antibody simultaneously targeting CD3 ⁇ CD137 ⁇ GPC3 Schematic structure of the specific antibody T2; C shows the schematic structure of the trispecific antibody Triad19, D shows the schematic structure of the bispecific antibody ZWB56.
  • Figure 2 shows the chromatographic pattern of affinity purification of fusion protein using Protein L, wherein A: chromatographic pattern of trispecific antibody T1 purification; B: chromatographic pattern of tri-specific antibody T2 purification.
  • Figure 3 shows the SDS-PAGE electrophoresis of the fusion protein expressed by CHO-K1 cells, wherein M: DNA Marker; 1: non-reducing condition; 2: reducing condition; A: T1 SDS-PAGE electrophoresis result of trispecific antibody; B : T2SDS-PAGE electrophoresis result of trispecific antibody.
  • Figure 4 shows the results of flow cytometry detection of fusion protein binding to cell surface antigens, wherein A1: binding of trispecific antibody T1 to Nalm-6 cells expressing CD19; A2: trispecific antibody T1 binding to CD3-expressing Binding of Jurkat cells; A3: Binding of trispecific antibodies T1 and T2 to HEK293-CD137 cells expressing CD137; B1: Binding of trispecific antibody T2 to Jurkat cells expressing CD3; B2: Trispecific antibody T2 Binding to HepG2 cells expressing GPC3.
  • Figure 5 shows the SDS-PAGE results of trispecific antibody Triad19 and bispecific antibody ZWB56, wherein A: SDS-PAGE results of bispecific antibody ZWB56; B: SDS-PAGE results of trispecific antibody Triad19, two panels Lanes 1-3 of the electropherogram are non-reducing conditions; DNA Marker and reducing conditions, respectively.
  • Figure 6 shows the results of detecting the functional activity of the antibody Triad19/ZWB56 by the CD3 functional cell line Jurkat Dual.
  • Figures 7 and 8 show the fitted drug killing curves for the trispecific antibody Triad19 and the bispecific antibody ZWB56, respectively.
  • Figure 9 shows the killing rate of cells by the antibody Triad19/ZWB56.
  • Figure 10 shows the quantitative results for each factor.
  • FIGS 11-15 show the results of T cell population analysis on T cells after T cell killing experiments.
  • the term "about” refers to ⁇ 10% of the recited number, eg, about 1% refers to the range of 0.9% to 1.1%.
  • fusion protein refers to the purposeful joining together of two or more segments of genes encoding functional proteins, thereby expressing the proteins.
  • the protein product obtained by linking the coding regions of two or more genes end-to-end under artificial conditions and expressing the genes controlled by the regulatory sequence is the fusion protein.
  • peptide linker refers to a short peptide used to link two functional proteins, which can be from 3 amino acids (aa) up to 76 amino acids in length.
  • the peptide linker can provide certain flexibility for each functional protein in the fusion protein, so that it can perform its own function.
  • the peptide linkers used in this application preferably contain only one cysteine, thereby enabling the formation of a stable disulfide bond between the two peptide linkers.
  • antibody refers to any form of antibody or fragment thereof that exhibits the desired biological activity. Accordingly, it is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, fusion proteins (eg, bispecific antibodies) and antibody fragments so long as they exhibit the desired biological activity. Therefore, those skilled in the art will also understand that the term “antibody” as used herein can also refer to fusion proteins of the same or different antibodies or fragments thereof in any form that can exhibit the desired biological activity, thereby achieving the function of a multispecific antibody.
  • antigen refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can also be used in animals to produce an antibody that binds an epitope of that antigen.
  • An antigen can have one or more epitopes.
  • Antigens described herein may include, but are not limited to, most proteins, bacteria, viruses, bacterial exotoxins, polysaccharides (eg, capsular polysaccharides of pneumococcus), lipids, and the like.
  • binding is a term well known in the art, and methods for determining such specific binding of an antibody to an antigen are also well known in the art.
  • “specifically binds” means that the antibody binds to the intended target, but does not significantly bind to other targets.
  • Antibodies bind the intended target epitope with significantly increased affinity and/or for a longer duration than binding to other epitopes.
  • antigen-binding fragment includes fragments or derivatives of antibodies that substantially retain their binding activity.
  • antigen-binding fragment refers to a portion of a full-length antibody, typically the antigen-binding or variable region thereof.
  • antigen-binding fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, single chain antibody molecules such as sc-Fv, and fusion proteins formed from antibody fragments. It is also contemplated that antigen-binding fragments may include conservative amino acid substitutions that do not substantially alter their binding activity.
  • Fab fragment as used herein includes the CH1 and variable regions of the light chain as well as the heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • Fab' fragment contains a light chain as well as a portion or fragment of a heavy chain that contains a VH domain and a CH1 domain and a region between the CH1 and CH2 domains such that between two Fabs An interchain disulfide bond can be formed between the two heavy chains of the 'fragment to form an F(ab')2 molecule.
  • F(ab')2 fragment contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that between the two heavy chains form interchain disulfide bonds.
  • the F(ab')2 fragment thus consists of two Fab' fragments linked together by disulfide bonds between the two heavy chains.
  • Fv fragment as used herein comprises variable regions from heavy and light chains, but lacks constant regions.
  • single-chain Fv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains exist as a single polypeptide chain.
  • Fv polypeptides also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • diabody refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable domain (VH) and a light chain variable domain linked thereto in the same polypeptide chain (VL) (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL polypeptide chain
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region comprises amino acid residues from "complementarity determining regions” or “CDRs” (eg residues 24-34 (LCDR-1), 50-56 (LCDR-2) and 89- 97 (LCDR-3) and residues 31-35 (HCDR-1), 50-65 (HCDR-2) and 95-102 (HCDR-3) in the heavy chain variable domain; Kabat et al, (1991 ) Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., and/or amino acid residues from "hypervariable loops" (i.e.
  • tumor-associated antigen refers to any molecule (eg, protein, peptide, lipid, carbohydrate, etc.) that is expressed solely or predominantly or overexpressed by tumor cells in order to associate the antigen with the tumor.
  • a tumor-associated antigen may be an antigen expressed by only one type of tumor, such that the tumor antigen is associated with or unique to only one type of tumor.
  • the tumor antigens may be tumor antigens associated or specific to various types of tumors.
  • tumor-associated antigens can be expressed by both breast cancer cells and colon cancer cells, but not by normal, non-tumor, or non-cancer cells.
  • Exemplary tumor-associated antigens are tumor cell surface antigens, which are more readily recognized by therapeutic and diagnostic antibodies.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population of antibodies are identical except for possible naturally occurring variations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single epitope.
  • the monoclonal antibodies disclosed herein are not limited to the source of the antibody or the manner in which it was prepared (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.).
  • the term includes intact immunoglobulins as well as fragments thereof and the like under the definition of "antibody”.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or by an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids incorporating recombinant polynucleotides, plasmids (eg, naked or contained in liposomes) and viruses (eg, lentiviruses, retroviruses, adenoviruses and adeno-associated virus).
  • the present application provides a fusion protein capable of targeting both CD3 and CD137, which connects an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD137 antibody or an antigen-binding fragment thereof through a linker such as a peptide linker.
  • a fusion protein comprising an anti-CD3 antibody or antigen-binding fragment thereof and an anti-CD137 antibody or antigen-binding fragment thereof can also be prepared by further linking another antibody or antigen-binding fragment thereof at the C-terminus or N-terminus of the fusion protein through a linker such as a peptide linker trispecific antibodies.
  • the present application cleverly utilizes the stabilization of disulfide bonds in the hinge region of the antibody, and uses the mutated antibody hinge as a linker to connect the antibodies in the fusion protein of the present application to each other, so that only the mutated antibody is between the heavy chain and the light chain. A pair of disulfide bonds are formed at the hinge region.
  • the application provides a fusion protein comprising:
  • first peptide linker is used to link the heavy chain of the anti-CD137 antibody or antigen-binding fragment thereof and the heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof
  • second peptide linker is used to link the the light chain of an anti-CD137 antibody or antigen-binding fragment thereof and the light chain of said anti-CD3 antibody or antigen-binding fragment thereof
  • the fusion protein further comprises:
  • the heavy chain of the first antibody or antigen-binding fragment thereof is linked to the heavy chain of the anti-CD3 antibody or antigen-binding fragment thereof through the third peptide linker
  • the light chain of the first antibody or antigen-binding fragment thereof is linked to the light chain of the anti-CD3 antibody or antigen-binding fragment thereof through the fourth peptide linker
  • the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker are each independently selected from the group consisting of sequences set forth in Seq ID NO. 1-2 (Seq ID NO. 1-2).
  • Seq ID NO. 1-2 Seq ID NO. 1-2
  • 1 Xaa Pro Pro Cys Pro Ala Pro Glu
  • Seq ID NO.2 Glu Pro Ala Pro Cys Pro Pro Pro Xaa, wherein Xaa can be any amino acid except Cys or absent) any one of the peptide linkers, where X represents any amino acid except Cys, or a deletion.
  • each of the first to fourth peptide linkers is a hinge region of a native antibody, wherein the hinge region has a deletion mutation that retains only one cysteine.
  • any of the first to fourth peptide linkers are independently selected from a C229 deletion mutated IgGl hinge region or a C229 deletion mutated IgGl hinge region with the hinge region D224-S242 inverted.
  • the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or single chain antibody molecules such as sc-Fv.
  • the fusion protein is a bispecific antibody or a trispecific antibody.
  • the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUCl, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met and GPC3.
  • the first antigen, CD137 molecule and CD3 molecule are independently derived from mammals, preferably non-human primates or humans.
  • the affinity constant of the first antibody or antigen-binding fragment thereof for binding to the first antigen is the affinity constant of the anti-CD137 antibody or antigen-binding fragment thereof for a CD137 molecule or an anti-CD3 antibody or antigen thereof 10-1000 times the affinity constant of the binding fragment to the CD3 molecule.
  • antibody affinity is essentially a non-covalent force. It reflects the ability of antibody molecules to bind to antigens, and methods for determining the affinity of antibodies to specific antigens are well known in the art, including but not limited to biofilm interference technology (BLI), solid-phase radioimmunoassay (SP-RIA), Equilibrium dialysis, combined antigen precipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), etc.
  • BBI biofilm interference technology
  • SP-RIA solid-phase radioimmunoassay
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • the first peptide linker and the second peptide linker are the same.
  • the first peptide linker and the second peptide linker are not the same.
  • the third peptide linker and the fourth peptide linker are the same.
  • the third peptide linker and the fourth peptide linker are not the same.
  • first peptide linker and the second peptide linker are the same, the third peptide linker and the fourth peptide linker are the same, however the first peptide linker is different from the third peptide linker.
  • all four of the first to fourth peptide linkers are the same.
  • first peptide linker and the second peptide linker are C229 deletion mutated IgG1 hinge regions
  • the third peptide linker and fourth peptide linker are C229 deletion mutated IgG1 hinges with an inverted hinge region D224-S242 Area.
  • first peptide linker and the second peptide linker are C229 deletion mutated IgG1 hinge regions with an inverted hinge region D224-S242, and the third and fourth peptide linkers are C229 deletion mutated IgG1 hinges Area.
  • the amino acid sequences of antibodies are numbered to identify equivalent positions, and a number of different numbering schemes currently exist for antibodies.
  • the Kabat protocol (Kabat et al., 1991) was developed based on the location of regions of high sequence variation between sequences of the same domain type. It is numbered differently for the heavy chain (VH) and light chain (V ⁇ and V ⁇ ) variable domains.
  • the Chothia scheme (Al-Lazikani, 1997) is the same as the Kabat scheme, but corrects the position where the annotation is inserted around the first VH complementarity determining region (CDR) to correspond to the structural loop.
  • Antibodies in this application are numbered according to the Kabat scheme.
  • the fusion protein described above may be a trispecific antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, the heavy chain of an anti-CD137 antibody or antigen-binding fragment thereof , a first peptide linker, a heavy chain of an anti-CD3 antibody or an antigen-binding fragment thereof, a third peptide linker, and a heavy chain of the first antibody or an antigen-binding fragment thereof, and the light chain sequentially comprises an anti-CD137 antibody or The light chain of its antigen-binding fragment, the second peptide linker, the light chain of the anti-CD3 antibody or its antigen-binding fragment, the fourth peptide linker, and the light chain of the first antibody or its antigen-binding fragment.
  • the fusion protein described above may be a trispecific antibody comprising a heavy chain and a light chain, wherein the heavy chain, in order from N-terminal to C-terminal, comprises the heavy chain of the first antibody or antigen-binding fragment thereof , a first peptide linker, a heavy chain of an anti-CD137 antibody or an antigen-binding fragment thereof, a third peptide linker, and a heavy chain of an anti-CD3 antibody or an antigen-binding fragment thereof, and the light chain sequentially comprises the first antibody or The light chain of the antigen-binding fragment thereof, the second peptide linker, the light chain of the anti-CD137 antibody or the antigen-binding fragment thereof, the fourth peptide linker, and the light chain of the anti-CD3 antibody or the antigen-binding fragment thereof.
  • the fusion protein described above may be a trispecific antibody comprising a heavy chain and a light chain, wherein the heavy chain, in order from N-terminal to C-terminal, comprises the heavy chain of the first antibody or antigen-binding fragment thereof , a first peptide linker, a heavy chain of an anti-CD3 antibody or an antigen-binding fragment thereof, a third peptide linker, and a heavy chain of an anti-CD137 antibody or an antigen-binding fragment thereof, and the light chain sequentially comprises the first antibody or The light chain of the antigen-binding fragment thereof, the second peptide linker, the light chain of the anti-CD3 antibody or its antigen-binding fragment, the fourth peptide linker, and the light chain of the anti-CD137 antibody or its antigen-binding fragment.
  • the fusion protein described above may be a trispecific antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, the heavy chain of an anti-CD3 antibody or antigen-binding fragment thereof , a first peptide linker, a heavy chain of an anti-CD137 antibody or an antigen-binding fragment thereof, a third peptide linker, and a heavy chain of the first antibody or an antigen-binding fragment thereof, and the light chain sequentially comprises an anti-CD3 antibody from the N-terminus to the C-terminus or the light chain of its antigen-binding fragment, the second peptide linker, the light chain of the anti-CD137 antibody or its antigen-binding fragment, the fourth peptide linker, and the light chain of the first antibody or its antigen-binding fragment.
  • the heavy chain comprises, in order from N-terminus to C-terminus, the heavy chain of an anti-CD3 antibody or antigen-binding fragment thereof ,
  • the peptide linkers described herein may be selected from a C229 deletion mutated IgGl hinge region or a C229 deletion mutated IgGl hinge region with the hinge region D224-S242 inverted.
  • the peptide linkers cross-linked by disulfide bonds are expected to be identical.
  • the first peptide linker and the second peptide linker are expected to be cross-linked by disulfide bonds, so both are chosen to be the same peptide linker.
  • the third peptide linker and the fourth peptide linker are expected to be cross-linked by disulfide bonds, so both are selected as the same peptide linker.
  • the first antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, and anti-CD3 antibody or antigen-binding fragment thereof may each be independently derived from monoclonal antibodies.
  • the monoclonal antibodies used in this application may be selected from one or more of the following: blincyto, adalimumab, secukinumab, Rituximab, Trastuzumab, Gemtuzumab-ozogamicin, Alemtuzumab, Bevacizumab, Silica Cetuximab, Panitumumab, Ofatumumab, Ipilimumab, Brentuximab vedotin, Denosumab Denosumab, Pertuzumab, Obinutuzumab, Ramucirumab, 3F8, abagovomab, adecatumumab, afutuzumab Afutuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab, betuximab ( bectumomab), belimumab, bevacizum
  • the antigens that the fusion protein of the present application can bind to can be cell-associated proteins, such as cell surface proteins on the membrane of cells (T cells, endothelial cells or tumor cells), or soluble proteins.
  • An antigen can also be any medically relevant protein, such as those proteins that are up-regulated during disease or infection, such as receptors and/or their corresponding ligands.
  • cell surface proteins include, but are not limited to, adhesion molecules such as integrins, E-selectin, P-selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CD69, CD134, ICOS, CD137, CD27, carcinoembryonic antigen (CEA), TCR, MHC class I and MHC class II antigens, VEGF, and receptors for these proteins.
  • adhesion molecules such as integrins, E-selectin, P-selectin or L-selectin
  • Soluble proteins include interleukins (eg, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-16, or IL-17), viral Antigens (eg, respiratory syncytial virus or cytomegalovirus antigens), immunoglobulins (eg, IgE), interferons (eg, interferon alpha, interferon beta, or interferon gamma), tumor necrosis factor-alpha (TNF ⁇ ), tumor necrosis Factor-beta, colony stimulating factor (eg G-CSF or GM-CSF) and platelet derived growth factor (eg PDGF-alpha and PDGF-beta) and their receptors (where appropriate).
  • Other antigens include bacterial cell surface antigens, bacterial toxins, viruses (eg, influenza virus, EBV, HepA, B and C), bioterrorism agents, radionuclides and heavy metals, and snake and spider
  • antigens that can be bound by the fusion proteins of the present application include serum carrier proteins, polypeptides that allow cell-mediated recruitment of effector functions, and radionuclide chelating proteins.
  • the antigens that can be bound by the fusion proteins of the present application are tumor-associated antigens, which include any one or more of the following: CD20, MESOTHELIN, EGFR, CD33, CD52, VEGF, ROR1, CD30, RANKL, MESOTHELIN, VEGF-R2, Her3, A33 antigen, CD5, CD19, CD22, CD23 (IgE receptor), CA242 antigen, 5T4, VEGFR-1, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74 , CD80, CD152, CD200, CD221, CCR4, NPC-1C, vimentin, insulin-like growth factor-1 receptor (IGF-1R), alpha-fetoprotein, carcinoembryonic antigen (CEA), integrin ⁇ v ⁇ 3 , Integrin ⁇ 5 ⁇ 1 , Fibroblast Activation Protein, FAP- ⁇ , TAG-72, MUC1, MUC16, Prostate Specific
  • the CD3 molecule consists of 4 subunits: ⁇ , ⁇ , ⁇ , ⁇ , its molecular weights are 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, and its lengths are 171, 207, 182, and 164 amino acid residues, respectively. Together they form 6 peptide chains, which are often tightly combined with the T cell receptor (TCR) to form a TCR-CD3 complex composed of 8 peptide chains. This complex has the ability to conduct T cell activation signals and stabilize the TCR structure. function.
  • the CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histo-compatibility complex) molecules, resulting in the conservation of the ITAM of CD3
  • ITAM immunoreceptor tyrosine-based activation motif
  • the tyrosine residues of the sequence are phosphorylated by the tyrosine protein kinase p56LCK in T cells, which can then recruit other SH2 (Scrhomology2) domain tyrosine protein kinases (eg, ZAP-70).
  • SH2 Strhomology2 domain tyrosine protein kinases
  • Phosphorylation of ITAM and binding to ZAP-70 is one of the important biochemical reactions in the early stage of T cell activation signaling process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by TCR recognition of the antigen.
  • CD137 (TNFRSF9 or 4-1BB) is a member of the tumor necrosis factor (TNF) receptor family and is a type I transmembrane protein composed of 255 amino acids.
  • the relative molecular mass of CD137 monomer is 30kDa, and it can also exist in the form of dimer, and the relative molecular mass is about 55kDa.
  • the CD137 molecules from the cell surface membrane fall off and enter the blood to form soluble CD137 (sCD137), which is almost absent in normal humans.
  • sCD137 soluble CD137
  • the activation of T lymphocytes not only requires the binding of the main MHC/Ag to TCR, but also requires the participation of second signals, such as the interaction of B7-CD28, CD137-CD137L, etc.
  • CD137 The role of CD137 is different from that of CD28.
  • CD28 is expressed on the surface of unactivated T lymphocytes and mainly affects the early immune response of cells, while CD137 is mainly expressed on activated T lymphocytes, so it mainly produces effects in the middle and late stages of T lymphocyte response.
  • the expression of CD137 on naive T cells is very low, but when T cells are activated, the expression of CD137 is up-regulated, mainly distributed on the surface of CD4+T and CD8+T cells.
  • the combination of CD137 and CD137L can produce a series of biological effects, including the induction of T cell activation, the release of chemokines and cytokines, and the aggravation of immune responses.
  • CD137-CD137L signaling is also involved in autoimmune diseases, such as type I autoimmune diabetes.
  • CD19 is one of the important membrane antigens involved in B cell activation and proliferation. It is a common surface marker for all B cells. It does not disappear after B cell activation. It is the most important B cell marker. A component of the body, the extracellular portion of CD19 binds to other membrane antigens for signal transduction. Elevated CD19 positive cells are found in malignant tumors of the B lymphocyte system, such as CD19 expression in 95% of acute pre-B lymphocytic leukemia cells and 94% of acute mature B lymphocytic leukemia cells, and also in chronic lymphocytic leukemia and Burkitt lymphoma, etc.
  • CD19 positive cells decreased in humoral immunodeficiency diseases, such as agammaglobulinemia, long-term use of immunosuppressants and so on. Therefore, CD19 detection can make a clear etiological diagnosis of the above diseases and provide a basis for differential diagnosis.
  • CD19 since CD19 is widely present on the surface of malignant tumor cells of the B lymphocyte system, it can be used as a cell surface target in the immunotherapy of leukemia and lymphoma.
  • GPC3 (Glypican-3, Glypican-3) is a member of the Glypican family and is a membranous heparan sulfate polysaccharide protein. During the embryonic period of mammals, it can play an important role in regulating the occurrence and growth of tissues and organs by affecting multiple molecular signaling pathways. Gene mutation and loss of function lead to overgrowth and aberration syndrome. The abnormal expression of GPC3 is closely related to the occurrence and development of various tumors. GPC3 is believed to play an important role in the diagnosis and treatment of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • PD-1 programmed death receptor 1
  • PD-1 programmed death receptor 1
  • PD-1 ECD extracellular region of PD-1
  • the extracellular region of PD-1, the PD-1 ECD serves as the binding moiety in the present application.
  • c-Met is a protein product encoded by the c-met proto-oncogene. It is a hepatocyte growth factor receptor with tyrosine kinase activity. It is related to a variety of oncogene products and regulatory proteins, and is involved in cell information transduction, cellular The regulation of cytoskeletal rearrangement is an important factor in cell proliferation, differentiation and motility. It is currently believed that c-Met is closely related to the occurrence and metastasis of various cancers.
  • the fusion proteins of the present application can incorporate CH2-CH3 domains.
  • the CH2-CH3 domain can be linked to the heavy chain of the first antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, anti-CD3 antibody or antigen-binding fragment thereof.
  • the CH2-CH3 domains are optionally mutated with KiH, introduce cysteine residues, introduce one or more salt bridge mutations to promote heterodimerization, such additions result in increased heterodimerization. stability.
  • Salt bridges herein include hydrogen bonds and electrostatic interactions, such as salt bridges that can occur between glutamic acid and lysine residues.
  • the heavy and light chains of native antibodies include variable regions (ie, V regions) and constant regions (ie, C regions), respectively.
  • the constant region of the heavy chain and the constant region of the light chain are called CH and CL, respectively.
  • the CL lengths of different types ( ⁇ or ⁇ ) of Ig are basically the same, but the CH lengths of different types of Ig are different.
  • IgG, IgA and IgD include CH1, CH2 and CH3
  • IgM and IgE include CH1, CH2, CH3 and CH4.
  • the application provides a fusion protein, which sequentially comprises from the N-terminus to the C-terminus:
  • disulfide bond can be formed between the first peptide linker and the second peptide linker, and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker, each independently is selected from the following: a peptide linker comprising any one of the sequences shown in Seq ID NO. 1-2, wherein X represents any amino acid except Cys, or deletion.
  • each of the first to fourth peptide linkers is a hinge region of a native antibody, wherein the hinge region has a deletion mutation that retains only one cysteine.
  • any of the first to fourth peptide linkers are independently selected from a C229 deletion mutated IgGl hinge region or a C229 deletion mutated IgGl hinge region with the hinge region D224-S242 inverted.
  • the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUCl, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met and GPC3.
  • the first antigen, the CD137 molecule and the CD3 molecule are independently derived from a mammal, preferably a non-human primate or a human.
  • the affinity constant of the Fab fragment for binding to the first antigen is the affinity constant of the anti-CD137 antibody or antigen-binding fragment thereof and the CD137 molecule or the affinity constant of the anti-CD3 antibody or antigen-binding fragment thereof and the CD3 molecule 10-1000 times the affinity constant.
  • the antigen-binding fragment is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or single chain antibody molecules such as sc-Fv.
  • the application provides a nucleic acid encoding the fusion protein of the first aspect or the second aspect.
  • the nucleic acid may be a codon-optimized nucleic acid suitable for expression in a host cell.
  • a codon-optimized nucleic acid suitable for expression in a host cell.
  • codons depending on the degeneracy of the codons, it still encodes the same protein.
  • Methods for codon optimization depending on the host cell used are well known to those skilled in the art.
  • the application provides an expression vector comprising the nucleic acid of the third aspect.
  • prokaryotic cloning vectors include plasmids from E. coli, such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4.
  • Prokaryotic vectors also include derivatives of bacteriophage DNA such as M13 and other filamentous single-stranded DNA bacteriophages.
  • An example of a vector that can be used in yeast is the 2 ⁇ plasmid.
  • Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences and those derived from functional mammalian vectors (such as those described above) and functional plasmids and Combinatorial shuttle vectors for phage DNA.
  • Expression vectors useful in the present application contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of cloned DNA sequences. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operon and promoter regions of bacteriophage lambda, the control regions of the fd coat protein, the glycolytic promoters of yeast, such as 3-phosphate Glycerate kinase promoters, yeast acid phosphatase promoters, such as Pho5, yeast alpha-mating factor promoters, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, such as SV40 Early and late promoters and other sequences known to control gene expression in prokaryotic or eukaryotic cells and their viruses or combinations thereof.
  • useful expression control sequences are the lac system, the trp system, the tac
  • the present application provides a host cell comprising the nucleic acid of the third aspect or the expression vector of the fourth aspect.
  • the host cell is a mammalian cell.
  • Mammalian cells can include, but are not limited to, CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select suitable host cells as required.
  • the application provides a method for preparing the fusion protein of the first aspect or the second aspect, comprising:
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the fifth aspect host cells, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the seventh aspect can be prepared into a desired dosage form by conventional methods in the pharmaceutical field.
  • the pharmaceutical composition is preferably in a liquid or suspension dosage form.
  • the pharmaceutically acceptable carrier is a carrier that does not impair the viability and function of immune cells, and does not affect the specific binding of the antibody or its antigen-binding fragment to the antigen, including but not limited to cell culture media, buffers, Physiological saline and balanced salt solutions, etc.
  • buffers include isotonic phosphates, acetates, citrates, borates, carbonates, and the like.
  • the pharmaceutically acceptable carrier is 1% serum in phosphate buffered saline.
  • fusion proteins disclosed herein and pharmaceutical compositions thereof can be used to treat, ameliorate or prevent tumors, autoimmune diseases or infectious diseases in an individual.
  • the pharmaceutical composition of the seventh aspect may further comprise a second agent for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease in an individual.
  • the present application provides the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect. Use in medicines for the treatment, amelioration or prevention of tumors, autoimmune diseases or infectious diseases.
  • the present application provides a method for treating, ameliorating or preventing a tumor, autoimmune disease or infectious disease in an individual, comprising administering to the individual a therapeutically effective amount of the first or second aspect
  • the fusion protein, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect or the host cell described in the fifth aspect comprising administering to the individual a therapeutically effective amount of the first or second aspect.
  • the method further comprises administering a second agent that treats, ameliorates, or prevents a tumor, an autoimmune disease, or an infectious disease.
  • the application provides the fusion protein of the first aspect or the second aspect, the nucleic acid of the third aspect, the expression vector of the fourth aspect or the host cell of the fifth aspect for use in therapy , the use of improving or preventing tumors, autoimmune diseases or infectious diseases.
  • Treatment refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (lessen) the target pathological state or disorder.
  • Individuals in need of treatment include those who already have the disorder, as well as those who will develop the disorder or whose disorder is to be prevented. Thus, the individual to be treated herein has been diagnosed with the disorder or is predisposed to or susceptible to the disorder.
  • the term "individual” as used herein refers to mammals including, but not limited to, primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice.
  • the mammal is a non-human primate or human.
  • a particularly preferred mammal is a human.
  • the tumor is a primary cancer or a metastatic cancer.
  • the tumor is selected from the group consisting of lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic cancers such as leukemia, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, B lymphocyte type non-Hodgkin Lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, head and neck cancers such as head and neck squamous cell carcinoma, glioblastoma, kidney cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreas Carcinoma, sarcoma, liver cancer, squamous cell carcinoma of the skin, thyroid cancer, cervical cancer, nasopharyngeal cancer, endometrial cancer, or metastatic cancer of the above tumors.
  • lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopo
  • the autoimmune disease may include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, systemic vasculitis, dermatomyositis, autoimmune hemolytic anemia, and the like.
  • infectious diseases include respiratory infectious diseases, digestive tract infectious diseases, blood infectious diseases, body surface infectious diseases, sexually transmitted diseases, and the like.
  • infectious diseases may include, but are not limited to, influenza, tuberculosis, mumps, measles, whooping cough, ascariasis, bacillary dysentery, hepatitis A, hepatitis B, malaria, encephalitis B Inflammation, filariasis, schistosomiasis, trachoma, rabies, tetanus, gonorrhea, syphilis, AIDS, etc.
  • the “therapeutically effective amount” used herein can be determined according to the specific situation, and can be easily grasped by those of ordinary skill in the art according to the actual required dosage, such as the patient's weight, age and disease condition.
  • Example 1 Preparation, expression and identification of trispecific antibodies targeting CD3 ⁇ CD137 ⁇ CD19
  • the VH and VL encoding nucleic acid sequences for the anti-CD3 and anti-CD19 antibodies were derived from Blincyto (Amgen).
  • the VH-encoding nucleic acid sequence of anti-CD19, the VH-encoding nucleic acid sequence of anti-CD3, and the encoding nucleic acid sequence of IgG1 CH1-Hinge mut were constructed into the plasmid pQKE3H by total synthesis (Universal Biotechnology).
  • VL coding nucleic acid sequence of anti-CD19 the VL coding nucleic acid sequence of anti-CD3 and the coding nucleic acid sequence of Kappa-Hinge mut (including the IgG1 hinge region of C229 deletion mutation) were constructed into plasmid pQKE3L by total synthesis China (General Biosystems (Anhui) Co., Ltd.).
  • Synthetic vector pUC57 IgG1 Hinge mut R-CD137 VH containing IgG1 Hinge mut R (C229 deletion mutation, and hinge region D224-S242 inversion) encoding nucleic acid sequence by DNA synthesis and anti-CD137 VH encoding nucleic acid in anti-CD137 monoclonal antibody Sequence see patent for sequence, patent number: US20190284292A1) (Universal Biosystems (Anhui) Co., Ltd.); synthetic vector pUC57 IgG1 Hinge mut R-CD137 VL, which contains IgG1 Hinge mut R (C229 deletion mutation) synthesized by DNA, and the hinge Region D224-S242 inversion) encoding nucleic acid sequence and anti-CD137 VL encoding nucleic acid sequence in anti-CD137 monoclonal antibody (sequence see patent, patent number: US20190284292A1) (Universal Biosystems (Anhui) Co., Ltd.).
  • the nucleotide sequences of IgG1 CH1-Hinge mut and Kappa-Hinge mut are as follows:
  • the nucleotide sequence of IgG1 Hinge mut R is as follows:
  • the synthesized vector plasmid pQKE3H was digested with restriction endonuclease EcoRI (NEB, R3101S), and the PCR amplification product obtained and the digested vector pQKE3H were digested with BM seamless cloning kit (Bomed Corporation) , according to the kit's instructions, carry out recombinant ligation to obtain the heavy chain expression vector pQKTriad1H (ligation sequence from 5' to 3'): anti-CD19 antibody VH-CH1-Hinge mut-anti-CD3 antibody VH-IgG1Hinge mut R-anti-CD137
  • PCR amplification primer pairs are as follows:
  • the synthesized vector plasmid pQKE3L was digested with restriction endonuclease EcoRI (NEB, R3101S), and the PCR amplification product and the digested vector pQKE3L were digested with BM seamless cloning kit (Bomed Corporation) , according to the instructions of the kit, carry out recombinant connection to obtain the light chain expression vector pQKTriad1L (the connection sequence is from 5' to 3': anti-CD19 antibody VL-Kappa-Hinge mut-anti-CD3 antibody VL-IgG1Hinge mut R-anti-CD137 antibody VL).
  • PCR amplification primer pairs are as follows:
  • the heavy chain expression vector pQKTriad1H and the light chain expression vector pQKTriad1L obtained above were transformed into E. coli TOP10, respectively. After single clones were picked and identified, they were cultured in LB medium containing ampicillin (final concentration of 100 mg/L) for 16 hours, and the culture conditions were 37° C., 200 rpm shaking culture. Bacteria were collected by centrifugation at 8000 x g for 20 minutes.
  • the plasmids were isolated and extracted, eluted with 1 mL of sterile ultrapure water, and finally the plasmid concentration was measured using a Nanodrop microspectrophotometer.
  • the heavy chain expression vector pQKTriad1H and the light chain expression vector pQKTriad1L were co-transfected into HEK293 cells for expression. Twenty - four hours before transfection, 1.5 x 106 HEK293 (ATCC, code: CRL-1573) cells were seeded in 500 mL shake flasks containing 100 mL of OPM-293 CD05 serum-free medium (Opmel, Cat: 81075-001). , the culture condition is 36.5 °C, 7.5% CO 2 , 120rpm suspension culture.
  • the recombinant plasmids pQKTriad1H and pQKTriad1L were mixed in 10 mL OPM-293 CD05 medium at a weight ratio of 1:1 (total DNA 100 ⁇ g), followed by 100 ⁇ L PEI (3 mg/mL), and vortexed quickly. Mix well and incubate at room temperature for 15 minutes. This mixture was then added to the above cell culture. The cells were incubated at 36.5[deg.]C, 7.5% CO2 , 120 rpm/min for 7 days to harvest the expressed antibody.
  • the antibody refers to the anti-CD19 ⁇ anti-CD3 ⁇ anti-CD137 trispecific antibody expressed by plasmids pQKTriad1H and pQKTriad1L, the antibody is named T1, and the structure is shown in A in FIG. 1 .
  • the harvested cell cultures were centrifuged at 3000 xg for 20 min, and the supernatant was collected and filtered through a 0.45 ⁇ m filter.
  • the filtered sample solution was loaded at a flow rate of 5 mL/min.
  • the Capto L affinity column was washed with a mixed buffer (pH 7.4) of 20 mM PB and 150 mM NaCl at a flow rate of 5 mL/min.
  • Antibodies purified by Capto L were checked for purity by HPLC (Agilent 1260II) SEC.
  • the chromatographic column was a Sepax water-soluble size exclusion chromatographic column, the mobile phase was 50 mm PB+300 mm NaCl pH 7.0, the sample loading was 10 ⁇ g, the flow rate was 1 mL/min, and the isocratic elution was 20 min. The results showed that the monomer purity was about 86% after one-step purification.
  • the affinity constant K D of the purified antibody was determined using the Molecular Interaction Instrument Fortebio Octet QK (Molecular Devices).
  • the T1 antibody was immobilized by the sensor of Fab-Ch1 at a concentration of 0.25 ⁇ M.
  • Antigen human CD19 (Yiqiao Shenzhou, Cat: 10084-HNAH), human CD3 (Yiqiao Shenzhou, Cat: 10977-H02H) and human CD137 (Yiqiao Shenzhou, Cat: 10041-H002H) according to 600nM, 300nM, 150nM and A concentration of 75 nM was loaded.
  • the results of affinity constant determination are shown in Table 1.
  • Nalm-6 cells (ATCC number: CRL-3273) that were recovered normally and subcultured for at least 3 passages were pipetted evenly and the cells were collected, centrifuged at 400g/min for 5min, resuspended and counted, and adjusted the cell density to 1 ⁇ 10 6 cells/ mL.
  • the groups were set as blank group, positive control group, secondary antibody group and experimental group (T1). First, 100 ⁇ l of the above cell suspension was added to the tubes of each group.
  • T1 antibody to the tube of the T1 group, incubate at room temperature for 30 min, wash with 3 mL of PBS buffer containing 2% FBS, centrifuge at 400 g/min for 5 min, and resuspend in 50 ⁇ l of PBS buffer containing 2% FBS; blank group Add 0.5 ⁇ l of water; add 0.5 ⁇ l of FITC anti-human CD19 (BioLegend, clone number: HIB19) to the positive control group; add 0.5 ⁇ l of APC anti-human Ig light chain K (BioLegend, clone number: TB28) to the T1 group and the secondary antibody group respectively -2).
  • FITC anti-human CD19 BioLegend, clone number: HIB19
  • APC anti-human Ig light chain K BioLegend, clone number: TB28
  • the groups were mixed and incubated at room temperature for 30 min in the dark. After incubation, the tubes of each group were washed with PBS buffer containing 2% FBS, centrifuged at 400 g/min for 5 min, resuspended in 100 ⁇ l PBS buffer containing 2% FBS, and loaded on a flow cytometer (Eisen, instrument model: NovoCyte). detection. The result is shown as A1 in Figure 4 .
  • Jurkat cells that were recovered normally and subcultured for at least 3 passages were pipetted evenly and the cells were collected, centrifuged at 400g/min for 5min, resuspended and counted, and adjusted the cell density to 1 ⁇ 10 6 cells /mL.
  • the groups were set as blank group, secondary antibody group and experimental group (T1). First, 100 ⁇ l of the above cell suspension was added to the tubes of each group.
  • T1 antibody 100 ng to the tube of the T1 group, incubate at room temperature for 30 min, wash with 3 mL of PBS buffer containing 2% FBS, centrifuge at 400 g/min for 5 min, and resuspend in 50 ⁇ l of PBS buffer containing 2% FBS; blank group 0.5 ⁇ l of water was added; 0.5 ⁇ l of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) was added to the T1 group and the secondary antibody group, respectively. The groups were mixed and incubated at room temperature for 30 min in the dark.
  • the normally recovered HEK293 cells (Concord Cell Resource Center, resource number: 3111C0001CCC000010) were subcultured for at least 3 passages. The cells were passaged 24 hours before transfection and seeded into 6-well plates. On the day of transfection, PEI (Sigma, Cat: 764647) and the synthetic plasmid pENTER CD137 (General Biosystems (Anhui) Co., Ltd.) were recovered to room temperature, and 5 ⁇ g of the plasmid was added to 500 ⁇ l of DMEM medium (gibco, REF: 11965-092 ), then added 15 ⁇ g of PEI, immediately vortexed for 15 minutes, then gently added the mixture dropwise to the cell culture medium, and placed it in the incubator for 24-48 hours.
  • DMEM medium gibco, REF: 11965-092
  • DMEM medium containing 10% FBS at 2 ⁇ g/mL puromycin was replaced after 48 h. After 3 days, the cells die on a large scale. You can gently tap the bottle wall and discard the supernatant. The cells with good adherence are possible stable transfected cell lines. After 8-10 days, depending on the growth state of the cells, the cells were digested and plated into 96-well plates, and the monoclonal cell lines were screened. During the period, pressurized DMEM medium containing 2ug/mL puromycin 10% FBS was used. The resulting cell line was named HEK293-CD137.
  • the human CD137 extracellular domain gene is transferred into the genome of this cell line, which can stably express the human CD137 extracellular domain protein and display it on the cell membrane.
  • HEK293-CD137 cells that were recovered and cultured for more than 3 passages were washed once with 10 mL of PBS, digested with 1 mL of 0.05% trypsin for 1 min, added 4 mL of DMEM medium containing 10% FBS, pipetted evenly, and then collected the cells, and centrifuged at 1000 rpm/min for 5 min , resuspend and count, adjust the cell density to 1 ⁇ 10 6 cells/mL.
  • the groups were set as blank group, positive control group, secondary antibody group and experimental group (T1).
  • the groups were mixed and incubated at room temperature for 30 min in the dark. After incubation, the tubes of each group were washed with PBS buffer containing 2% FBS, centrifuged at 1000 rpm/min for 5 min, resuspended in 100 ⁇ l PBS buffer containing 2% FBS, and loaded on a flow cytometer (Eisen, instrument model: NovoCyte). detection. The result is shown as A3 in Figure 4.
  • the nucleic acid sequence encoding GPC3 scFv was used to amplify the anti-GPC3 VH fragment; the plasmid pQKTriad1 H (see Example 1) was used as a template to amplify the CH1-Hinge mut-CD3 VH-CD137 VH fragment, and the total synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.) carried out double digestion with SapI and EcoRI, recovered the corresponding PCR products and digestion products, carried out recombinase ligation, and obtained the final recombinant plasmid named pQKTriad2H (ligation sequence from 5' to 3' is : anti-GPC3 antibody VH-CH1-Hinge mut-anti-CD3 antibody VH-IgG1Hinge mut R-anti-CD137 antibody VH).
  • the synthetic vector pUC57 GPC3 scFv (see the patent for the sequence, patent number: US7919086B2) (General Biosystems (Anhui) Co., Ltd.), which contains the anti-GPC3 scFv-encoding nucleic acid sequence synthesized by DNA, is used to amplify the anti-GPC3 VL fragment; pQKTriad1 L (see Example 1) was used as the template to amplify the Kappa-Hinge mut-CD3 VL-CD137 VL fragment, and the fully synthetic vector pQKX2 (General Biosystems (Anhui) Co., Ltd.) was double digested with SapI and EcoRI, Recover the corresponding PCR products and digestion products, carry out recombinant enzyme ligation, and obtain the final recombinant plasmid named pQKTriad2L (the connection sequence is from 5' to 3': anti-GPC3 antibody VL-Kappa-Hinge mut-anti-CD3
  • PCR amplification primer pairs are as follows:
  • the transfection was carried out with reference to the procedure described in Example 1, wherein the transfected cells were HEK293 (ATCC number: CRL-1573), and the transfection volume was 100 mL.
  • the transfected cells were suspended and cultured in a 500 mL shake flask for 7 days, and the antibody was harvested under the culture conditions of 36.5° C., 7.5% CO 2 , and 120 rpm/min.
  • the obtained antibody refers to the anti-GPC3 ⁇ anti-CD3 ⁇ anti-CD137 trispecific antibody expressed by plasmids pQKTriad2H and pQKTriad2L, the antibody is named T2, and the structure is shown in B in FIG. 1 .
  • Example 1 Purification was carried out with reference to the procedure described in Example 1 (B in Figure 2, B in Figure 3). After buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer, and the calculated protein yield was 50 mg/L.
  • the purity of antibody T2 was determined by HPLC, and its monomer purity was about 76% after one-step purification.
  • T2 antibody was immobilized by the sensor of Fab-Ch1 at a concentration of 0.25 ⁇ M.
  • Antigens human GPC3 (Yiqiao Shenzhou, Cat: 10088-H08H), human CD3 (Yiqiao Shenzhou, Cat: 10977-H02H) and human CD137 (Yiqiao Shenzhou, Cat: 10041-H002H) according to 600nM, 300nM, 150nM and A concentration of 75 nM was loaded.
  • the results of affinity constant determination are shown in Table 2.
  • Example 2 For specific materials and operation procedures, see 1.5.2 in Example 1, wherein the experimental group (T2) antibody is T2. The result is shown as B1 in Figure 4 .
  • Example 1 For specific materials and operation procedures, see 1.5.3 in Example 1, wherein the experimental group (T2) antibody is T2. The result is shown as A3 in Figure 4.
  • HepG2 cells Concord Cell Resource Center, resource number: 3111C0001CCC000035
  • the groups were set as blank group, secondary antibody group and experimental group (T2).
  • T2 secondary antibody group and experimental group
  • T2TsAb 100 ng of T2TsAb to the tube of the T2 group, incubate for 30 min at room temperature, wash with 3 mL of PBS buffer containing 2% FBS, centrifuge at 1000 rpm/min for 5 min, and resuspend in 50 ⁇ l of PBS buffer containing 2% FBS; the blank group added 0.5 ⁇ l of water; 0.5 ⁇ l of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) was added to the T2 group and the secondary antibody group, respectively. The groups were mixed and incubated at room temperature for 30 min in the dark.
  • Example 3 Preparation, expression and identification of trispecific antibody targeting CD3 ⁇ CD137 ⁇ c-Met and bispecific antibody targeting CD3 ⁇ c-Met
  • Example 1 for the specific operation flow of expression vector construction and plasmid amplification.
  • the VH and VL encoding nucleic acid sequences of the anti-c-Met antibody were derived from Onartuzumab (MetMAb) (patent number: US7615529B2).
  • the synthetic vector pUC57 c-Met scFv (Universal Biosystems (Anhui) Co., Ltd.), which contains the anti-c-Met scFv-encoding nucleic acid sequence synthesized by DNA, is used to amplify the anti-c-Met VH fragment; with plasmid pQKTriad1H (see Embodiment 1) is a template, used to amplify IgG1CH1-Hingemut-CD3VH-IgG1HingemutR-CD137 VH fragment, carry out SapI and EcoRI double to fully synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.).
  • pQKTriad19H ligation sequence from 5' to 3' is: anti-c-Met antibody VH-IgG1 CH1-Hinge-mut - anti-CD3 antibody VH-IgG1 Hinge mut R-anti-CD137 antibody VH).
  • the synthetic vector pUC57 c-Met scFv (Universal Biosystems (Anhui) Co., Ltd.), which contains the anti-c-Met scFv-encoding nucleic acid sequence synthesized by DNA, is used to amplify the anti-c-Met VL fragment; with plasmid pQKTriad1 L (see Embodiment 1) is a template, used to amplify Kappa-Hinge mut-CD3 VL-IgG1 Hinge mut R-CD137 VL fragment, carry out SapI and EcoRI double digestion to the fully synthetic vector pQKX2 (General Biosystems (Anhui) Co., Ltd.) , recover the corresponding PCR products and digestion products, carry out recombination enzyme connection, and obtain the final recombinant plasmid named pQKTriad19L (the connection sequence is from 5' to 3': anti-c-Met antibody VL-Kappa-Hinge mut-anti-CD3
  • PCR amplification primer pairs are as follows:
  • Example 1 for the specific operation flow of expression vector construction and plasmid amplification.
  • the VH and VL-encoding nucleic acid sequences of the anti-c-Met antibody were derived from Onartuzumab (MetMAb) (patent number: US7615529B2).
  • the pQKTriad19H plasmid (see Example 3.1.1) was used as a template to amplify the c-Met VH-IgG1 CH1-Hinge mut-CD3 VH fragment, and SapI was performed on the fully synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.).
  • Double digestion with EcoRI recover the corresponding PCR products and digestion products, carry out recombinase ligation, and obtain the final recombinant plasmid named pQKZWB56H (the connection sequence is from 5' to 3': anti-c-Met antibody VH-IgG1CH1-Hinge -mut-anti-CD3 antibody VH); using the pQKTriad19L plasmid (see Example 3.1.1) as a template, it was used to amplify the c-Met VL-Kappa-Hinge mut-CD3 VL fragment, and the fully synthetic vector pQKX2 (General Biosystems) was used as a template.
  • pQKZWB56L the connection sequence from 5' to 3' is: anti-c - Met antibody VL-Kappa-Hinge-mut-anti-CD3 antibody VL.
  • the bispecific antibody ZWB56 lacks only the IgG1 Hingemut R-anti-CD137 antibody VH and the IgG1 Hingemut R-anti-CD137 antibody VL ( Figure 1C and Figure 1D).
  • PCR amplification primer pairs are as follows:
  • transfection was carried out with reference to the procedure described in Example 1, wherein the transfected cells were HEK293 (ATCC number: CRL-1573), and the transfection volume was 100 mL.
  • the transfected cells were suspended and cultured in a 500 mL shake flask for 7 days, and the antibody was harvested under the culture conditions of 36.5° C., 7.5% CO 2 , and 120 rpm/min.
  • the obtained antibody is an anti-c-Met ⁇ anti-CD3 ⁇ anti-CD137 trispecific antibody expressed by plasmids pQKTriad19H and pQKTriad19L
  • the antibody is named Triad19, and its structure is shown in C in Figure 1; the obtained antibody is expressed by plasmids pQKZWB56H and pQKZWB56L
  • the antibody was named ZWB56, and the structure is shown in D in Figure 1.
  • Example 2 Purification was performed according to the procedure described in Example 1. After buffer exchange, the protein concentration was measured using a Nanodrop microspectrophotometer, and the protein yield was calculated, where Triad19 was 9.8 mg/L and ZWB56 was 125 mg/L.
  • Select SDS-PAGE gel for protein electrophoresis add reducing and non-reducing buffers respectively, and identify the size of molecular bands in reduced and non-reducing states of protein. After Coomassie brilliant blue staining, the accuracy and purity of the protein were analyzed according to the protein marker, and the results are shown in Figure 5. The protein electrophoresis was analyzed, and the bands showed that the electrophoresis was consistent with the expected protein size, and the protein purity was high.
  • the kinetic parameters of the binding of ZWB56/Triad19 to c-Met protein, CD3 protein and CD137 protein were determined by Fortebio molecular interaction instrument. Specifically, c-Met protein, CD3 protein, CD137 protein were captured and immobilized with His or Fc sensors, and after equilibration in PBS, they bound to ZWB56/Triad19. ZWB56/Triad19 was diluted with PBS to 4 concentrations, 500nM, 250nM, 125nM, 62.5nM, respectively, and dissociated in PBS. The results of affinity constant determination are shown in Table 3 and Table 4.
  • Target cell processing prepare a bottle of target cells U87 MG in T75 (Concord Cell Resource Center, resource number: 3111C0001CCC000208), digest with 0.25% trypsin, centrifuge at 850 rpm for 5 min at room temperature, discard the supernatant, and use working culture The cells were resuspended in DMEM medium containing 10% FBS (inactivated), and the cell density and viability were counted with an automatic cell counter, and then the cell suspension was diluted to 5 ⁇ 10 4 cells/mL.
  • Jurkat Dual cell treatment Take a bottle of Jurkat Dual cells in T75 (Concord Cell Resource Center, resource number: 3111C0001CCC000075), centrifuge at 850 rpm for 5 min, discard the supernatant, and use working medium (containing 10% FBS (inactivated)) 1640 medium), resuspend and count, and adjust the cells to 2 ⁇ 10 6 cells/mL.
  • working medium containing 10% FBS (inactivated) 1640 medium
  • Triad19/ZWB56 Dilute Triad19/ZWB56, the highest concentration point of Triad19 is 20 ⁇ g/mL, add 50 ⁇ L/well, and the final concentration of the corresponding drug is 10 ⁇ g/mL, that is, the final concentration is 100 nM.
  • the highest concentration point of ZWB56 is 15 ⁇ g/mL, which is added at 50 ⁇ L/well, corresponding to a final drug concentration of 7.5 ⁇ g/mL, that is, the final concentration is 100 nM.
  • Control wells or wells without target cells were supplemented to 200 ⁇ l with complete medium.
  • the target cells are 5000 cells/well and the effector T cells are 25000 cells/well
  • the sorted T cells are diluted to 5 ⁇ 10 5 cells/mL.
  • Triad19 and ZWB56 drug concentration gradient dilution take the highest drug concentration point as 5nM, the corresponding final concentration of Triad19 is 0.5 ⁇ g/mL, the final concentration of ZWB56 is 0.375 ⁇ g/mL, and the drug preparation concentration is 4 times the final concentration, namely Triad19 2 ⁇ g /mL, 1.5 ⁇ g/mL for ZWB56.
  • T cells After the T cell killing experiment, conduct T cell grouping analysis, including CD4, CD8, CD45RO, CCR7, PD-1, Granzyme B, analyze the proportion of CD4 and CD8 cells, central memory T cells and effector Analysis of the proportion of memory T cells, and the proportion of Granzyme B with drug concentration, as shown in Figure 11- Figure 15.
  • GraphPad Prism 8 was selected for the above mapping analysis.
  • the results of the T cell killing experiment showed that the drug IC50 was obtained by fitting the drug killing curve, and the killing ability of Triad19 was one order of magnitude higher than that of ZWB56.
  • Analysis of the cell killing rate fitting curve at each concentration point shows that ZWB56 is weaker than Triad19 in terms of killing power corresponding to each concentration point.
  • the analysis results of IL2, IL6, IFN- ⁇ cytokines showed that the expression of each cytokine showed a significant drug dose relationship, the higher the drug concentration, the higher the expression of each factor. Similarly, at the same drug concentration, ZWB56 is weak.
  • Triad19 has stronger ability to activate T cells and kill tumor cells than ZWB56 under the comparison of equimolar administration, including the comparison of cytokines and the analysis of cell populations by flow cytometry.

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Abstract

本申请提供了一种融合蛋白，其包含：能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；以及第一肽接头和第二肽接头。本申请还提供了编码所述融合蛋白的核酸、包含所述核酸的表达载体、包含所述核酸或所述表达载体的宿主细胞、制备所述融合蛋白的方法，包含所述融合蛋白的药物组合物以及所述融合蛋白的用途。

Description

一种同时靶向CD3和CD137的融合蛋白及其制备方法和用途 技术领域
本申请通常涉及抗体领域。更具体地，本申请涉及一种同时靶向CD3和CD137的融合蛋白及其制备方法和用途。
发明背景
20世纪80年代中期，研究人员设计出有两个结合模块的抗体，一个与肿瘤细胞抗原结合，另一个与T细胞表面的CD3蛋白相匹配。1985年，Pilar Perez等人(Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody,Pilar Perez,Robert W.Hoffman,Stephen Shaw,Jeffrey A.Bluestone&David M.Segal Nature volume 316,pages354–356(1985)Cite this article)报道了这种双特异性抗体可以破坏培养皿中的癌细胞并能缩小小鼠体内的肿瘤。2000年，Peter Kufer和Gert Riethmüller推出一种简化的双特异性抗体，其两个模块通过一种柔性肽而不是传统的抗体骨架连接。简化的设计使得抗体的制造更容易，但由于抗体骨架的缺失，肾脏会在2小时内将其从血液中清除。这种类型的分子也被称为双特异性T细胞衔接器(bispecific T cell engager，
Figure PCTCN2021130015-appb-000001
)。
BITE分子虽然展示了强大的抗肿瘤能力，但其产量仍然较低，而且这种双特异性抗体有时也会引发严重的副作用，包括肝损伤和过度免疫反应，其中白细胞会分泌大量有毒的细胞因子信号。这样的细胞因子“风暴”会导致发烧，严重时还会导致器官损伤。
为了解决双特异性抗体的制造问题，近年来涌现了大量不同的分子模式，用于双特异性抗体的分子设计与制造。但如何设计使两个或者更多个结合模块以实现多靶向以及协同作用，仍然面临诸多挑战。
发明概述
为解决上述技术问题，本文中提供一种新的融合蛋白，具体为一种 新的多特异性抗体，所述多特异性抗体能够同时靶向活化T细胞上的CD3和CD137，且其与CD3和CD137抗原的亲和力均较低(KD在10-1000nM)，但分子结合T细胞的亲合力不低于传统的CD3双特异性抗体。因此，在提高了有效T细胞招募的同时，所述多特异性抗体还具有减少由靶向CD3的高亲和力分子所导致的T细胞过度活化，降低潜在的不良反应风险；此外，这种新的多特异性抗体具有更高的分子量，能够防止过度肾清除从而增加其体内半衰期。
在第一方面，本申请提供了一种融合蛋白，其包含：
a)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；以及
c)第一肽接头和第二肽接头，
其中所述第一肽接头用于连接所述抗CD137抗体或其抗原结合片段的重链以及所述抗CD3抗体或其抗原结合片段的重链，并且所述第二肽接头用于连接所述抗CD137抗体或其抗原结合片段的轻链和所述抗CD3抗体或其抗原结合片段的轻链，
所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键。
在一些实施方案中，所述融合蛋白还包含：
d)能特异性结合第一抗原的第一抗体或其抗原结合片段，以及
e)第三肽接头和第四肽接头，
其中所述第一抗体或其抗原结合片段的重链通过所述第三肽接头连接所述抗CD3抗体或其抗原结合片段的重链，并且
所述第一抗体或其抗原结合片段的轻链通过所述第四肽接头连接所述抗CD3抗体或其抗原结合片段的轻链，
所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键。
在一些实施方案中，所述第一肽接头、第二肽接头、第三肽接头和第四肽接头各自独立地选自以下：包含Seq ID NO.1-2所示序列(Seq ID NO.1：Xaa Pro Pro Cys Pro Ala Pro Glu；Seq ID NO.2：Glu Pro Ala Pro Cys Pro Pro Xaa，其中Xaa可以为除Cys之外的任何氨基酸或不存在)中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失。
在一些实施方案中，所述第一肽接头至第四肽接头中的每一个为天 然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变。
在一些实施方案中，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在一些实施方案中，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体或单链抗体分子如sc-Fv。
在一些实施方案中，所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met和GPC3。
在一些实施方案中，所述第一抗原、CD137分子和CD3分子独立地来源于哺乳动物，优选非人类的灵长类或者人类。
在一些实施方案中，所述第一抗体或其抗原结合片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍。
在第二方面，本申请提供了一种融合蛋白，其从N端至C端依次包含：
a)能特异性结合第一抗原的第一抗体的Fab片段；
b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；
c)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
其中所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的重链依次通过第一肽接头和第三肽接头连接，并且所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的轻链依次通过第二肽接头和第四肽接头连接，
其中所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键，并且所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键，各自独立地选自以下：包含Seq ID NO.1-2所示序列中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失。
在一些实施方案中，所述第一肽接头至第四肽接头中的每一个为天 然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变。
在一些实施方案中，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在一些实施方案中，所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met和GPC3。
在一些实施方案中，所述第一抗原、所述CD137分子和所述CD3分子独立地来源于哺乳动物，优选非人类的灵长类或者人类。
在一些实施方案中，所述Fab片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍。
在第三方面，本申请提供了编码第一方面或第二方面所述的融合蛋白的核酸。
在第四方面，本申请提供了包含第三方面所述的核酸的表达载体。
在第五方面，本申请提供了一种宿主细胞，其包含第三方面所述的核酸或第四方面所述的表达载体。
在一些实施方案中，所述宿主细胞为哺乳动物细胞。哺乳动物细胞可以包括但不限于CHO细胞、NS0细胞、SP2/0细胞、HEK293细胞、COS细胞和PER.C6细胞。
在第六方面，本申请提供了制备第一方面或第二方面所述的融合蛋白的方法，其包括：
a)培养第五方面所述的宿主细胞；和
b)从所述宿主细胞中或所述宿主细胞的培养物上清中回收所述融合蛋白。
在第七方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞，以及药学上可接受的载体。
在第八方面，本申请提供了第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主 细胞在制备用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的药物中的用途。
在第九方面，本申请提供了治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的方法，其包括向有需要的个体施用第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞。
在第十方面，本申请提供了第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的用途。
附图说明
图1显示了本申请中构建的融合蛋白的结构示意图，其中A显示了同时靶向CD3×CD137×CD19的三特异性抗体T1的结构示意图，B显示了同时靶向CD3×CD137×GPC3的三特异性抗体T2的结构示意图；C显示了三特异性抗体Triad19的结构示意图，D显示了双特异性抗体ZWB56的结构示意图。
图2显示了采用Protein L亲和纯化融合蛋白的层析图谱，其中A：三特异性抗体T1纯化层析图谱；B：三特异型性抗体T2纯化层析图谱。
图3显示了采用CHO-K1细胞表达的融合蛋白的SDS-PAGE电泳图，其中M:DNA Marker；1:非还原条件；2:还原条件；A：三特异性抗体T1SDS-PAGE电泳结果；B：三特异性抗体T2SDS-PAGE电泳结果。
图4显示了融合蛋白与细胞表面抗原结合的流式细胞术检测结果，其中A1：三特异性抗体T1与表达CD19的Nalm-6细胞的结合情况；A2：三特异性抗体T1与表达CD3的Jurkat细胞的结合情况；A3:三特异性抗体T1、T2与表达CD137的HEK293-CD137细胞结合情况；B1：三特异性抗体T2与表达CD3的Jurkat细胞的结合情况；B2：三特异性抗体T2与表达GPC3的HepG2细胞的结合情况。
图5显示了三特异性抗体Triad19和双特异性抗体ZWB56的SDS-PAGE结果，其中A：双特异性抗体ZWB56 SDS-PAGE电泳结果；B：三特异性抗体Triad19 SDS-PAGE电泳结果，两幅电泳图的第1-3泳 道分别非还原条件；DNA Marker和还原条件。
图6显示了通过CD3功能细胞株Jurkat Dual检测抗体Triad19/ZWB56功能活性的结果。
图7和8分别显示了三特异性抗体Triad19和双特异性抗体ZWB56的拟合药物杀伤曲线。
图9显示了抗体Triad19/ZWB56对细胞的杀伤率。
图10显示了各因子的定量结果。
图11-15显示了对T细胞杀伤实验后的T细胞进行T细胞分群分析的结果。
发明的详细描述
提供以下定义和方法以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，本申请的术语按照相关领域普通技术人员的常规用法理解。
定义
本文使用的术语“约”指所记载的数字的±10％，例如约1％指的0.9％至1.1％的范围。
本文使用的术语“融合蛋白(fusion protein)”是指有目的地把两段或更多段编码功能蛋白的基因连接在一起，进而表达所述蛋白。这种通过人工条件下将两个或更多个基因的编码区首尾连接，由调控序列控制的基因表达后所得的蛋白质产物即为融合蛋白。
本文所用的术语“肽接头”在本申请的背景下是指用于连接两个功能蛋白之间的短肽，长度可以从3个氨基酸(aa)高至76个氨基酸。肽接头可为融合蛋白中的各功能蛋白提供一定的柔性，使其能够发挥各自的功能。本申请中所用的肽接头优选只含有一个半胱氨酸，从而能够在两个肽接头之间形成一个稳定的二硫键。
本文所用的术语“抗体”是指能显示期望的生物活性的任何形式的抗体或其片段。因此，它以最广义的含义使用，具体覆盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体、融合蛋白(例如双特异性抗体)和抗体片段，只要它们能显示期望的生物活性。因此，本领域技术人员也 可以理解，本文所用的术语“抗体”还可以指能显示期望的生物活性的任何形式的相同或不同抗体或其片段的融合蛋白，从而实现多特异性抗体的功能。
本文所用的术语“抗原”是指能被选择性结合剂如抗体结合的分子或分子部分，还能用于动物中以制备能结合该抗原的表位的抗体。抗原可以具有一个或多个抗原表位。本文所述的抗原可以包括但不限于大多数蛋白质、细菌、病毒、细菌外毒素，多糖(如肺炎球菌的荚膜多糖)和类脂等。
本文所用的术语“特异性结合”是本领域熟知的术语，并且测定抗体与抗原此类特异性结合的方法也为本领域所熟知。例如，在一些实施方案中，“特异性结合”是指抗体与预期的靶标结合，但不与其他靶标显著结合。相比与其他表位的结合，抗体以明显增加的亲和力和/或以更长的持续时间结合预期的靶表位。
本文所用的术语“抗原结合片段”包括抗体的基本上保留其结合活性的片段或衍生物。因此，术语“抗原结合片段”指全长抗体的一部分，通常是其抗原结合区或可变区。抗原结合片段的实例包括但不限于：Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体、单链抗体分子如sc-Fv以及从抗体片段形成的融合蛋白。还认为，抗原结合片段可包括不会实质上改变其结合活性的保守氨基酸置换。
本文所用的术语“Fab片段”包含轻链以及重链的CH1和可变区。Fab分子的重链不能与另一个重链分子形成二硫键。
本文所用的术语“Fab’片段”含有轻链以及重链的部分或片段，所述部分或片段含有VH结构域和CH1结构域以及在CH1和CH2结构域之间的区域，使得在2个Fab’片段的两条重链之间可以形成链间二硫键，以形成F(ab’)2分子。
本文所用的术语“F(ab’)2片段”含有两条轻链和两条重链，所述重链含有在CH1和CH2结构域之间的恒定区的一部分，使得在两条重链之间形成链间二硫键。F(ab’)2片段因而由两个Fab’片段组成，而两个Fab’片段通过两条重链之间的二硫键连接在一起。
本文所用的术语“Fv片段”包含来自重链和轻链的可变区，但是缺少 恒定区。
本文所用的术语“单链Fv”或“scFv”，是指包含抗体的VH结构域和VL结构域的抗体片段，其中这些结构域以单一多肽链形式存在。通常，Fv多肽还包含VH结构域和VL结构域之间的多肽接头，所述接头使scFv能够形成期望的结构以进行抗原结合。
本文所用的术语“双体”指具有两个抗原结合位点的小抗体片段，所述片段在同一多肽链中包含重链可变结构域(VH)和与之连接的轻链可变结构域(VL)(VH-VL或VL-VH)。通过使用短得不能让同一链上的两个结构域之间发生配对的接头，各结构域被迫与另一条链的互补结构域发生配对，从而产生两个抗原结合位点。
本文所用的术语“超变区”指抗体的负责抗原结合的氨基酸残基。超变区包含来自“互补决定区”或“CDR”的氨基酸残基(例如轻链可变结构域中的残基24-34(LCDR-1)、50-56(LCDR-2)和89-97(LCDR-3)和重链可变结构域中的残基31-35(HCDR-1)、50-65(HCDR-2)和95-102(HCDR-3)；Kabat等人,(1991)Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.，和/或来自“超可变环”的氨基酸残基(即轻链可变结构域中的残基26-32(L1)、50-52(L2)和91-96(L3)和重链可变结构域中的残基26-32(H1)、53-55(H2)和96-101(H3)；Chothia和Lesk,(1987)J.Mol.Biol.196:901-917。“框架区”或“FR”残基，是指本文定义为CDR残基的超变区残基之外的那些可变结构域残基。
本文使用的术语“肿瘤相关抗原”指肿瘤细胞单独表达或主要表达或过表达的任何分子(例如蛋白、肽、脂质、碳水化合物等)，以使所述抗原与肿瘤相关。肿瘤相关抗原可以是仅一种类型的肿瘤表达的抗原，以使所述肿瘤抗原仅与一种类型的肿瘤相关或者仅是一种类型的肿瘤所特有的。可选地，肿瘤抗原可以是多种类型肿瘤相关或者特有的肿瘤抗原。例如，肿瘤相关抗原可以被乳腺癌细胞和结肠癌细胞都表达，但不被正常的、非肿瘤或非癌细胞所表达。示例性的肿瘤相关抗原为肿瘤细胞表面抗原，这类抗原更利于为治疗性和诊断性抗体所识别。
本文所述的术语“单克隆抗体”指从基本同质的抗体群中获得的抗体，即构成该群抗体的单个抗体之间是相同的，除了可以少量存在的可能自然发生的变异外。单克隆抗体高度特异性的针对单一抗原表位。本文公开的单克隆抗体不限于抗体来源或其制备方式(例如，通过杂交瘤、噬菌体挑选、重组表达、转基因动物等)。该术语包括在“抗体”定义下的完整免疫球蛋白以及其片段等。
本文使用的术语“表达载体”是指包含重组多核苷酸的载体，所述重组多核苷酸包含与待表达的核苷酸序列可操作地连接的表达控制序列。表达载体包含用于表达的足够的顺式作用元件；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域已知的所有那些，如掺入重组多核苷酸的粘粒，质粒(例如，裸露的或包含在脂质体中)和病毒(如慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
具体实施方式
本申请提供了一种能够同时靶向CD3和CD137的融合蛋白，其通过接头例如肽接头将抗CD3抗体或其抗原结合片段以及抗CD137抗体或其抗原结合片段连接起来。包含抗CD3抗体或其抗原结合片段以及抗CD137抗体或其抗原结合片段的融合蛋白还可以通过接头例如肽接头在融合蛋白的C端或N端进一步连接另一抗体或其抗原结合片段，从而制备出三特异性抗体。
本申请巧妙地利用抗体铰链区中二硫键的稳定作用，将突变的抗体铰链作为接头将本申请的融合蛋白中的各抗体相互连接起来，使得重链和轻链之间仅在突变的抗体铰链区处形成一对二硫键。通过本申请，不仅可以很容易地获得稳定的高纯度的目标多特异性抗体，例如三特异性抗体，而且所获得的三特异性抗体相比双特异性抗体，能以更高的结合亲和力结合其靶抗原。
在第一方面，本申请提供了一种融合蛋白，其包含：
a)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；以及
c)第一肽接头和第二肽接头，
其中所述第一肽接头用于连接所述抗CD137抗体或其抗原结合片段的重链以及所述抗CD3抗体或其抗原结合片段的重链，并且所述第二肽接头用于连接所述抗CD137抗体或其抗原结合片段的轻链和所述抗CD3抗体或其抗原结合片段的轻链，
所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键。
在一些实施方案中，所述融合蛋白还包含：
d)能特异性结合第一抗原的第一抗体或其抗原结合片段，以及
e)第三肽接头和第四肽接头，
其中所述第一抗体或其抗原结合片段的重链通过所述第三肽接头连接所述抗CD3抗体或其抗原结合片段的重链，并且
所述第一抗体或其抗原结合片段的轻链通过所述第四肽接头连接所述抗CD3抗体或其抗原结合片段的轻链，
所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键。
在一些实施方案中，所述第一肽接头、第二肽接头、第三肽接头和第四肽接头各自独立地选自以下：包含Seq ID NO.1-2所示序列(Seq ID NO.1：Xaa Pro Pro Cys Pro Ala Pro Glu；Seq ID NO.2：Glu Pro Ala Pro Cys Pro Pro Xaa，其中Xaa可以为除Cys之外的任何氨基酸或不存在)中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失。
在一些实施方案中，所述第一肽接头至第四肽接头中的每一个为天然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变。
在一些实施方案中，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在一些实施方案中，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体或单链抗体分子如sc-Fv。
在一些实施方案中，所述融合蛋白是双特异性抗体或三特异性抗体。
在一些实施方案中，所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met和GPC3。
在一些实施方案中，所述第一抗原、CD137分子和CD3分子独立地 来源于哺乳动物，优选非人类的灵长类或者人类。
在一些实施方案中，所述第一抗体或其抗原结合片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍。
在本文中，抗体和抗原之间的结合力称为抗体亲和力，其本质是一种非共价作用力。其体现了抗体分子和抗原结合的能力，测定抗体对特定抗原的亲和力的方法是本领域内公知的，包括但不限于生物膜干涉技术(BLI)、固相放射免疫法(SP-RIA)、平衡透析法、结合抗原沉淀法、放射免疫法(RIA)、酶联免疫吸附实验(ELISA法)、表面等离子共振法(SPR)等。抗体亲和力的大小可以用亲和力常数K D表示，亲和力常数K D越高，则抗体结合抗原的能力越强。
在一些实施方案中，第一肽接头和第二肽接头是相同的。
在一些实施方案中，第一肽接头和第二肽接头是不相同的。
在一些实施方案中，第三肽接头和第四肽接头是相同的。
在一些实施方案中，第三肽接头和第四肽接头是不相同的。
在优选的实施方案中，第一肽接头和第二肽接头是相同的，第三肽接头和第四肽接头是相同的，然而第一肽接头不同于第三肽接头。
在优选的实施方案中，第一肽接头至第四肽接头中的四种都是相同的。
在具体的实施方案中，第一肽接头和第二肽接头为C229缺失突变的IgG1铰链区，并且第三肽接头和第四肽接头为C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在具体的实施方案中，第一肽接头和第二肽接头为C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区，并且第三肽接头和第四肽接头为C229缺失突变的IgG1铰链区。
对抗体的氨基酸序列进行编号以鉴定等同位置，目前针对抗体存在多种不同的编号方案。Kabat方案(Kabat等，1991)是基于相同结构域类型的序列之间的高序列变异区域的位置而开发的。其对抗体重链(VH)和轻链(Vλ和Vκ)可变结构域的编号不同。Chothia方案(Al-Lazikani， 1997)与Kabat方案相同，但校正了在第一个VH互补决定区(CDR)周围插入注释的位置，使其对应于结构环。本申请中的抗体是按照Kabat方案来编号的。
在一些实施方案中，上文所述的融合蛋白可以为三特异性抗体，其包含重链和轻链，其中重链从N端至C端依次包含抗CD137抗体或其抗原结合片段的重链、第一肽接头、抗CD3抗体或其抗原结合片段的重链、第三肽接头以及第一抗体或其抗原结合片段的重链，并且轻链从N端至C端依次包含抗CD137抗体或其抗原结合片段的轻链、第二肽接头、抗CD3抗体或其抗原结合片段的轻链、第四肽接头以及第一抗体或其抗原结合片段的轻链。
在一些实施方案中，上文所述的融合蛋白可以为三特异性抗体，其包含重链和轻链，其中重链从N端至C端依次包含第一抗体或其抗原结合片段的重链、第一肽接头、抗CD137抗体或其抗原结合片段的重链、第三肽接头以及抗CD3抗体或其抗原结合片段的重链，并且轻链从N端至C端依次包含第一抗体或其抗原结合片段的轻链、第二肽接头、抗CD137抗体或其抗原结合片段的轻链、第四肽接头以及抗CD3抗体或其抗原结合片段的轻链。
在一些实施方案中，上文所述的融合蛋白可以为三特异性抗体，其包含重链和轻链，其中重链从N端至C端依次包含第一抗体或其抗原结合片段的重链、第一肽接头、抗CD3抗体或其抗原结合片段的重链、第三肽接头以及抗CD137抗体或其抗原结合片段的重链，并且轻链从N端至C端依次包含第一抗体或其抗原结合片段的轻链、第二肽接头、抗CD3抗体或其抗原结合片段的轻链、第四肽接头以及抗CD137抗体或其抗原结合片段的轻链。
在一些实施方案中，上文所述的融合蛋白可以为三特异性抗体，其包含重链和轻链，其中重链从N端至C端依次包含抗CD3抗体或其抗原结合片段的重链、第一肽接头、抗CD137抗体或其抗原结合片段的重链、第三肽接头以及以及第一抗体或其抗原结合片段的重链，并且轻链从N端至C端依次包含抗CD3抗体或其抗原结合片段的轻链、第二肽接头、抗CD137抗体或其抗原结合片段的轻链、第四肽接头以及以及第一抗体 或其抗原结合片段的轻链。
在优选的实施方案中，本文所述的肽接头可以选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在优选的实施方案中，预期通过二硫键交联的肽接头是相同的。例如，在最终的融合蛋白中，第一肽接头和第二肽接头预期会发生二硫键交联，则将二者选择为相同的肽接头。同理，第三肽接头和第四肽接头预期会发生二硫键交联，则将二者选择为相同的肽接头。
在本申请中，第一抗体或其抗原结合片段、抗CD137抗体或其抗原结合片段、抗CD3抗体或其抗原结合片段可以各自独立地来源于单克隆抗体。
在一些实施方案中，本申请中所用的单克隆抗体可以选自以下的一种或多种：博纳吐单抗(blincyto)、阿达木单抗(adalimumab)、苏金单抗(secukinumab)、利妥昔单抗(Rituximab)、曲妥珠单抗(Trastuzumab)、吉妥珠单抗-奥佐米星(Gemtuzumab ozogamicin)、阿仑单抗(Alemtuzumab)、贝伐单抗(Bevacizumab)、西妥昔单抗(Cetuximab)、帕尼单抗(Panitumumab)、奥法木单抗(Ofatumumab)、伊匹单抗(Ipilimumab)、贝伦妥单抗-维多汀(Brentuximab vedotin)、地诺单抗(Denosumab)、帕妥珠单抗(Pertuzumab),Obinutuzumab、雷莫芦单抗(Ramucirumab)、3F8、阿巴伏单抗(abagovomab)、阿德木单抗(adecatumumab)、阿夫土珠单抗(afutuzumab)、培化阿珠单抗(alacizumab(pegol))、阿麦妥昔(amatuximab)、阿泊珠单抗(apolizumab)、巴维昔单抗(bavituximab)、贝妥莫单抗(bectumomab)、贝利木单抗(belimumab)、贝伐珠单抗(bivatuzumab)、莫-坎妥珠单抗(cantuzumab mertansine)、拉-坎妥珠单抗(cantuzumab(ravtansine))、卡罗单抗-喷地肽(capromab(pendetide))、卡妥索单抗(catumaxomab)、泊-西他珠单抗(citatuzumab(bogatox))、西妥木单抗(cixutumumab)、clivatuzumab(tetraxetan)、可那木单抗(conatumumab)、达西珠单抗(dacetuzumab)、达洛珠单抗(dalotuzumab)、地莫单抗(Detumomab)、drozitumab、依美昔单抗(ecromeximab)、依决洛单抗(edrecolomab)、埃罗妥珠单抗(elotuzumab)、enavatuzumab、恩司昔单抗(ensituximab)、依帕珠单抗(epratuzumab)、厄马索单抗 (ertumaxomab)、伊瑞西珠(etaracizumab)、法利珠单抗(farletuzumab)、FBTA05、flanvotumab、加利昔单抗(galiximab)、吉妥珠单抗(gemtuzumab)、ganitumab、吉瑞昔单抗(girentuximab)、格莱木单抗-维多汀(glembatumumab(vedotin))、替-伊莫单抗(ibritumomab tiuxetan)、icrucumab、伊戈伏单抗(igovomab)、拉-英达西单抗(indatuximab ravtansine)、英妥木单抗(intetumumab)、伊珠单抗-奥佐米星(inotuzumab ozogamicin)、伊匹木单抗(ipilimumab)(MDX-101)、伊妥木单抗(iratumumab)、拉贝珠单抗(labetuzumab)、来沙木单抗(lexatumumab)、林妥珠单抗(lintuzumab)、莫-洛伏珠单抗(lorvotuzumab(mertansine))、鲁卡木单抗(lucatumumab)、鲁昔单抗(lumiliximab)、马帕木单抗(mapatumumab)、马妥珠单抗(matuzumab)、米拉珠单抗(milatuzumab)、米妥莫单抗(mitumomab)、莫加珠单抗(mogamulizumab)、moxetumomab(pasudotox)、他那可单抗(nacolomab(tafenatox))、他那莫单抗(naptumomab(estafenatox))、narnatumab、奈昔木单抗(necitumumab)、尼妥珠单抗(nimotuzumab)、nivolumab、NR-LU-10、olaratumab、莫奥珠单抗(oportuzumab(monatox))、奥戈伏单抗(oregovomab)、帕尼单抗(panitumumab)、帕妥珠单抗(pertuzumab)、普立木单抗(pritumumab)、雷妥莫单抗(racotumomab)、radretumab、、罗妥木单抗(robatumumab)、奥马珠单抗(omalizumab)、西罗珠单抗(sibrotuzumab)、司妥昔单抗(siltuximab)、帕他普莫单抗(taplitumomab(paptox))、替妥莫单抗(tenatumomab)、替妥木单抗(teprotumumab)、替西木单抗(ticilimumab)、曲美木单抗(tremelimumab)、替加珠单抗(tigatuzumab)、西莫白介素单抗(tucotuzumab(celmoleukin))、ublituximab、乌瑞鲁单抗(urelumab)、维妥珠单抗(veltuzumab)、伏洛昔单抗(volociximab)、伏妥昔单抗(votumumab)和扎鲁木单抗(zalutumumab)。
本申请的融合蛋白能结合的抗原可以是细胞相关蛋白，例如细胞(T细胞、内皮细胞或肿瘤细胞)膜上的细胞表面蛋白，也可以是可溶性蛋白。抗原还可以是任何医学上相关的蛋白，例如在疾病或感染期间上调的那些蛋白，例如受体和/或它们相应的配体。细胞表面蛋白的具体实例包括但不限于粘附分子例如整合素、E-选择蛋白、P-选择蛋白或L-选择蛋白、 CD2、CD3、CD4、CD5、CD7、CD8、CD11a、CD11b、CD18、CD19、CD20、CD23、CD25、CD33、CD38、CD40、CD45、CD69、CD134、ICOS、CD137、CD27、癌胚抗原(CEA)、TCR、MHCI类和MHCII类抗原、VEGF以及这些蛋白的受体。可溶性蛋白包括白细胞介素(例如IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-8、IL-12、IL-16或IL-17)，病毒抗原(例如呼吸道合胞病毒或巨细胞病毒抗原)，免疫球蛋白(例如IgE)，干扰素(例如干扰素α、干扰素β或干扰素γ)，肿瘤坏死因子-α(TNFα)，肿瘤坏死因子-β，集落刺激因子(例如G-CSF或GM-CSF)和血小板源性生长因子(例如PDGF-α和PDGF-β)以及它们的受体(适当时)。其他抗原包括细菌细胞表面抗原、细菌毒素、病毒(例如流感病毒、EBV、HepA、B和C)、生物恐怖试剂、放射性核素和重金属、及蛇和蜘蛛毒和毒素。
可以被本申请的融合蛋白结合的其他抗原包括血清载体蛋白，允许细胞介导的效应子功能招募的多肽，和核素螯合蛋白。
在一些实施方案中，可以被本申请的融合蛋白结合的抗原为肿瘤相关抗原，其包括下述中的任何一种或多种：CD20、MESOTHELIN、EGFR、CD33、CD52、VEGF、ROR1、CD30、RANKL、MESOTHELIN、VEGF-R2、Her3、A33抗原、CD5、CD19、CD22、CD23(IgE受体)、CA242抗原、5T4、VEGFR-1、CD33、CD37、CD40、CD44、CD51、CD52、CD56、CD74、CD80、CD152、CD200、CD221、CCR4、NPC-1C、波形蛋白、胰岛素样生长因子-1受体(IGF-1R)、甲胎蛋白、癌胚抗原(CEA)、整合素α vβ 3、整合素α 5β 1、成纤维细胞活化蛋白、FAP-α、TAG-72、MUC1、MUC16、前列腺特异性膜抗原(PMSA)、EGP40泛癌抗原、糖蛋白EpCAM、程序性死亡-1、肝再生磷酸酶3(PRL-3)、Lewis-Y抗原、GD2、磷脂酰肌醇聚糖-3(GPC3)和间皮素。
CD3分子由4个亚基组成：δ、ε、γ、ζ，其分子量分别为18.9kDa、23.IkDa、20.5kDa、18.7kDa，其长度分别有171、207、182、164个氨基酸残基。它们一起组成6条肽链，常与T细胞受体(T cell recptor，TCR)紧密结合形成由8条肽链组成的TCR-CD3复合体，此复合体具有传导T细胞活化信号、稳定TCR结构的功能。CD3胞质段含有免疫受体酪氨酸活化基序(immunoreceptortyrosine-based activation motif，ITAM)，TCR 识别并结合由MHC(major histo-compatibility complex)分子提呈的抗原肽，导致CD3的ITAM的保守序列的酪氨酸残基被T细胞内的酪氨酸蛋白激酶p56LCK磷酸化，然后可募集其他SH2(Scrhomology2)结构域的酪氨酸蛋白激酶(如ZAP-70)。ITAM的磷酸化和与ZAP-70的结合是T细胞活化信号传导过程早期阶段的重要生化反应之一。因此，CD3分子的功能是转导TCR识别抗原所产生的活化信号。
CD137(TNFRSF9或4-1BB)属于肿瘤坏死因子(tumor necrosis factor,TNF)受体家族成员，是由255个氨基酸组成的Ⅰ型跨膜蛋白。CD137单体的相对分子质量为30kDa，同时它也能以二聚体的形式存在，相对分子质量约为55kDa。细胞表面膜CD137分子脱落后进入血液能够形成可溶性CD137(sCD137)，正常人体内几乎不存在。在免疫反应中，T淋巴细胞的激活除了需要主要MHC/Ag与TCR结合，还需要第二信号的参与，如B7-CD28、CD137-CD137L等的相互作用。CD137的作用不同于CD28，CD28表达于未激活T淋巴细胞表面，主要影响细胞早期免疫应答，而CD137主要在活化的T淋巴细胞上表达，因此主要在T淋巴细胞应答的中晚期产生效应。CD137在初始T细胞上表达量很低，但是当T细胞激活后CD137表达上调，主要分布于CD4+T、CD8+T细胞表面。CD137与CD137L结合可产生一系列生物学效应，包括诱导T细胞激活，释放趋化因子、细胞因子，加剧免疫应答。此外，CD137-CD137L信号也参与自身免疫性疾病，如Ⅰ型自身免疫性糖尿病等。
CD19是参与B细胞活化与增殖的重要膜抗原之一，是所有B细胞共有的表面标志，B细胞活化后不消失，是最重要的B细胞标志物，同时CD19也是B细胞表面的传达信号复合体的构成部分，CD19的细胞外部分同其他膜抗原结合进行信号传导。CD19阳性细胞升高见于B淋巴细胞系统的恶性肿瘤，如CD19在95％急性前B淋巴细胞白血病细胞和94％急性成熟B淋巴细胞白血病细胞表达，也见于慢性淋巴细胞性白血病和Burkitt淋巴瘤等；CD19阳性细胞降低见于体液免疫缺陷病，如无丙种球蛋白血症、长期使用免疫抑制剂者等。故CD19检测可对上述疾病进行明确的病因诊断，并为鉴别诊断提供依据。此外由于CD19广泛存在于B淋巴细胞系统恶性肿瘤细胞表面，故可在白血病及淋巴瘤的免疫疗法中 作为细胞表面的靶点。
GPC3(Glypican-3，磷脂酰肌醇聚糖-3)为Glypican家族中的一员，属膜性硫酸乙酰肝素多糖蛋白。在哺乳动物的胚胎期可通过影响多个分子信号通路而对组织器官的发生和生长发挥重要调控作用。基因突变和功能丧失时导致过度生长和畸变综合征。GPC3异常表达与多种肿瘤的发生、发展关系密切。认为GPC3对肝细胞癌(HCC)的诊断和治疗方面有重要作用。
PD-1(程序性死亡受体1)，是一种重要的免疫抑制分子，为免疫球蛋白超家族，是一个268氨基酸残基的膜蛋白。以PD-1为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义。其配体PD-L1也可作为靶点，相应的抗体也可以起到相同的作用。PD-1或PD-L1可以充当在本申请中的结合部分，例如第一结合部分和/或第二结合部分。优选地，PD-1的胞外区，即PD-1ECD充当本申请中的结合部分。
c-Met是一种由c-met原癌基因编码的蛋白产物，为肝细胞生长因子受体，具有酪氨酸激酶活性，与多种癌基因产物和调节蛋白相关，参与细胞信息传导、细胞骨架重排的调控，是细胞增殖、分化和运动的重要因素。目前认为，c-Met与多种癌的发生和转移密切相关。
本申请的融合蛋白可以引入CH2-CH3结构域。CH2-CH3结构域可以与第一抗体或其抗原结合片段、抗CD137抗体或其抗原结合片段、抗CD3抗体或其抗原结合片段的重链连接。所述CH2-CH3结构域，任选地，以KiH突变、引入半胱氨酸残基、引入一个或多个盐桥突变以促进异二聚化，这样的添加导致提高的异二聚体的稳定性。本文中的盐桥包括氢键和静电相互作用，如可以在谷氨酸和赖氨酸残基之间发生的盐桥。
天然抗体的重链和轻链分别包括可变区(即V区)和恒定区(即C区)。重链的恒定区和轻链的恒定区分别称为CH和CL。不同型(κ或λ)Ig的CL长度基本一致，但是不同类Ig的CH长度不同，例如IgG、IgA和IgD包括CH1、CH2和CH3，而IgM和IgE则包括CHl、CH2、CH3和CH4。
在第二方面，本申请提供了一种融合蛋白，其从N端至C端依次包 含：
a)能特异性结合第一抗原的第一抗体的Fab片段；
b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；
c)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
其中所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的重链依次通过第一肽接头和第三肽接头连接，并且所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的轻链依次通过第二肽接头和第四肽接头连接，
其中所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键，并且所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键，各自独立地选自以下：包含Seq ID NO.1-2所示序列中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失。
在一些实施方案中，所述第一肽接头至第四肽接头中的每一个为天然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变。
在一些实施方案中，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
在一些实施方案中，所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met和GPC3。
在一些实施方案中，所述第一抗原、所述CD137分子和所述CD3分子独立地来源于哺乳动物，优选非人类的灵长类或者人类。
在一些实施方案中，所述Fab片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍。
在一些实施方案中，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体或单链抗体分子如sc-Fv。
在第三方面，本申请提供了编码第一方面或第二方面所述的融合蛋白的核酸。
在优选的实施方案中，所述核酸可以是适合在宿主细胞中表达的密码子优化的核酸。例如根据密码子的简并性，其仍然编码同样的蛋白质。根据所用宿主细胞进行密码子优化的方法是本领域技术人员公知的。
在第四方面，本申请提供了包含第三方面所述的核酸的表达载体。
可以使用任何合适的表达载体。例如，原核克隆载体包括来自大肠杆菌的质粒，如colEl、pCRl、pBR322、pMB9、pUC、pKSM和RP4。原核载体还包括噬菌体DNA如M13和其它丝状单链DNA噬菌体的衍生物。可用于酵母的载体的实例是2μ质粒。用于在哺乳动物细胞中表达的合适载体包括以下众所周知的衍生物：SV-40、腺病毒、逆转录病毒衍生的DNA序列以及衍生自功能性哺乳动物载体(如上述那些)和功能性质粒和噬菌体DNA的组合的穿梭载体。
另外的真核表达载体为本领域已知的(例如，P J.Southern&P.Berg,J.Mol.Appl.Genet,1:327-341(1982)；Subramani等人,Mol.Cell.Biol,1:854-864(1981)；Kaufinann&Sharp,"Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene,"J.Mol.Biol,159:601-621(1982)；Kaufhiann&Sharp,Mol.Cell.Biol,159:601-664(1982)；Scahill等人,"Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"Proc.Nat'l Acad.Sci USA,80:4654-4659(1983)；Urlaub&Chasin,Proc.Nat'l Acad.Sci USA,77:4216-4220,(1980)，将其全部通过引用并入本文)。
可用于本申请的表达载体含有至少一个表达控制序列，其与待表达的DNA序列或片段可操作连接。将控制序列插入载体中以控制和调节克隆的DNA序列的表达。有用的表达控制序列的实例是lac系统，trp系统，tac系统，trc系统，噬菌体λ的主要操纵子和启动子区，fd外壳蛋白的控制区，酵母的糖酵解启动子，例如3-磷酸甘油酸激酶的启动子，酵母酸性磷酸酶的启动子，例如Pho5，酵母α-交配因子的启动子，以及来源于多瘤病毒、腺病毒、逆转录病毒和猿猴病毒的启动子，例如SV40的早期和晚期启动子和已知控制原核或真核细胞及其病毒或其组合的基因表达的其它序列。
在第五方面，本申请提供了一种宿主细胞，其包含第三方面所述的核酸或第四方面所述的表达载体。
在一些实施方案中，所述宿主细胞为哺乳动物细胞。哺乳动物细胞可以包括但不限于CHO细胞、NS0细胞、SP2/0细胞、HEK293细胞、COS细胞和PER.C6细胞。本领域技术人员能够根据需要选择适合的宿主细胞。
在第六方面，本申请提供了制备第一方面或第二方面所述的融合蛋白的方法，其包括：
a)培养第五方面所述的宿主细胞；和
b)从所述宿主细胞中或所述宿主细胞的培养物上清中回收所述融合蛋白。
在第七方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞，以及药学上可接受的载体。
第七方面的药物组合物可按制药领域的常规方法制备成所需的剂型。在一些实施方案中，所述药物组合物优选为液体或悬浮液剂型。
在一些实施方案中，所述药学上可接受的载体为不减弱免疫细胞活力以及功能、不影响抗体或其抗原结合片段与抗原特异性结合的载体，包括但不限于细胞培养基、缓冲液、生理盐水和平衡盐溶液等。缓冲液的实例包括等渗磷酸盐、醋酸盐、柠檬酸盐、硼酸盐以及碳酸盐等。在具体的实施方案中，所述药学上可接受的载体为含1％血清的磷酸盐缓冲液。
本文公开的融合蛋白及其药物组合物能够用于治疗、改善或预防个体的肿瘤、自身免疫性疾病或传染性疾病。
第七方面所述的药物组合物还可以包含用于治疗、改善或预防个体的肿瘤、自身免疫性疾病或传染性疾病的第二药剂。
在第八方面，本申请提供了第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞在制备用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的药物中的用途。
在第九方面，本申请提供了用于治疗、改善或预防个体的肿瘤、自身免疫性疾病或传染性疾病的方法，其包括向个体施用治疗有效量的第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞。
在一些实施方案中，所述方法还包括施用治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的第二药剂。
在第十方面，本申请提供了第一方面或第二方面所述的融合蛋白，第三方面所述的核酸，第四方面所述的表达载体或第五方面所述的宿主细胞用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的用途。
“治疗”既指治疗性处理，也指预防性或防止性的措施，其目的就是预防或减缓(减轻)目标病理状态或病症。需要治疗的个体包括那些已经存在所述病症的个体，还包括那些将发展为该病症的或欲对其病症进行预防的个体。因此，本文中欲被治疗的个体已经被诊断为患有该病症或倾向于或易患该病症。
本文使用的术语“个体”是指哺乳动物，包括但不限于灵长类动物、牛、马、猪、绵羊、山羊、狗、猫以及诸如大鼠和小鼠的啮齿类动物。优选地，哺乳动物为非人类的灵长类或者人类。特别优选的哺乳动物是人。
在某些实施方案中，所述肿瘤为原发性癌症或转移性癌症。在具体的实施方案中，肿瘤选自肺癌例如非小细胞肺癌、结直肠癌、膀胱癌、造血系统癌症例如白血病、乳腺癌、胃癌、胃食管结合部腺癌、B淋巴细胞型非霍奇金淋巴瘤、霍奇金淋巴瘤，间变大细胞淋巴瘤、头颈癌例如头颈部鳞状细胞癌、恶性胶质瘤，肾癌、黑色素瘤、前列腺癌、骨癌、骨巨细胞瘤、胰腺癌、肉瘤、肝癌、皮肤鳞癌、甲状腺癌、宫颈癌、鼻咽癌、子宫内膜癌，或上述肿瘤的转移癌。
在某些实施方案中，所述自身免疫性疾病可以包括系统性红斑狼疮、类风湿关节炎、硬皮病、系统性血管炎、皮肌炎和自身免疫性溶血性贫血等。
在某些实施方案中，所述传染性疾病包括呼吸道传染病、消化道传染病、血液传染病、体表传染病和性传染病等。在具体的实施方案中， 传染性疾病可以包括但不限于流行性感冒，肺结核，腮腺炎，麻疹，百日咳、蛔虫病，细菌性痢疾，甲型肝炎、乙型肝炎，疟疾，流行性乙型脑炎，丝虫病、血吸虫病，沙眼，狂犬病，破伤风、淋病、梅毒、艾滋病等。
本文中所用的“治疗有效量”可以根据具体情况而定，本领域普通技术人员根据实际所需药量可以很容易地掌握，如可根据患者体重、年龄和病症情况来确定。
本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。
上述公开内容总体上描述了本申请，通过下面的实施例进一步示例本申请。描述这些实施例仅为说明本申请，而不是限制本申请的范围。尽管本文中使用了特殊的术语和值，这些术语和值同样被理解为示例性的，并不限定本申请的范围。除非特别指明，本说明书中的实验方法和技术为本领域常规的方法和技术。对于其它没有特别注明厂家的材料和设备等，其通常是可通过商业途径常规获得的。
实施例
以下实施例用于说明本申请，但不用来限制本申请的范围。在不背离本申请精神和实质的情况下，对本申请方法、步骤或条件所作的修改或替换，均属于本申请的范围。
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1：靶向CD3×CD137×CD19的三特异性抗体的制备、表达与鉴定材料
抗CD3抗体和抗CD19抗体的VH和VL编码核酸序列来源于Blincyto(Amgen)。其中抗CD19的VH编码核酸序列、抗CD3的VH 编码核酸序列以及IgG1 CH1-Hinge mut(即包括C229缺失突变的IgG1铰链区)的编码核酸序列通过全合成的方式构建进质粒pQKE3H中(通用生物系统(安徽)有限公司)，抗CD19的VL编码核酸序列、抗CD3的VL编码核酸序列以及Kappa-Hinge mut(包括C229缺失突变的IgG1铰链区)编码核酸序列通过全合成的方式构建进质粒pQKE3L中(通用生物系统(安徽)有限公司)。合成载体pUC57 IgG1 Hinge mut R-CD137 VH，其含有通过DNA合成的IgG1 Hinge mut R(C229缺失突变，且铰链区D224-S242倒转)编码核酸序列以及抗CD137单克隆抗体中的抗CD137 VH编码核酸序列(序列参见专利，专利号：US20190284292A1)(通用生物系统(安徽)有限公司)；合成载体pUC57 IgG1 Hinge mut R-CD137 VL，其含有通过DNA合成的IgG1 Hinge mut R(C229缺失突变，且铰链区D224-S242倒转)编码核酸序列和抗CD137单克隆抗体中的抗CD137 VL编码核酸序列(序列参见专利，专利号：US20190284292A1)(通用生物系统(安徽)有限公司)。
IgG1 CH1-Hinge mut和Kappa-Hinge mut的核苷酸序列如下：
Figure PCTCN2021130015-appb-000002
IgG1 Hinge mut R核苷酸序列如下：
Figure PCTCN2021130015-appb-000003
1.1靶向CD3×CD137×CD19的三特异性抗体表达载体的制备
1.1.1三特异性抗体重链表达载体pQKTriad1H的构建
以pUC57 IgG1 CH1 Hinge mut R-CD137VH为模板，使用金牌Mix PCR试剂盒(TSINGKE公司)，按照试剂盒的说明书，扩增IgG1 Hinge mut R-CD137 VH片段，其扩增产物大小约为0.4kb；同时以限制性内切酶EcoRI(NEB,R3101S)对合成的载体质粒pQKE3H进行酶切，将所得的PCR扩增产物和酶切后的载体pQKE3H以BM无缝克隆试剂盒(博迈德公司)，按照试剂盒的说明书，进行重组连接以获得重链表达载体pQKTriad1H(连接顺序从5’到3’)：抗CD19抗体VH-CH1-Hinge mut-抗CD3抗体VH-IgG1Hinge mut R-抗CD137抗体VH)。
PCR扩增引物对如下：
Figure PCTCN2021130015-appb-000004
1.1.2三特异性抗体轻链表达载体pQKTriad1 L的构建
以pUC57 IgG1 Hinge mut R-CD137 VL为模板，使用金牌Mix PCR试剂盒(TSINGKE公司)，按照试剂盒的说明书，扩增IgG1 Hinge mut R-CD137 VL片段，其扩增产物大小约为0.4kb；同时以限制性内切酶EcoRI(NEB,R3101S)对合成的载体质粒pQKE3L进行酶切，将所得的PCR扩增产物和酶切后的载体pQKE3L以BM无缝克隆试剂盒(博迈德公司)，按照试剂盒的说明书，进行重组连接以获得轻链表达载体pQKTriad1L(连接顺序从5’到3’为：抗CD19抗体VL-Kappa-Hinge mut-抗CD3抗体VL-IgG1Hinge mut R-抗CD137抗体VL)。
PCR扩增引物对如下：
Figure PCTCN2021130015-appb-000005
1.1.3重组质粒的扩增和制备
将如上获得的重链表达载体pQKTriad1H和轻链表达载体pQKTriad1L分别转化至大肠杆菌(E.coli)TOP10中。挑取单克隆并鉴定后，在含有氨苄青霉素(终浓度为100mg/L)的LB培养基中培养16小时， 培养条件为37℃，200rpm振荡培养。以8000×g离心20分钟收集细菌。使用NucleoBond Xtra Midi试剂盒(Macherey-nagel)，按照试剂盒的说明书，对质粒进行分离提取，以1mL的无菌超纯水进行洗脱，最后使用Nanodrop微量分光光度计测定质粒浓度。
1.2抗体的表达
将重链表达载体pQKTriad1H和轻链表达载体pQKTriad1L共同转染HEK293细胞进行表达。转染前24小时，将1.5×10 6的HEK293(ATCC，编号：CRL-1573)细胞接种于含有100mL OPM-293 CD05无血清培养基(奥浦迈，Cat：81075-001)的500mL摇瓶中，培养条件为36.5℃，7.5％CO 2，120rpm悬浮培养。转染时，将重组质粒pQKTriad1H和pQKTriad1L按照1:1的重量比(DNA总量为100μg)于10mL OPM-293 CD05培养基中混合，随后加入100μL PEI(浓度为3mg/mL)，迅速涡旋混匀，室温静止孵育15分钟。然后将该混合物加入至上述细胞培养物中。细胞在36.5℃，7.5％CO 2，120rpm/min条件下继续培养7天以收获表达的抗体。该抗体指由质粒pQKTriad1H和pQKTriad1L表达的抗CD19×抗CD3×抗CD137三特异性抗体，抗体命名为T1，结构如图1中的A所示。
1.3抗体的纯化
将收获的细胞培养物于3000×g离心20min，收集上清并用0.45μm过滤器过滤。用20mM PB和150mM NaCl的混合缓冲液(pH 7.4)平衡5mL Capto L亲和层析柱(GE)，流速5mL/min，体积大于5CV。将过滤后的样品液以5mL/min流速上样。上样完成后，用20mM PB和150mM NaCl的混合缓冲液(pH 7.4)洗涤Capto L亲和层析柱，流速5mL/min。用50mM柠檬酸(pH 3.0)缓冲液进行洗脱，流速5mL/min，收集完整洗脱峰，同时用1M Tris HCl(pH 9.0)缓冲液调节收集到的洗脱液的pH至7.0左右(图2中的A)。纯化产物经超滤管超滤，将Tris-柠檬酸缓冲液置换成商品化的PBS缓冲液。将所获蛋白用SDS-PAGE和考马斯亮蓝染色检测(图3中的A)，使用Nanodrop微量分光光度计测定蛋白浓度，计算蛋白产量为230mg/L。
1.4抗体的鉴定
1.4.1 HPLC测定抗体纯度
经Capto L纯化的抗体，通过HPLC(安捷伦1260II)SEC检测其纯度。色谱柱为Sepax水溶性体积排阻色谱柱，流动相为50mm PB+300mm NaCl pH 7.0，上样量为10μg，流速1mL/min，等度洗脱20min。结果表明一步纯化后其单体纯度为约86％。
1.4.2 Fortebio测定抗体亲和力
纯化的抗体使用分子互作仪Fortebio Octet QK(Molecular Devices公司)测定其亲和力常数K D。通过Fab-Ch1的传感器(sensor)固定T1抗体，固定浓度为0.25μM。抗原人CD19(义翘神州，Cat：10084-HNAH)、人CD3(义翘神州，Cat：10977-H02H)和人CD137(义翘神州，Cat：10041-H002H)分别按照600nM，300nM，150nM和75nM的浓度上样。亲和力常数测定结果如表1所示。
表1：亲和力常数测定结果
抗体-抗原 亲和力常数K D(M)
T1/CD19Ag 6.76E-09
T1/CD3εAg 1.12E-07
T1/CD137Ag 7.2E-08
1.5流式细胞术检测抗体与细胞结合活性
1.5.1抗体与Nalm-6细胞结合活性
对正常复苏并至少传代培养3代以上的Nalm-6细胞(ATCC编号：CRL-3273)吹打均匀并收集细胞，400g/min离心5min，重悬计数，调整细胞密度为1×10 6个细胞/mL。组别设为空白组，阳性对照组，二抗组和实验组(T1)，先向各组的管中加入100μl的上述细胞悬液。在T1组的管中加入100ng的T1抗体，室温孵育30min，用3mL含有2％FBS的PBS缓冲液清洗，400g/min离心5min，以50μl的含有2％FBS的PBS缓冲液重悬；空白组加入0.5μl的水；阳性对照组加入FITC抗人CD19(BioLegend，克隆号：HIB19)0.5μl；T1组和二抗组分别加入0.5μl的APC抗人Ig轻链K(BioLegend，克隆号：TB28-2)。将各组混匀后于室 温避光孵育30min。孵育完成后以含有2％FBS的PBS缓冲液清洗各组的管，400g/min离心5min，以100μl含有2％FBS的PBS缓冲液重悬，上流式细胞仪(艾森，仪器型号：NovoCyte)检测。结果如图4中的A1所示。
1.5.2抗体与Jurkat细胞结合活性
对正常复苏并至少传代培养3代以上的Jurkat细胞(协和细胞资源中心，资源编号：3111C0001CCC000075)吹打均匀并收集细胞，400g/min离心5min，重悬计数，调整细胞密度为1×10 6个细胞/mL。组别设为空白组，二抗组和实验组(T1)，先向各组的管中加入100μl的上述细胞悬液。在T1组的管中加入100ng的T1抗体，室温孵育30min，用3mL含有2％FBS的PBS缓冲液清洗，400g/min离心5min，以50μl的含有2％FBS的PBS缓冲液重悬；空白组加入0.5μl的水；T1组和二抗组分别加入0.5μl的APC抗人Ig轻链K(BioLegend，克隆号：TB28-2)。将各组混匀后于室温避光孵育30min。孵育完成后以含有2％FBS的PBS缓冲液清洗各组的管，400g/min离心5min，以100μl含有2％FBS的PBS缓冲液重悬，上流式细胞仪(艾森，仪器型号：NovoCyte)检测。结果如图4中的A2所示。
1.5.3抗体与HEK293-CD137细胞结合活性
正常复苏的HEK293细胞(协和细胞资源中心，资源编号：3111C0001CCC000010)至少传代培养3代以上，转染前24h对细胞进行传代，并接种到6孔板中。转染当天将PEI(Sigma，Cat：764647)和合成质粒pENTER CD137(通用生物系统(安徽)有限公司)复苏至室温，取5μg质粒加入至500μl的DMEM培养基中(gibco，REF：11965-092)，再加入15μg的PEI，立即旋涡振荡15min，然后将混合物轻轻滴加至细胞培养液中，放置与培养箱中继续培养24-48h。48h后更换含有为2μg/mL嘌呤霉素的10％FBS的DMEM培养基。3天后细胞出现大规模死亡，可以轻轻拍瓶壁，将上清弃去，贴壁良好的细胞即为可能的稳转细胞株。8-10天后视细胞生长状态，消化细胞并铺到96孔板中，筛选单克隆细胞 株。期间一直用含有2ug/mL嘌呤霉素10％FBS的DMEM培养基加压培养。最终得到的细胞株被命名为HEK293-CD137。此细胞株基因组中转入了人CD137胞外区基因，其可以稳定表达人CD137胞外区蛋白并且展示在细胞膜上。将复苏并培养了超过3代的HEK293-CD137细胞以10mL PBS洗1次，1mL 0.05％胰酶消化1min，加入4mL含有10％FBS的DMEM培养基，吹打均匀后收集细胞，1000rpm/min离心5min，重悬计数，调整细胞密度为1×10 6个细胞/mL。组别设为空白组，阳性对照组，二抗组和实验组(T1)，先向各组的管中加入10μl的上述细胞悬液。在T1组的管中加入100ng的Triad1TsAb，室温孵育30min，用3mL含有2％FBS的PBS缓冲液清洗，1000rpm/min离心5min，以50μl的含有2％FBS的PBS缓冲液重悬；空白组加入0.5μl的水；阳性对照组加入抗CD137抗体(抗体参见专利，专利号：US-2019-0284292-A1)0.5μl；T1组和二抗组分别加入0.5μl的APC抗人Ig轻链K(BioLegend，克隆号：TB28-2)。将各组混匀后于室温避光孵育30min。孵育完成后以含有2％FBS的PBS缓冲液清洗各组的管，1000rpm/min离心5min，以100μl含有2％FBS的PBS缓冲液重悬，上流式细胞仪(艾森，仪器型号：NovoCyte)检测。结果如图4中的A3所示。
实施例2：靶向CD3×CD137×GPC3的三特异性抗体的制备、表达与鉴定
2.1靶向CD3×CD137×GPC3的三特异性抗体表达载体的制备
表达载体构建及质粒扩增的具体操作流程参照实施例1，其中合成载体pUC57 GPC3 scFv(序列参见专利，专利号：US7919086B2)(通用生物系统(安徽)有限公司)，其含有通过DNA合成的抗GPC3 scFv编码核酸序列，用来扩增抗GPC3 VH片段；以质粒pQKTriad1 H(见实施例1)为模板，用来扩增CH1-Hinge mut-CD3 VH-CD137 VH片段，对全合成载体pQKX1(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒命名为pQKTriad2H(连接顺序从5’到3’为：抗GPC3抗体VH-CH1-Hinge mut-抗CD3抗体VH-IgG1Hinge mut R-抗CD137抗体 VH)。合成载体pUC57 GPC3 scFv(序列参见专利，专利号：US7919086B2)(通用生物系统(安徽)有限公司)，其含有通过DNA合成的抗GPC3 scFv编码核酸序列，用来扩增抗GPC3 VL片段；以质粒pQKTriad1 L(见实施例1)为模板，用来扩增Kappa-Hinge mut-CD3 VL-CD137 VL片段，对全合成载体pQKX2(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒命名为pQKTriad2L(连接顺序从5’到3’为：抗GPC3抗体VL-Kappa-Hinge mut-抗CD3抗体VL-IgG1 Hinge mut R-抗CD137抗体VL)。
PCR扩增引物对如下：
Figure PCTCN2021130015-appb-000006
Figure PCTCN2021130015-appb-000007
2.2抗体的表达
参照实施例1所描述的流程进行转染，其中转染细胞为HEK293(ATCC编号：CRL-1573)，转染体积为100mL。转染后的细胞于500mL摇瓶中悬浮培养7天后收获抗体，培养条件为36.5℃，7.5％CO 2，120rpm/min。所得抗体指由质粒pQKTriad2H和pQKTriad2L表达的抗GPC3×抗CD3×抗CD137三特异性抗体，抗体命名为T2，结构如图1中的B所示。
2.3抗体的纯化
参照实施例1所描述的流程进行纯化(图2中的B，图3中的B)，经缓冲液置换后使用Nanodrop微量分光光度计测定蛋白浓度，计算蛋白产量为50mg/L。
2.4抗体的鉴定
2.4.1 HPLC测定抗体纯度
参照实施例1以HPLC测定抗体T2的纯度，一步纯化后其单体纯度为约76％。
2.4.2 Fortebio测定抗体亲和力
具体流程参照实施例1。通过Fab-Ch1的传感器来固定T2抗体，固定浓度为0.25μM。抗原人GPC3(义翘神州，Cat：10088-H08H)、人CD3(义翘神州，Cat：10977-H02H)和人CD137(义翘神州，Cat：10041-H002H)分别按照600nM，300nM，150nM和75nM的浓度上样。亲和力常数测定结果如表2所示。
表2：亲和力常数测定结果
抗体-抗原 亲和力常数K D(M)
T2/GPC3Ag 1.04E-08
T2/CD3εAg 7.4E-08
T2/CD137Ag 4.4E-08
2.5流式细胞术检测抗体与细胞结合活性
2.5.1抗体与Jurkat细胞结合活性
具体材料及操作流程参见实施例1中的1.5.2，其中实验组(T2)抗体为T2。结果如图4中的B1所示。
2.5.2抗体与HEK293-CD137细胞结合活性
具体材料及操作流程参见实施例1中的1.5.3，其中实验组(T2)抗体为T2。结果如图4中的A3所示。
2.5.3抗体与HepG2细胞结合活性
对正常复苏并至少传代培养3代以上的HepG2细胞(协和细胞资源中心，资源编号：3111C0001CCC000035)吹打均匀并收集细胞，1000rpm/min离心5min，重悬计数，调整细胞密度为1×10 6个细胞/mL。组别设为空白组，二抗组和实验组(T2)，先向各组的管中加入100μl的上述细胞悬液。在T2组的管中加入100ng的T2TsAb，室温孵育30min，用3mL含有2％FBS的PBS缓冲液清洗，1000rpm/min离心5min，以50μl的含有2％FBS的PBS缓冲液重悬；空白组加入0.5μl的水；T2组和二抗组分别加入0.5μl的APC抗人Ig轻链K(BioLegend，克隆号：TB28-2)。将各组混匀后于室温避光孵育30min。孵育完成后以含有2％FBS的PBS缓冲液清洗各组的管，1000rpm/min离心5min，以100μl含有2％FBS的PBS缓冲液重悬，上流式细胞仪(艾森，仪器型号：NovoCyte)检测。结果如图4中的B2所示。
实施例3：靶向CD3×CD137×c-Met三特异性抗体及靶向CD3×c-Met双特异性抗体的制备、表达与鉴定
3.1.1靶向CD3×CD137×c-Met的三特异性抗体Triad19表达载体的制备
表达载体构建及质粒扩增的具体操作流程参照实施例1。抗c-Met抗体的VH和VL编码核酸序列来源于Onartuzumab(MetMAb)(专利号：US7615529B2)。合成载体pUC57 c-Met scFv(通用生物系统(安徽)有限公司)，其含有通过DNA合成的抗c-Met scFv编码核酸序列，用来扩增抗c-Met VH片段；以质粒pQKTriad1 H(见实施例1)为模板，用来扩增IgG 1 CH1-Hinge mut-CD3 VH-IgG1 Hinge mut R-CD137 VH片段，对全合成载体pQKX1(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒命名为pQKTriad19H(连接顺序从5’到3’为：抗c-Met抗体VH-IgG1 CH1-Hinge-mut-抗CD3抗体VH-IgG1 Hinge mut R-抗CD137抗体VH)。合成载体pUC57 c-Met scFv(通用生物系统(安徽)有限公司)，其含有通过DNA合成的抗c-Met scFv编码核酸序列，用来扩增抗c-Met VL片段；以质粒pQKTriad1 L(见实施例1)为模板，用来扩增Kappa-Hinge mut-CD3 VL-IgG1 Hinge mut R-CD137 VL片段，对 全合成载体pQKX2(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒命名为pQKTriad19L(连接顺序从5’到3’为：抗c-Met抗体VL-Kappa-Hinge mut-抗CD3抗体VL-IgG1Hinge mut R-抗CD137抗体VL)。
PCR扩增引物对如下：
Figure PCTCN2021130015-appb-000008
Figure PCTCN2021130015-appb-000009
3.1.2靶向CD3×c-Met的双特异性抗体ZWB56表达载体的制备
表达载体构建及质粒扩增的具体操作流程参照实施例1。抗c-Met抗体的VH和VL编码核酸序列来源于Onartuzumab(MetMAb)(专利号：US7615529B2)。以pQKTriad19H质粒(见实施例3.1.1)为模板，用来扩增c-Met VH-IgG1 CH1-Hinge mut-CD3 VH片段，对全合成载体pQKX1(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒命名为pQKZWB56H(连接顺序从5’到3’为：抗c-Met抗体VH-IgG1CH1-Hinge-mut-抗CD3抗体VH)；以pQKTriad19L质粒(见实施例3.1.1)为模板，用来扩增c-Met VL-Kappa-Hinge mut-CD3 VL片段，对全合成载体pQKX2(通用生物系统(安徽)有限公司)进行SapI和EcoRI双酶切，回收相应的PCR产物和酶切产物，进行重组酶连，获得最终的重组质粒 命名为pQKZWB56L(连接顺序从5’到3’为：抗c-Met抗体VL-Kappa-Hinge-mut-抗CD3抗体VL)。相较于三特异性抗体Triad19，双特异性抗体ZWB56仅缺少了IgG1 Hinge mut R-抗CD137抗体VH和IgG1 Hinge mut R-抗CD137抗体VL(图1C和图1D)。
PCR扩增引物对如下：
Figure PCTCN2021130015-appb-000010
3.2抗体的表达
参照实施例1所描述的流程进行转染，其中转染细胞为HEK293(ATCC编号：CRL-1573)，转染体积为100mL。转染后的细胞于500mL摇瓶中悬浮培养7天后收获抗体，培养条件为36.5℃，7.5％CO 2，120rpm/min。其中所得抗体为质粒pQKTriad19H和pQKTriad19L表达的抗c-Met×抗CD3×抗CD137三特异性抗体时，抗体命名为Triad19，结构如图1中的C所示；所得抗体为质粒pQKZWB56H和pQKZWB56L表达的抗c-Met×抗CD3双特异性抗体时，抗体命名为ZWB56，结构如图1中的D所示。
3.3抗体的纯化
参照实施例1所描述的流程进行纯化，经缓冲液置换后使用Nanodrop微量分光光度计测定蛋白浓度，计算蛋白产量，其中Triad19为9.8mg/L，ZWB56为125mg/L。
3.4抗体的鉴定
3.4.1 SDS-PAGE测定抗体大小及纯度
选择SDS-PAGE胶进行蛋白电泳，分别加入还原及非还原缓冲液，对蛋白还原及非还原状态分子条带大小进行鉴定。考马斯亮蓝染色后， 根据蛋白Marker分析蛋白的准确性及纯度，结果如图5所示。分析蛋白电泳，条带显示电泳与预期蛋白大小一致，且蛋白纯度较高。
3.4.2 Fortebio测定抗体亲和力
使用Fortebio分子相互作用仪分别测定ZWB56/Triad19与c-Met蛋白，CD3蛋白、CD137蛋白的结合的动力学参数。具体地，用His或Fc传感器捕获固定c-Met蛋白，CD3蛋白、CD137蛋白，在PBS中平衡后，与ZWB56/Triad19结合。将ZWB56/Triad19用PBS稀释为4个浓度，分别为500nM、250nM、125nM、62.5nM，于PBS中解离。亲和力常数测定结果如表3和表4所示。
表3：T19和各靶点亲和力常数测定结果
抗体-抗原 亲和力常数K D(M)
T19/c-Met Ag 3.73E-09
T19/CD3εAg 1.04E-07
T19/CD137Ag 1.02E-07
表4：B56和各靶点亲和力常数测定结果
抗体-抗原 亲和力常数K D(M)
B56/c-Met Ag 2.37E-09
B56/CD3εAg 1.16E-07
3.5通过CD3功能细胞株Jurkat Dual检测抗体功能活性
1).靶细胞处理：准备一瓶T75中的靶细胞U87 MG(协和细胞资源中心，资源编号：3111C0001CCC000208)，用0.25％胰酶消化，室温下850rpm离心5min，弃上清后，用工作培养基(含10％FBS(灭活)的DMEM培养基)重悬细胞，并用自动细胞计数仪计数细胞密度和活率，然后稀释细胞悬液至5×10 4个细胞/mL。
2).将稀释好的U87 MG细胞按照100μL/孔加入到96孔板中。放入5％CO 2的37℃细胞培养箱孵育6h，待细胞贴壁。
3).Jurkat Dual细胞处理：取一瓶T75中的Jurkat Dual细胞(协和 细胞资源中心，资源编号：3111C0001CCC000075)，850rpm离心5min，弃上清，用工作培养基(含10％FBS(灭活)的1640培养基)重悬计数，将细胞调整至2×10 6个细胞/mL。
4).对Triad19/ZWB56进行稀释，Triad19最高浓度点为20μg/mL，按照50μL/孔加入，对应药物终浓度为10μg/mL，即终浓度为100nM。ZWB56最高浓度点为15μg/mL，按照50μL/孔加入，对应药物终浓度为7.5μg/mL，即终浓度为100nM。三倍梯度稀释，共计10个浓度点。
5).按照板孔设计，向96孔板中，分别向每孔加入50μL不同浓度Triad19/ZWB56稀释液及50μL稀释好的Jurkat Dual细胞，以使每孔中含1×10 5个Jurkat Dual细胞。
6).对照孔或者没有靶细胞的孔用完全培养基补液至200μl。
7).将96孔板移入恒温细胞培养箱(37℃、5％CO 2)中培养18～24h。
8).培养结束后，室温条件下，300～400g离心5min。
9).取新96孔板(黑色透明底)，吸取40μl细胞培养上清，每孔加入50μl QUANTI-Luc底物，混匀后上多功能酶标仪检测信号强度值。
10).将检测结果用GraphPad Prism 8软件分析，统计结果。
结果如图6显示，加入表达c-Met的靶细胞U87 MG时，Triad19/ZWB56的CD3部分功能信号明显加强，同时相较ZWB56，Triad19的CD3功能较强。无靶细胞作用下，单纯抗体药物对Jurkat Dual细胞无功能激活作用。
实施例四：RTCA分析抗体作用下T细胞杀伤实验
实验步骤如下：
1.取表达c-Met的U87 MG细胞，消化后离心，用完全培养基(DMEM/1640+10％FBS+1％GluMax)重悬计数，按照5×10 4个细胞/mL浓度进行稀释。
2.取RTCA(ACEA)配套板Eplate16，按照RTCA仪器操作，首先将RTCA板加入50μL完全培养基，将板置于仪器上走预活化步骤。
3.板预活化后，将U87 MG细胞按照100μL/孔细胞加入板中，静置半小时后开始程序。
4.观察细胞生长曲线，待细胞处于生长指数期时，复苏PBMC(澳赛尔斯生物技术(上海)有限公司)，采用Stemcell公司的EasySep TMHuman T Cell Isolation Kit试剂盒从人PBMC中分选T细胞，用工作培养基(含1％GluMax，10％FBS(灭活)的1640培养基)重悬细胞并计数。
5.按照效靶比5：1的比例，即靶细胞是5000个细胞/孔，效应T细胞为25000个细胞/孔，将分选的T细胞稀释到5×10 5个细胞/mL。
6.Triad19及ZWB56药物浓度梯度稀释，取药物最高浓度点为5nM，对应Triad19终浓度为0.5μg/mL，ZWB56终浓度为0.375μg/mL，药物配制浓度为终浓度4倍量，即Triad19 2μg/mL，ZWB56为1.5μg/mL。
7.暂停RTCA程序，将板孔中各细胞弃掉50μL上清，取稀释好的T细胞每孔中加入50μL，Triad19/ZWB56按照浓度对应加入50μL，对照孔中加入完全培养基补至200μL。
8.待板孔于RTCA仪器上静置30min后，开始程序。观察杀伤实验中细胞生长曲线，分析后拟合药物杀伤曲线，如图7、图8所示，Triad19拟合IC50值在亚nM水平，ZWB56拟合EC50值在nM水平。并通过GraphPad Prism 8分析药物各浓度下对细胞的杀伤率，如图9所示。
9.取96孔板同步进行平行实验，目的在于给药后杀伤16-20h取细胞上清，检测IL-2/IL-6细胞因子。另等杀伤实验终止时，取平行孔细胞上清进行IFN-γ因子检测。
10.采用人IL-2/IL-6/IFN-γELISA试剂盒(联科生物)，进行细胞因子ELISA检测，对各样本的因子进行定量，酶标仪检测OD值后，通过GraphPad Prism 8分析相关数据，结果汇总于图10。
11.收集T细胞杀伤实验后的T细胞，进行T细胞分群分析，包括CD4、CD8、CD45RO、CCR7、PD-1、Granzyme B，对CD4及CD8细胞比例进行分析，中枢记忆性T细胞和效应记忆性T细胞比例分析，以及Granzyme B随药物浓度比例变化，如图11-图15所示。以上作图分析均选择GraphPad Prism 8进行。
结果分析，T细胞杀伤实验结果显示，拟合药物杀伤曲线获得药物IC50，Triad19杀伤能力高于ZWB56一个数量级。分析每个浓度点下细胞杀伤率拟合曲线来看，对应每个浓度点，ZWB56相较于Triad19杀伤力偏弱。IL2、IL6、IFN-γ细胞因子分析结果显示，各细胞因子的表达量呈现明显的药物剂量关系，药物浓度越高，各因子表达量越高。同样，同等药物浓度下，ZWB56偏弱。
细胞流式分析结果显示，Triad19/ZWB56给药后，随药物剂量升高，CD3T细胞中CD8比例上调，对应CD4比例下调。CD4或CD8效应记忆性T细胞(TEM)比例变化伴随药物浓度升高有升高趋势，中枢记忆性T细胞(TCM)基本无变化。Granzyme B指标，Triad19在高浓度点明显高于ZWB56，这也刚好对应ZWB56杀伤偏弱的原因。PD-1在一定程度上也可以作为T细胞活化的一个指标，分析来看，随药物浓度升高，CD4T细胞及CD8T细胞上PD-1均有升高趋势，且具有一定的规律性。
总结来看，在等摩尔给药的对比下，Triad19较ZWB56具有较强的T细胞活化及杀伤肿瘤细胞的能力，包括各细胞因子比较及流式检测各细胞分群分析。
虽然，上文中已经用一般性说明及具体实施方案对本申请的技术方案作了详尽的描述，但在这些技术方案的基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本申请精神的基础上所做的这些修改或改进，均属于本申请要求保护的范围。

Claims (15)

  1. 一种融合蛋白，其包含：
    a)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
    b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；以及
    c)第一肽接头和第二肽接头，
    其中所述第一肽接头用于连接所述抗CD137抗体或其抗原结合片段的重链以及所述抗CD3抗体或其抗原结合片段的重链，并且所述第二肽接头用于连接所述抗CD137抗体或其抗原结合片段的轻链和所述抗CD3抗体或其抗原结合片段的轻链，
    所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键。
  2. 如权利要求1所述的融合蛋白，其还包含：
    d)能特异性结合第一抗原的第一抗体或其抗原结合片段，以及
    e)第三肽接头和第四肽接头，
    其中所述第一抗体或其抗原结合片段的重链通过所述第三肽接头连接所述抗CD3抗体或其抗原结合片段的重链，并且
    所述第一抗体或其抗原结合片段的轻链通过所述第四肽接头连接所述抗CD3抗体或其抗原结合片段的轻链，
    所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键。
  3. 如权利要求2所述的融合蛋白，其中所述第一肽接头、第二肽接头、第三肽接头和第四肽接头各自独立地选自以下：包含Seq ID NO.1-2所示序列中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失，任选地，所述第一肽接头至第四肽接头中的每一个为天然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变，任选地，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区，
    任选地，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体或单链抗体分子如sc-Fv。
  4. 如权利要求2或3所述的融合蛋白，其中所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met和GPC3，任选地，所述第一抗原、CD137分子和CD3分子独立地来源于哺乳动物，优选非人类的灵长类或者人类，任选地，所述第一抗体或其抗原结合片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍。
  5. 一种融合蛋白，其从N端至C端依次包含：
    a)能特异性结合第一抗原的第一抗体的Fab片段；
    b)能特异性结合CD3分子的抗CD3抗体或其抗原结合片段；
    c)能特异性结合CD137分子的抗CD137抗体或其抗原结合片段；
    其中所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的重链依次通过第一肽接头和第三肽接头连接，并且所述Fab片段、所述抗CD3抗体或其抗原结合片段、所述抗CD137抗体或其抗原结合片段各自的轻链依次通过第二肽接头和第四肽接头连接，
    其中所述第一肽接头和所述第二肽接头之间仅能形成一个二硫键，并且所述第三肽接头和所述第四肽接头之间仅能形成一个二硫键，各自独立地选自以下：包含Seq ID NO.1-2所示序列中的任一种的肽接头，其中X代表除Cys的任意氨基酸，或者缺失，任选地，所述第一肽接头至第四肽接头中的每一个为天然抗体的铰链区，其中铰链区发生了仅保留一个半胱氨酸的缺失突变，任选地，所述第一肽接头至第四肽接头中的任一个独立地选自C229缺失突变的IgG1铰链区或C229缺失突变，且铰链区D224-S242倒转的IgG1铰链区。
  6. 如权利要求5所述的融合蛋白，其中所述第一抗原选自MESOTHELIN、EGFR、PSMA、GD2、CEA、MUC1、FAP、BCMA、EphA2、CD19、CD22、EpCAM、CEA、PD-L1、B7H3、ROR1、c-Met 和GPC3，任选地，所述第一抗原、所述CD137分子和所述CD3分子独立地来源于哺乳动物，优选非人类的灵长类或者人类，任选地，所述Fab片段与所述第一抗原结合的亲和力常数是所述抗CD137抗体或其抗原结合片段与CD137分子的亲和力常数或者抗CD3抗体或其抗原结合片段与CD3分子的亲和力常数的10-1000倍，任选地，所述抗原结合片段选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、双体或单链抗体分子如sc-Fv。
  7. 一种核酸，其编码权利要求1-6中任一项所述的融合蛋白。
  8. 一种表达载体，其包含权利要求7所述的核酸。
  9. 一种宿主细胞，其包含权利要求7所述的核酸或权利要求8所述的表达载体，任选地，所述宿主细胞为哺乳动物细胞，所述哺乳动物细胞选自CHO细胞、NS0细胞、SP2/0细胞、HEK293细胞、COS细胞和PER.C6细胞。
  10. 制备权利要求1-6中任一项所述的融合蛋白的方法，其包括：
    a)培养权利要求9所述的宿主细胞；和
    b)从所述宿主细胞中或所述宿主细胞的培养物上清中回收所述融合蛋白。
  11. 药物组合物，其包含权利要求1-6中任一项所述的融合蛋白，权利要求7所述的核酸，权利要求8所述的表达载体或权利要求9所述的宿主细胞，以及药学上可接受的载体。
  12. 权利要求1-6中任一项所述的融合蛋白，权利要求7所述的核酸，权利要求8所述的表达载体或权利要求9所述的宿主细胞在制备用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的药物中的用途。
  13. 治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的方法，其包括向有需要的个体施用权利要求1-6中任一项所述的融合蛋白，权利要求7所述的核酸，权利要求8所述的表达载体或权利要求9所述的宿主细胞，任选地，其还包括施用治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的第二药剂。
  14. 权利要求1-6中任一项所述的融合蛋白，权利要求7所述的核酸，权利要求8所述的表达载体或权利要求9所述的宿主细胞用于治疗、改善或预防肿瘤、自身免疫性疾病或传染性疾病的用途。
  15. 如权利要求12-14中任一项所述的用途或方法，其中所述肿瘤选自肺癌、结直肠癌、膀胱癌、白血病、乳腺癌、胃癌、胃食管结合部腺癌、B淋巴细胞型非霍奇金淋巴瘤、霍奇金淋巴瘤，间变大细胞淋巴瘤、头颈癌、恶性胶质瘤，肾癌、黑色素瘤、前列腺癌、骨癌、胰腺癌、肉瘤、肝癌、皮肤鳞癌、宫颈癌、鼻咽癌、子宫内膜癌，或上述肿瘤的转移癌，任选地，所述自身免疫性疾病选自系统性红斑狼疮、类风湿关节炎、系统性血管炎和自身免疫性溶血性贫血，任选地，所述传染性疾病选自流行性感冒、乙型肝炎、狂犬病、梅毒、艾滋病。
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