WO2023169349A1 - 一种融合蛋白、制备方法及其应用 - Google Patents

一种融合蛋白、制备方法及其应用 Download PDF

Info

Publication number
WO2023169349A1
WO2023169349A1 PCT/CN2023/079786 CN2023079786W WO2023169349A1 WO 2023169349 A1 WO2023169349 A1 WO 2023169349A1 CN 2023079786 W CN2023079786 W CN 2023079786W WO 2023169349 A1 WO2023169349 A1 WO 2023169349A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
ligand
fusion protein
binding
seq
Prior art date
Application number
PCT/CN2023/079786
Other languages
English (en)
French (fr)
Inventor
梁耀极
刘杰
Original Assignee
厦门柏慈生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门柏慈生物科技有限公司 filed Critical 厦门柏慈生物科技有限公司
Priority to CN202380014988.4A priority Critical patent/CN118339194A/zh
Publication of WO2023169349A1 publication Critical patent/WO2023169349A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the invention relates to the field of biomedicine. Specifically, the present invention relates to a fusion protein containing a ligand protein and its application. The invention also relates to derivatives, conjugates, pharmaceutical combinations, nucleic acid molecules, expression vectors and host cells of the fusion protein as well as methods for preparing the fusion protein.
  • Ligand is a type of molecule that can bind to its receptor protein and transmit signals.
  • the ligands described in the present invention refer to protein ligands, including: cytokines, ligands on cell membranes, protein hormones, etc.
  • Cytokine (CK) is a very important, free ligand and a low molecular weight soluble protein produced by cells. According to its function, it can be divided into interleukins, interferons, tumor necrosis factor superfamily, and colony-stimulating factors. , chemokines, growth factors, etc. Cytokines mediate intracellular signal transduction by binding to corresponding cytokine receptors on the cell surface and exert a variety of biological functions, including immune regulation, blood cell production, cell growth, and repair of damaged tissue. Cytokine-based drugs are therefore an important area of drug development. However, many natural cytokines are not suitable as drugs. Because a cytokine can usually bind to multiple receptors expressed on different cell surfaces, transmitting different signals.
  • the same receptor can also bind to different cytokines, which results in the pleiotropic effects of cytokines. , overlap, antagonism, synergy and other physiological characteristics. In clinical treatment, it is necessary to specifically activate a specific signal involving cytokines, without activating unnecessary signals, to further perform the expected function, and to achieve the goal of good therapeutic effect with minimal side effects.
  • Most of the cytokine receptors are transmembrane proteins, consisting of an extracellular domain, a transmembrane region and a cytoplasmic region.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • IL-2 is a 15.5kDa globular glycoprotein with a length of 133 amino acids.
  • the structure of IL-2 consists of four antiparallel, amphipathic ⁇ -helices and some linking sequences (loops) (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)).
  • IL-2 is mainly derived from activated CD4+T cells, activated CD8+T cells, natural killer (NK) cells, dendritic cells and macrophages, and can be regulated by binding to IL-2 receptors on the cell surface. its role.
  • the IL-2 receptor is a complex composed of three subunits, namely IL-2R ⁇ (CD25), IL-2R ⁇ (CD122) and IL-2R ⁇ (CD132).
  • the expression of these three subunits and their affinity for IL-2 vary.
  • the heterodimeric IL-2 receptor formed by IL-2R ⁇ subunit and IL-2R ⁇ subunit is mainly expressed by cytotoxic CD8+ T cells and NK cells. It binds to IL-2 with medium affinity and is called medium affinity IL-2. 2 receptor (IL-2R ⁇ ).
  • the heterotrimeric IL-2 receptor formed by IL-2R ⁇ subunit, IL-2R ⁇ subunit and IL-2R ⁇ subunit is mainly expressed on regulatory T cells (Treg) (Byman, 0.
  • IL-2R ⁇ high-affinity IL-2 receptor
  • CD25 IL-2 receptor ⁇
  • IL-2R ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • IL-2R ⁇ is not necessary for signaling, but can promote the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ .
  • IL-2R ⁇ is expressed in all immune cells; IL-2R ⁇ is expressed in CD8+ T cells, NK cells, and regulatory T cells, and the expression level increases after T cells are activated; IL-2R ⁇ Regulatory T cells continue to be highly expressed, and will be briefly expressed in activated CD8+ T cells, and then the expression level will be reduced. Since resting effector T cells and NK cells do not have IL-2R ⁇ on their cell surface, they are relatively insensitive to IL-2. Treg cells always express the highest level of IL-2R ⁇ in the body. Therefore, under normal circumstances, IL-2 will preferentially stimulate the proliferation of Treg cells.
  • IL-2 has the function of expanding lymphocyte populations and improving the effector functions of these cells in the body, especially the proliferation and activation of CD8+ T cells and NK cells, which endows IL-2 with anti-tumor capabilities.
  • IL-2 is the first cytokine used in tumor immunotherapy in history, and its anti-tumor effect has been clinically proven.
  • High-dose IL-2 therapy is already approved for patients with metastatic renal cell carcinoma and malignant melanoma. After years of clinical application, people have a deeper understanding of IL-2, and the clinical application of IL-2 has also brought many problems.
  • VLS vascular leak syndrome
  • VLS may be related to the binding of IL-2 to IL-2RA expressed by endothelial cells (Kriegetal., ProcNatAcadSciUSA107, 11906-11 (2010)).
  • IL-2 binds to the high-affinity IL-2 receptor highly expressed on suppressive regulatory T cells (Treg)
  • Treg cells suppress effector T cells from destroying their targets; either by inhibiting T cell assistance and activation through cell-cell contact; or by attenuating IL-2 induction through the release of immunosuppressive cytokines, such as IL-10 or TGF- ⁇ .
  • Inhibiting the binding of IL-2 to the high-affinity IL2 receptor (IL-2R ⁇ ), retaining or enhancing the binding to the medium-affinity IL2 receptor (IL-2R ⁇ ), and extending its half-life are ways to overcome the problems of IL-2 in tumor immunotherapy key.
  • IL-15 another member of the IL-2 family with anti-tumor activity. Its receptor consists of three receptor subunits: IL-15 receptor ⁇ (IL15RA or IL-15R ⁇ ), IL-2 receptor body beta (IL-2R ⁇ , also known as IL-15R ⁇ or CD122) and ⁇ c (also known as CD132). IL-15 and IL-2 are very similar in structure and belong to the spiral cytokine family. The heterotrimeric receptor for IL-15 shares IL-2R ⁇ /IL-15R ⁇ (CD122) and the common ⁇ c chain (CD132) with the IL-2 receptor.
  • IL-15R ⁇ as a unique component of the IL-15 receptor complex, is mainly expressed on monocytes and dendritic cells. Different from other ⁇ c family cytokines, IL-15 as a cytokine first binds to IL-15R ⁇ expressing cells, and then the IL-15/IL-15R ⁇ complex is presented to IL-15 on activated T cells or NK cells. 2 ⁇ /15R ⁇ and ⁇ c. This limits the activity of IL-15.
  • IL-15 mutant (IL-15N72D) and IL-15R ⁇ Su/Fc dimeric protein have been shown to have excellent anti-tumor activity in mouse models. However, it still has shortcomings such as high clearance rate and toxicity caused by activating peripheral immune cells. For clinical use, it is necessary to have a biased IL-15 that can directly bind to the IL-15R ⁇ receptor and directly activate T cells/NK cells while avoiding stimulation of regulatory T cells (Treg).
  • IL-2 mutants can be used to alter the affinity specificity of IL-2 for different receptors.
  • Merck's mutants R38W, F42K, WO2008003473A2 reduce the interaction with ⁇ -receptor subunits to achieve effector T cell activation to enhance efficacy; while Roche's IL-2 mutants (F42A, Y45A and L72G , US2016/0208017A1), which does not bind to ⁇ receptors, but can bind to ⁇ and ⁇ receptor subunit complexes normally and can exert effects. It is currently in clinical use.
  • IL-2R ⁇ medium-affinity IL2 receptor
  • the object of the present invention is to construct a fusion protein including a ligand and its binding protein.
  • the fusion protein contains two polypeptide chains, connecting the ligand and the ligand-binding protein to the heavy chain constant domain CH1 and light chain of the antibody respectively.
  • the present invention provides a fusion protein that fully utilizes the potential of a ligand-binding protein to regulate its function by binding to a ligand (such as a naturally occurring receptor that can antagonize the ligand and An antagonist of its own interaction; or an antibody targeting the target ligand or its antigen-binding protein, which has the potential to block the interaction between the ligand and its receptor or potentially activate the function of the ligand, etc.), through The ligand and its binding protein are constructed into the fusion protein, so that the ligand-binding protein can regulate the function of the ligand and then exert the specific function of the ligand.
  • a ligand such as a naturally occurring receptor that can antagonize the ligand and An antagonist of its own interaction; or an antibody targeting the target ligand or its antigen-binding protein, which has the potential to block the interaction between the ligand and its receptor or potentially activate the function of the ligand, etc.
  • the fusion protein contains a first polypeptide and a second polypeptide. These two peptide chains are modified based on the heavy chain constant domain CH1 and the light chain constant domain CL of the antibody.
  • the first polypeptide and the second polypeptide of the fusion protein respectively have the following polypeptide structural formulas from the N-terminus to the C-terminus:
  • the first polypeptide and the second polypeptide of the fusion protein respectively have the following polypeptide structural formulas from the N-terminus to the C-terminus:
  • the first polypeptide C1-L1-A (Formula III)
  • A is a ligand
  • B is a ligand-binding protein that can interact with A; it can also be that A is a ligand-binding protein, and B is a ligand that can interact with A;
  • the ligand includes wild-type Ligands or variants thereof;
  • the variants include mutants, transformations and truncations of the ligand, as well as the extracellular domain of the ligand or mutants, transformations and truncations of the extracellular domain of the ligand;
  • the described ligand-binding protein includes wild-type ligand-binding protein or variants thereof;
  • the described variants include mutants, transformations and truncations of the ligand-binding protein, as well as the extracellular domain of the ligand-binding protein or the ligand-binding protein. Mutants, modifications, and truncations of the extracellular domain of ligand-binding proteins;
  • C1 is the antibody light chain constant domain CL
  • C2 is the antibody heavy chain constant domain CH1
  • a disulfide bond is formed between C1 and C2; thus the two peptide chains have a heterodimer form
  • the CL is a CL of a human antibody (including the constant region of the kappa light chain (IGKC) and the constant region of the lambda light chain (IGLC)) or has the same structure as the human CL
  • An amino acid sequence that has at least 80% (preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) sequence identity to the CH1 of a human antibody or has at least An amino acid sequence with 80% (preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) sequence identity
  • the CL is human IGKC, which The amino acid sequence is shown in SEQ ID NO: 1; the CH1 is human CH1, and its amino acid sequence is shown in SEQ ID NO: 2.
  • L1 and L2 are independent joint elements (in some embodiments, there may be no joint elements L1 and L2).
  • Optional joint elements include but are not limited to the following sequences:
  • n is a positive integer (such as 2, 3, 4, 5 or 6, etc.);
  • the fusion protein also includes the Fc region of an antibody, and the Fc region is connected to the first polypeptide or the second polypeptide; further increasing the molecular weight of the fusion protein, thereby extending its half-life in the body.
  • the Fc region can be from an immunoglobulin of different subtypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.
  • mutations can be introduced into the wild-type Fc sequence to alter the associated Fc-mediated activity. The mutations include, but are not limited to: a). Mutations that alter Fc-mediated CDC activity; b).
  • mutations can be introduced into the Fc sequence, thereby making the mutated Fc more likely to form homodimers or heterodimers.
  • the knob-hole model mentioned in Ridgway, Presta et al. 1996 and Carter 2001 utilizes the spatial interaction of amino acid side chain groups at the Fc contact interface, making it easier to form heterodimers between different Fc mutations; another example is CN102558355 or CN103388013A.
  • the immunoglobulin Fc region is preferably a human immunoglobulin Fc region, more preferably an Fc region of human IgG1.
  • the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO: 5.
  • the Fc region is connected to the C-terminus of the first polypeptide, so that the first polypeptide forms a polypeptide structural formula A-L1-C1-L3-Fc or C1-L1-A-L3-Fc.
  • the Fc region is connected to the C-terminus of the second polypeptide, so that the second polypeptide forms a polypeptide structural formula B-L2-C2-L3-Fc or C2-L2-B-L3-Fc.
  • the Fc region is connected to the N-terminus of the first polypeptide, so that the first polypeptide forms the polypeptide structural formula Fc-L3-A-L1-C1 or Fc-L3-C1-L1-A.
  • the Fc region is connected to the N-terminus of the second polypeptide, so that the second polypeptide forms the polypeptide structural formula Fc-L3-B-L2-C2 or Fc-L3-C2-L2-B.
  • the fusion protein can also form an immunoglobulin-like tetramer structure, which includes two first polypeptide chains and two second polypeptide chains.
  • L3 is an independent linker element (in some embodiments, there may be no linker element L3).
  • Optional linker elements include but are not limited to the following sequences:
  • n is a positive integer (such as 2, 3, 4, 5 or 6, etc.);
  • derivatives of the fusion protein are also included.
  • the ligand is a cytokine (including interleukin, interferon, tumor necrosis factor superfamily, colony-stimulating factor, chemokine, growth factor, etc.), protein hormone molecule, Ligands on the cell membrane or their variants, modifications, and truncations.
  • the ligand-binding protein is a receptor for the ligand or a variant of the receptor.
  • the variants include: mutants, transformations, truncations of the receptor, as well as the extracellular domain of the receptor and the receptor. Mutants, modifications, and truncations of the receptor extracellular domain.
  • Receptors as naturally occurring antagonists that can antagonize the interaction between ligands and themselves, are important potential molecules that can regulate the function of ligands.
  • receptors as proteins that naturally antagonize cytokines (for example, CD25 is a protein that naturally antagonizes the binding of IL-2 to CD25, CD122 is a protein that naturally antagonizes the binding of IL-2 to CD122, etc.)
  • cytokines and receptors are constructed into a fusion In the protein, the ligand-binding protein can regulate the function of the ligand, thereby exerting the specific function of the ligand.
  • the ligand-binding protein is an antibody of the ligand or an antigen-binding fragment thereof; blocking the interaction between the ligand and its receptor or potentially activating the ligand through the antibody or antigen-binding protein thereof The function of the ligand, etc., and then exert the specific function of the ligand.
  • Single-domain antibodies and single-chain antibodies are the smallest functional units that retain the antigen specificity of the original antibody, and both perform their functions in the form of a single peptide chain.
  • the antibody or antigen-binding fragment thereof is a single domain antibody or a single chain antibody.
  • Single-domain antibodies and single-chain antibodies have unique advantages in molecular modification and fusion protein construction due to their simple structure (only one peptide chain can perform antibody-related functions) and easy engineering.
  • Single domain antibody refers to a fragment that contains a single variable domain in an antibody. Like intact antibodies, it can selectively bind to specific antigens. Compared with the mass of 150-160kDa of intact antibodies, single domain antibodies are much smaller, only about 12-15kDa.
  • the first single domain antibodies were modified from heavy chain antibodies found in camelids (Hamers-Casterman C, et.al (1993) 6428:446-448). Camels and cartilaginous fish (shark) have natural heavy chain antibodies (Heavy Chain Antibody, HcAb) that lack the light chain. As natural antibody molecules that lack the light chain, heavy chain antibodies are genetically engineered to be different from traditional IgG antibody molecules. Have greater advantages.
  • VHH single domain antibody Variable domain of heavy chain of heavy chain antibody
  • cartilaginous fish also have heavy chain antibodies (IgNAR, immune Immunoglobulin new antigen receptor (abbreviation of Immunoglobulin new antigen receptor), single domain antibodies (Variable domain of Ig new antigen receptor) called "V-NAR segments” can also be produced from this type of antibody.
  • Single domain antibodies have the same heavy chain Antibodies have considerable structural stability and antigen-binding activity. They are the smallest units currently known that can bind target antigens. Their molecular weight is only 15KD, only one-tenth of traditional antibodies, so they are also called nanobodies. ).
  • single domain antibodies Compared with ordinary antibodies, single domain antibodies have small molecular weight, simple structure, easy genetic modification, small size, good antigen specificity, strong tissue penetration, high stability, and have broad application prospects in the diagnosis and treatment of diseases. .
  • single-domain antibodies are one of the important choices as ligand-binding proteins for constructing the fusion proteins of the present invention because of their small molecular weight and simple structure.
  • Single-chain antibody variable fragment is composed of antibody heavy chain variable domain (heavy chain variable domain, VH) and light chain variable domain (light chain variable domain, VL) through a 10-25 It is a recombinant antibody form (about 27kDa) composed of flexible short peptides (linkers) connected by amino acids.
  • scFv is a fusion protein and retains the antigen specificity of the original immunoglobulin.
  • the smaller molecular size of scFv brings advantages such as strong intra-tumor penetration, rapid degradation in the blood, and small negative feedback in the human body, which also lays the foundation for the clinical application of scFv.
  • single-chain antibodies are also one of the important choices as ligand-binding proteins for constructing the fusion proteins of the present invention because they have only one peptide chain and simple structure.
  • the ligand is IL-2
  • the ligand-binding protein is the extracellular domain of CD25
  • a CD25/IL-2 fusion is formed through the first polypeptide chain and the second polypeptide chain.
  • Protein (Ab-CD25/IL-2-Complex) the fusion protein or its derivative has at least one of the following characteristics:
  • the ligand is IL-2
  • the ligand-binding protein is the extracellular domain of CD122
  • a CD122/IL-2 fusion is formed through the first polypeptide chain and the second polypeptide chain.
  • protein (Ab-CD122/IL-2-Complex). Because killer T cells, NK cells, etc. mainly express CD122/CD132 dimer receptors, and regulatory T cells (Treg) mainly express CD25-containing trimer receptors, the Ab-CD122/IL-2-Complex has It has the potential to specifically activate regulatory T cells without activating the function of killer T or NK cells, thereby promoting the body to restore immune balance, and has a potentially important role in the treatment of autoimmune diseases.
  • the fusion protein or its derivative has at least one of the following characteristics:
  • the ligand is IL-2
  • the ligand-binding protein is an anti-IL-2 VHH
  • a VHH/IL-2 fusion is formed via the first polypeptide chain and the second polypeptide chain.
  • Protein (Ab-VHH/IL-2-Complex) the fusion protein or its derivative has at least one of the following characteristics:
  • the ligand is IL-15
  • the ligand-binding protein is the extracellular domain of IL15RA
  • an IL15RA/IL-15 fusion protein is formed through the first polypeptide chain and the second polypeptide chain ( Ab-IL15RA/IL-15-Complex);
  • the fusion protein or its derivative has at least one of the following characteristics indivual:
  • the present invention provides a conjugate, including the above-mentioned fusion protein or a derivative thereof, which is connected to other modules directly or indirectly through a linker element.
  • the other modules include antigen-binding modules, cytotoxins, radioactive isotopes, bioactive proteins, detectable markers, drugs, toxins, cytokines, enzyme gold nanoparticles/nanorods, nanomagnetic particles, and viruses.
  • Coat protein or VLP any one or combination thereof.
  • the antigen-binding moiety is an antibody or antigen-binding fragment; most preferably, the antibody or antigen-binding fragment targets an antigen presented on a tumor cell or in the environment of a tumor cell.
  • the antigen-binding moiety targets an antigen presented on tumor cells or in the environment of tumor cells. In some embodiments, the antigen-binding moiety targets an antigen on functional cells (eg, CD8+ T cells, NK cells, CIK cells, TIL cells, macrophages, DC cells, etc.).
  • functional cells eg, CD8+ T cells, NK cells, CIK cells, TIL cells, macrophages, DC cells, etc.
  • the fusion protein is linked to at least one other module.
  • the fusion protein and other modules form a fusion protein, that is, the fusion protein shares peptide bonds with other modules.
  • the fusion protein is linked to at least one other module, such as a first and a second other module.
  • the other modules are antigen-binding modules.
  • the fusion protein shares an amino- or carboxyl-terminal peptide bond with the first antigen-binding module
  • the second antigen-binding module shares an amino- or carboxyl-terminal peptide bond with: i) the fusion protein or ii) the first antigen-binding module.
  • the fusion protein shares a carboxy-terminal peptide bond with the first other module and an amino-terminal peptide bond with the second other module.
  • the other module is an antigen-binding module.
  • the antigen-binding module may be an antibody or antigen-binding fragment, including but not limited to an immunoglobulin molecule (eg, an IgG (eg, IgG1) class immunoglobulin molecule), an antibody, or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment is selected from the group consisting of polypeptide complexes comprising an antibody heavy chain variable region and an antibody light chain variable region, Fab, Fv, sFv, F(ab')2, linear antibodies, single chain Antibodies, scFv, sdAb, sdFv, Nanobodies, peptide antibodies peptibody, domain antibodies, multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), receptor binding region and interacting protein binding regions.
  • each antigen-binding module can be independently selected from various forms of antibodies and antigen-binding fragments, e.g., the first antigen-binding module may be a Nanobody molecule, and the second antigen binding module may be a scFv molecule, or the first and second antigen binding modules may each be a Nanobody molecule, or each of the first and second antigen binding modules may be Fab molecules.
  • the antigen targeted by each antigen-binding module can be selected independently, e.g., the first and second antigen-binding modules.
  • the second antigen binding module is directed to a different antigen or to the same antigen.
  • the antigen bound by the antigen-binding module may be selected from the group consisting of the A1 domain of tenascin C (TNCA1), the A2 domain of tenascin C (TNCA2), and the extradomain of fibronectin (ExtraDomainB) ( EDB), carcinoembryonic antigen (CEA), and melanoma-related chondroitin sulfate protein White polysaccharide (MCSP).
  • TNCA1 domain of tenascin C TNCA1
  • TNCA2 domain of tenascin C TNCA2
  • EDB extradomain of fibronectin
  • CEA carcinoembryonic antigen
  • MCSP melanoma-related chondroitin sulfate protein White polysaccharide
  • tumor antigens include, but are not limited to, MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b.
  • DPPIV dipeptidyl peptidase IV
  • ADAbp adenosine deaminase binding protein
  • cyclophilin b include, but are not limited to, MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b.
  • CRC Colorectal-related antigen
  • CEA carcinoembryonic antigen
  • CAP-1 and CAP-2 immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and Its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3-zeta chain
  • MAGE family of tumor antigens such as MAGE-A1, MAGE- A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2) , MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGEB4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE family of tumor antigens
  • non-limiting examples of viral antigens include influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIVgp160, and HIVgp120.
  • ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin , EGF-type laminin, lectin, fibronectin, notch, tenascin and matrixin.
  • the present invention discloses a pharmaceutical composition, including the above-mentioned fusion protein or its derivative or the above-mentioned conjugate and a pharmaceutically acceptable diluent, carrier or auxiliary agent.
  • the pharmaceutical composition may be a lyophilized preparation or an injectable solution.
  • the present invention provides a nucleic acid molecule encoding the above-mentioned fusion protein or a derivative thereof.
  • the nucleic acid molecule includes two polynucleotide modules, the first polynucleotide encodes the first polypeptide chain as described in the first aspect of the present invention, and the second polynucleotide encodes the first polypeptide chain as described in the first aspect of the present invention. the second polypeptide chain.
  • the nucleic acid of the invention can be RNA, DNA or cDNA.
  • the present invention provides an expression vector containing the nucleic acid sequence of the above-mentioned fusion protein or a derivative thereof.
  • the vector can be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
  • the expression vector is a viral vector that can produce viruses with physiological functions, such as some common oncolytic viruses: herpes simplex virus (HSV), adenovirus, vaccinia virus, reovirus, etc.
  • HSV herpes simplex virus
  • adenovirus vaccinia virus
  • reovirus reovirus
  • the invention relates to a host cell expressing or capable of expressing one or more fusion proteins of the invention and/or containing a nucleic acid or vector of the invention.
  • Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include, but are not limited to, Gram-negative bacterial strains (e.g. Escherichiacoli strains, Proteus strains and Pseudomonas strains) and Gram-positive bacterial strains (e.g. Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains) cells.
  • Gram-negative bacterial strains e.g. Escherichiacoli strains, Proteus strains and Pseudomonas strains
  • Gram-positive bacterial strains e.g. Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains
  • Suitable fungal cells include, but are not limited to, species of the genera Trichoderma, Neurospora, and Aspergillus. cells of the species; or include Saccharomyces (such as Saccharomycescerevisiae), Schizosaccharomyces (such as Schizosaccharomycespombe), Pichia (such as Pichia) Cells of species of Pichia pastoris and Pichiamethanolica and Hansenula.
  • Saccharomyces such as Saccharomycescerevisiae
  • Schizosaccharomyces such as Schizosaccharomycespombe
  • Pichia such as Pichia
  • Suitable mammalian cells include, but are not limited to, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.
  • amphibian cells insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins may also be used in the present invention.
  • the host cell is a functional cell capable of expressing the fusion protein of the invention or its derivatives (such as: CAR-T, CAR-NK, CD8+T cells, NK cells, CIK cells, TIL cells, Macrophages, DC cells, etc.), the functional cells have any one or more of the following physiological functions: tumor killing, pathogen clearance, immune effects, etc.
  • the functional cells have any one or more of the following physiological functions: tumor killing, pathogen clearance, immune effects, etc.
  • the present invention provides the fusion protein or derivatives thereof, nucleic acid molecules, host cells, immunoconjugates and pharmaceutical compositions of the present invention for preparation and treatment of related diseases (such as proliferative diseases, immune diseases, etc.) , Use in drugs that regulate T cell-mediated immune responses and stimulate an individual's immune system.
  • the proliferative disease may be a tumor or cancer (eg, metastatic tumor or carcinoma), or may be a solid tumor (eg, metastatic renal cell carcinoma and malignant melanoma).
  • the fusion protein or its derivatives, immunoconjugates, and pharmaceutical combinations disclosed in the present invention can be used to treat disease conditions that benefit from stimulating the host's immune system, especially conditions where it is desired to enhance cellular immune responses, which can include Disease situations in which the host immune response is insufficient or defective.
  • disease conditions for which fusion proteins or derivatives or immunoconjugates thereof are administered include tumors or infections in which cellular immune response is a key mechanism of specific immunity, such as cancer (e.g., renal cell carcinoma or melanoma), immune Deficiencies (e.g. in HIV-positive patients, immunosuppressed patients), chronic infections, etc.
  • enhancing cellular immune response may include any one or more of the following: general increase in immune function, increase in T cell function, increase in B cell function, recovery of lymphocyte function, IL-2 receptor expression Increased T cell responsiveness, increased natural killer cell activity or lymphokine-activated killer (LAK) cell activity, etc.
  • the disease for which the fusion protein or its derivatives, immunoconjugates, or drug combinations disclosed in the present invention are used to treat is a proliferative disorder, such as cancer.
  • a proliferative disorder such as cancer.
  • cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, Rectal, stomach, prostate, blood, skin, squamous cell, bone and kidney cancers.
  • neoplasms located in: abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands) , pituitary gland, testis, ovary, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Also included are precancerous conditions or injuries and cancer metastasis.
  • the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer.
  • other cell proliferative disorders may also be treated with the fusion proteins of the present disclosure or derivatives thereof, including but not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura ( purpura), sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other organ listed above Cell proliferative disorders other than neoplasia in the system.
  • the disease involves autoimmunity, transplant rejection, post-traumatic immune response, and infectious disease (eg, HIV).
  • At least 2 times a day, at least 1 time a day, at least 1 time every 48 hours, at least once every 72 hours, weekly A method of administering a fusion protein or a derivative thereof or an immunoconjugate to a subject at least once, at least once every 2 weeks, at least once every month, at least once every 2 months, or at least once every 3 months.
  • the fusion protein or its derivatives or immunoconjugates can be administered by any effective route.
  • the fusion protein or derivative thereof, immunoconjugate is administered by parenteral injection, including subcutaneous injection.
  • agents comprising a pharmaceutically acceptable amount of a fusion protein or derivative thereof, an immunoconjugate (e.g., a therapeutically effective amount), including those described above, together with one or more pharmaceutically acceptable diluents , pharmaceutical compositions with carriers or excipients (eg, isotonic injection solutions).
  • the pharmaceutical composition is generally a pharmaceutical composition suitable for human administration.
  • the pharmaceutical compositions comprise at least one additional prophylactic or therapeutic agent.
  • Some embodiments contain a sterile container containing one of the pharmaceutical compositions described above and optionally one or more additional components.
  • kits which includes the fusion protein of the present invention, its derivatives, conjugates, pharmaceutical compositions, nucleic acid molecules, expression vectors or host cells, and instructions for use.
  • Kits generally include labels indicating the intended use of the kit contents.
  • the term label includes any written or recorded material on or provided with the kit or otherwise provided with the kit.
  • the present invention provides a method for constructing and producing a fusion protein or a derivative thereof, including using the aforementioned nucleic acid molecule or using the aforementioned expression vector under conditions suitable for expressing the fusion protein or a derivative thereof, Or use the aforementioned host cells for expression.
  • the fusion protein of the present invention has a biased ligand function.
  • a functionally biased ligand can be constructed to overcome the problems in the drug production process of related ligand molecules (such as cytokines). It weakens/eliminates the functions of the original ligand protein in certain aspects, retains/enhances the functions of other specific aspects, and then exerts specific functions, which can be used in clinical drugs.
  • Figure 1 is a schematic structural diagram 1 of the Ab-CD25/IL-2-Complex fusion protein of the present invention
  • Figure 2 is a schematic structural diagram 2 of the Ab-CD25/IL-2-Complex fusion protein of the present invention
  • Figure 3 is a schematic structural diagram 3 of the Ab-CD25/IL-2-Complex fusion protein of the present invention.
  • Figure 4 shows the expression and purification of Ab-CD25/IL-2-Complex fusion protein
  • Figure 5 shows the binding of Ab-CD25/IL-2-Complex fusion protein to CD25
  • Figure 6 shows the binding of Human IL-2, Human IL-2-Fc and CD25;
  • Figure 7 shows the binding of Ab-CD25/IL-2-Complex fusion protein and CD122 (bar graph);
  • Figure 8 shows the binding of Ab-CD25/IL-2-Complex fusion protein and CD122 (curve graph);
  • Figure 9 shows how Ab-CD25/IL-2-Complex fusion protein stimulates NK92 cells to activate p-STAT5;
  • Figure 10 shows how Ab-CD25/IL-2-Complex fusion protein promotes immune cells to kill tumor cells
  • Figure 11 shows the combination of Anti-IL-2-VHH-Fc and IL-2
  • Figure 12 shows the situation of Anti-IL-2-VHH-Fc antagonizing the binding of IL-2 and CD25;
  • Figure 13 is a schematic structural diagram of the Ab-VHH/IL-2-Complex fusion protein of the present invention.
  • Figure 14 shows the expression and purification of Ab-VHH/IL-2-Complex fusion protein
  • Figure 15 shows the binding of Ab-VHH/IL-2-Complex fusion protein to CD25
  • Figure 16 shows the binding of Ab-VHH/IL-2-Complex fusion protein to CD122 (bar graph);
  • Figure 17 shows how Ab-VHH/IL-2-Complex fusion protein stimulates NK92 cells to activate p-STAT5;
  • Figure 18 is a schematic structural diagram of the Ab-IL15RA/IL-15-Complex fusion protein of the present invention.
  • Figure 19 shows the expression and purification of Ab-IL15RA/IL-15-Complex fusion protein
  • Figure 20 shows the binding of Ab-IL15RA/IL-15-Complex fusion protein and IL15RA
  • Figure 21 shows the binding of Ab-IL15RA/IL-15-Complex fusion protein and IL15R ⁇ (CD122) (bar graph);
  • Figure 22 shows the binding of Ab-IL15RA/IL-15-Complex fusion protein and IL15R ⁇ (CD122) (curve graph);
  • Figure 23 shows how Ab-IL15RA/IL-15-Complex fusion protein stimulates NK92 cells to activate p-STAT5;
  • Figure 24 shows how Ab-IL15RA/IL-15-Complex fusion protein promotes immune cells to kill tumor cells
  • Figure 25 shows the expression of Ab-IL15RA/IL-15-Complex12-14 fusion protein
  • Figure 26 shows the expression of Ab-IL15RA/IL-15-Complex21-24 fusion protein
  • Figure 27 shows the binding of Ab-IL15RA/IL-15-Complex13-14, 21-24 fusion protein and IL15RA;
  • Figure 28 shows the binding of Ab-IL15RA/IL-15-Complex13-14, 21-24 fusion protein and IL15R ⁇ (CD122);
  • Figure 29 shows the expression of three Ab-Complex fusion proteins.
  • Ligand is a type of molecule that can bind to its receptor protein and transmit signals.
  • the ligands described in the present invention refer to protein ligands, including: cytokines, ligands on cell membranes, protein hormones, etc.
  • the ligand of the present invention also encompasses variants of the ligand, and the variants include mutants, transformations and truncations of the ligand, as well as the extracellular domain of the ligand and mutations in the extracellular domain of the ligand. Body, modified body, truncated body.
  • Receptor is intended to be interpreted broadly and refers to a protein molecule capable of binding a ligand and conducting a signal.
  • the receptors described in the present invention also include variants of the receptor, and the variants include mutants, transformations and truncations of the receptor, as well as the extracellular domain of the receptor and mutations of the extracellular domain of the receptor. Body, modified body, truncated body.
  • Cytokine (CK) is intended to be interpreted broadly. It is a low molecular weight soluble protein produced by cells. According to its function, it can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, and chemokines. factors, growth factors, etc. Cytokine is a protein polypeptide, Plays an important role in cell signaling pathways. The term encompasses unprocessed cytokines as well as any form of cytokine derived from processing in the cell. The term also encompasses naturally occurring cytokine variants, such as splice variants or allelic variants. The term also encompasses artificially engineered cytokine variants, modifications, and truncations that have similar functions.
  • Receptor ectodomain is intended to be construed broadly and includes receptor ectodomains of ligands, cytokines, as described in the term above.
  • the term encompasses the unprocessed receptor extracellular domain as well as any form of receptor extracellular domain derived from processing in the cell.
  • the term also encompasses naturally occurring variants of the extracellular domain of the receptor, such as splice variants or allelic variants.
  • the term also encompasses variants, modifications, and truncations of artificially engineered receptor extracellular domains that have similar functions.
  • Interleukin-2 refers to any natural IL-2 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in the cell.
  • the term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants.
  • the term also encompasses IL-2 variants, modifications, and truncations with similar functions.
  • the amino acid sequence of an exemplary wild-type human IL-2 is shown in SEQ ID NO: 6.
  • Raw human IL-2 additionally contains an N-terminal 20 amino acid signal peptide (see UniProt entry number: P60568) that is lacking in the mature IL-2 molecule.
  • "interleukin-2”, “interleukin 2”, “interleukin-2”, “interleukin 2”, “IL2” and “IL-2” can be used interchangeably.
  • CD25 or "alpha subunit of the IL-2 receptor” refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "Full-length” unprocessed CD25, as well as any form of CD25 derived from processing in a cell, also includes naturally occurring CD25 variants, such as splice variants or allelic variants. The term also covers CD25 variants, modifications, and truncations with similar functions.
  • CD25 is human CD25 (see UniProt entry number: P01589), an exemplary sequence of an extracellular domain fragment of which is set forth in SEQ ID NO: 8-9.
  • "CD25”, "IL-2R ⁇ " and " ⁇ subunit of IL-2 receptor” in the present invention can be used interchangeably.
  • Interleukin-15 refers to any natural IL-15 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-15 as well as any form of IL-15 derived from processing in cells.
  • the term also encompasses naturally occurring IL-15 variants, such as splice variants or allelic variants.
  • the term also encompasses IL-15 variants, modifications, and truncations with similar functions.
  • the amino acid sequence of an exemplary wild-type human IL-15 is shown in SEQ ID NO: 14.
  • Raw human IL-15 additionally contains an N-terminal signal peptide (see UniProt entry number: P40933) that is lacking in the mature IL-15 molecule.
  • N-terminal signal peptide see UniProt entry number: P40933
  • "interleukin-15”, “interleukin 215", “interleukin-15”, “interleukin 15”, “IL15” and “IL-15” can be used interchangeably.
  • IL15RA or "alpha subunit of IL-15 receptor” refers to any native IL15RA from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "Full-length” unprocessed IL15RA, as well as any form of IL15RA derived from processing in a cell, also includes naturally occurring IL15RA variants, such as splice variants or allelic variants. The term also covers IL15RA variants, modifications, and truncations with similar functions.
  • IL15RA is human IL15RA (see UniProt entry number: Q13261), an exemplary sequence of an extracellular domain fragment of which is set forth in SEQ ID NO: 15-17.
  • IL15RA "IL-15R ⁇ ” and “ ⁇ subunit of IL-15 receptor” in the present invention can be used interchangeably.
  • Derivatives are intended to be interpreted broadly to include any product related to the target protein. Including but not limited to human and non-human target protein homologues, Fragments or truncations, fusion proteins (such as fusion with signal peptide or other active or inactive components, active components such as antibodies or antigen-binding fragments thereof), modified forms (such as PEGylation, glycosylation, albumin conjugation/ Fusion, Fc conjugation/fusion, hydroxyethylation, etc.), and conservatively modified proteins, etc.
  • fusion proteins such as fusion with signal peptide or other active or inactive components, active components such as antibodies or antigen-binding fragments thereof
  • modified forms such as PEGylation, glycosylation, albumin conjugation/ Fusion, Fc conjugation/fusion, hydroxyethylation, etc.
  • an “immunoconjugate” is a specific conjugate comprising at least one fusion protein or derivative thereof and at least one antigen-binding moiety.
  • the immunoconjugate comprises at least one fusion protein or derivative thereof and at least two antigen-binding moieties.
  • Particular immunoconjugates according to the invention essentially consist of a fusion protein or derivative thereof and an antigen-binding moiety linked by one or more linker sequences.
  • the antigen-binding moiety can be linked to a fusion protein or derivative thereof through a variety of interactions and in a variety of configurations.
  • Immunoglobulin also known as Antibody (Ab) is a large Y-shaped protein mainly secreted by plasma cells and used by the immune system to identify and neutralize foreign substances such as bacteria, viruses and other pathogens.
  • Conventional immunoglobulins are composed of four peptide chains, including light chain (L chain) and heavy chain (H chain). The light chain and the heavy chain are connected by a disulfide bond (-S-S-). The heavy chain and the light chain have variable regions (V regions) and constant regions (C regions) respectively. The heavy chain and the light chain The V regions are called VH and VL respectively, and the C regions of the heavy chain and light chain are called CH (CH1, CH2, CH3, etc.) and CL respectively.
  • Antibody is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the desired antigen-binding activity. specific antibodies) and antigen-binding fragments.
  • Antibodies may include murine antibodies, human antibodies, camel antibodies, shark antibodies, humanized antibodies, chimeric antibodies, heavy chain antibodies, Nanobodies, single domain antibodies, etc.
  • the antibody may be an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain and ⁇ chain respectively. , ⁇ chain and ⁇ chain.
  • IgM immunoglobulins
  • IgD immunoglobulins
  • IgG immunoglobulins
  • IgA immunoglobulin heavy chain constant chain
  • IgE immunoglobulins
  • heavy chains are ⁇ chain, ⁇ chain and ⁇ chain respectively.
  • ⁇ chain and ⁇ chain respectively.
  • the same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into kappa or lambda chains through differences in constant regions.
  • Antigen-binding fragment refers to Fab fragments, Fab’ fragments, F(ab’)2 fragments, single-chain Fv (scFv), Nanobodies (i.e. VHH), and VH/VL domains with antigen-binding activity.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antigen-binding fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Different linkers can also be used to connect two antibody variable regions into a polypeptide chain, called a single chain antibody (single chain antibody) or single chain Fv (scFv).
  • Single domain antibody is intended to be interpreted broadly and refers to fragments that comprise a single variable domain of an antibody. It includes variable regions of heavy chain antibodies from organisms such as camels and cartilaginous fishes, as well as variable regions of artificially modified antibody heavy and light chains, such as heavy chain antibodies produced by artificially modified mice. Variable area.
  • Single chain antibody is intended to be interpreted broadly and includes an antibody heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a flexible short peptide (linker) Recombinant antibodies (approximately 27kDa), and other antibodies that can function as single peptide chains.
  • VH antibody heavy chain variable domain
  • VL light chain variable domain
  • linker flexible short peptide
  • amino acid difference refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in the reference sequence compared to the other sequence.
  • substitution will preferably be a conservative amino acid substitution, which means that an amino acid residue is replaced by another amino acid residue with a similar chemical structure and its impact on the function, activity or other biological properties of the polypeptide Small or essentially no effect.
  • the conservative amino acid substitution is well known in the art.
  • the conservative amino acid substitution is preferably one amino acid in the following groups (i)-(v) is replaced by another amino acid residue in the same group: (i) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) Polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) Polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Replacement; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is often measured using sequence analysis software. For example, identity can be determined using the BLAST program of the NCBI database.
  • sequence identity For the determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988, Biocomputing: Informatics, and, Genome, Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data , Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MS Stockton Press, New York, 199 1 .
  • Vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is called an expression vector when it permits the expression of a protein encoded by a polynucleotide inserted therein.
  • the vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 bacteriophage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • phages such as lambda phage or M13 bacteriophage and animal viruses.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papopaviruses. (such as SV40).
  • Vectors can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain an origin of replication.
  • Host cell and “host cell line” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which includes the initially transformed cell and progeny derived therefrom (regardless of passage number). The offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cell are included herein.
  • “Host cells” include, but are not limited to, prokaryotic cells such as E.
  • coli or Bacillus subtilis eukaryotic cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and Mammalian cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • eukaryotic cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • Mammalian cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the ligand is IL-2 (SEQ ID NO: 6) or an IL-2 variant (SEQ ID NO: 7), so
  • the ligand-binding protein is an extracellular domain fragment of CD25 (SEQ ID NO: 8-9).
  • the extracellular domain of CD25 is a protein that naturally antagonizes the binding of IL-2 to CD25.
  • We hope to obtain biased IL-2 by constructing a CD25/IL-2 fusion protein as described in the present invention: reducing the binding to CD25 or not binding to CD25. Binds and retains binding/activation of medium affinity IL-2 receptors.
  • the Ab-CD25/IL-2-Complex fusion protein designed according to the first polypeptide of "Formula I" and the second polypeptide of "Formula II” is shown in Figure 1A and B Shown; the Ab-CD25/IL-2-Complex fusion protein designed according to the first polypeptide of "Formula III” and the second polypeptide of "Formula IV” is shown in Figure 2A and B.
  • the fusion proteins in Figures 1 and 2 do not contain the Fc region. Furthermore, we designed the Ab-CD25/IL-2-Complex fusion protein containing the Fc region as shown in Figure 3.
  • the Fc-containing Ab-CD25/IL-2-Complex fusion protein can form an immunoglobulin-like tetramer complex with a higher molecular weight, which is beneficial to extending the half-life of the target protein in the body
  • Figure 3 Structural formula clone the nucleotide sequences that can express the first polypeptide chain and the second polypeptide chain into the vector (Ab-Vector) used by Xiamen Baici Biotechnology Co., Ltd. for expression in Expi293F cells to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide chain are shown in Table 2.
  • the transfection process is as follows: Expi293F cells are cultured in a 37°C, 8% CO2 incubator with shaking at 135 rpm at 135 rpm to 4 to 5 ⁇ 10 ⁇ 6 cells/ml, and the cells are diluted with fresh culture medium to 3 ⁇ 10 ⁇ 6 cells/ml. Concentration, 30ml/bottle.
  • the CD25 recombinant protein was coated on the ELISA plate at an amount of 400ng/well, kept at room temperature for 2 hours, and blocked with 5% skimmed milk powder at room temperature. After blocking, wash 3 times with washing buffer, then add 100ul Human IL-2, Ab-CD25/IL-2-C1, Ab-CD25/IL-2-C2 or Ab-CD25/IL-2-C3 to each well. Gradient dilution series (0.625nM to 1000nM), react at room temperature for 2 hours, wash buffer 3 times. Add Alpaca-VHH-Fc-Anti-IL-2 antibody and react at room temperature for 1 hour, washing buffer 3 times.
  • Fc-fused IL-2 protein Human IL-2-Fc
  • the binding ability of Fc-fused IL-2 protein (Human IL-2-Fc) to CD25 is similar to that of wild-type Human IL-2, indicating that Fc will not significantly affect the binding of IL-2 and CD25, further indicating that Ab -The extremely significantly weakened binding reactions of CD25/IL-2-C1, Ab-CD25/IL-2-C2 and Ab-CD25/IL-2-C3 with CD25 are specific effects of CD25/IL-2 fusion proteins.
  • CD122 recombinant protein in the ELISA plate at an amount of 400ng/well, and then add equimolar amounts of the negative control IGG-Isotype, Human IL-2, Ab-CD25/IL-2-C1, and Ab-coupled with Biotin.
  • CD25/IL-2-C2 or Ab-CD25/IL-2-C3, HRP-Streptavidin was used to detect the binding of each protein to CD122, as shown in Figure 7.
  • Ab-CD25/IL-2-C1, Ab-CD25/IL-2-C2 and Ab-CD25/IL-2-C3 to CD122 Ab-CD25/IL-2-Complex fusion Protein gradient dilution series (0.625nM to 1000nM) binding assay to detect three fusion proteins and CD122
  • the binding curve is shown in Figure 8. It can be seen from Figure 8 that Ab-CD25/IL-2-C1, Ab-CD25/IL-2-C2 and Ab-CD25/IL-2-C3 can bind to CD122 well, suggesting that the fusion protein retains binding /Ability to activate medium affinity IL-2 receptors.
  • NK92 cells (ATCC, CRL-2407) were subcultured using TBDNK92KIT medium from Tianjin Haoyang Biotechnology.
  • NK92 cells were cultured in IL2-free medium for 2-3 days. According to the required sample volume, NK92 cells cultured without IL-2 were seeded in 24-well plates. Use PBS (negative control), Human IL-2, Ab-CD25/IL-2-C1, Ab-CD25/IL-2-C2 and Ab-CD25/IL-2-C3 and IGG-Isotype (negative control) respectively.
  • the above IL-2 starved NK92 cells were treated. Treatment time: 20min, 60min; treatment concentration: 15.625nM; 1000rcf, 5min after stimulation, collect cell pellet. Cells were lysed with RIPAlysisbuffer, 5 ⁇ Loadingbuffer was added, and P-STAT5 and Actin were detected by westernblot, as shown in Figure 9.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • the above isolated PBMC were added to a 24-well cell culture dish.
  • the cell culture dish was pre-coated with 10 ⁇ g/mL anti-CD3 (novoprotein, GMP-A018) and anti-CD28 (novoprotein, GMP-A063) and kept overnight at 4°C.
  • CD3 and anti-CD28 antibodies can promote T cell activation and proliferation.
  • PBMC were cultured in RPMI 1640 containing 10% fetal calf serum containing 1:100 penicillin-streptomycin.
  • CD25-Fc control
  • IGG-Isotype control
  • Human IL-2 Ab-CD25/IL-2-C1, Ab-CD25/IL -2-C2 or Ab-CD25/IL-2-C3.
  • Half of the cells were passaged every other day, equal amounts of fresh culture medium were added, and corresponding stimulants were added: CD25-Fc (control), IGG-Isotype (control), Human IL-2, Ab-CD25/IL-2-C1, Ab -CD25/IL-2-C2 or Ab-CD25/IL-2-C3, the proliferated and activated immune cells will be used for subsequent tumor killing experiments.
  • the steps are as follows: Add 50 ⁇ L of 1640 complete culture medium to the E-Plate 96-well culture plate, equilibrate at 37 degrees for 1 hour, and detect the impedance baseline. catch Seed 5,000 tumor cells (human lung squamous cell carcinoma H226) per well. Place the well plate horizontally at room temperature for 30 minutes to allow the cells to settle evenly to the bottom of the well plate. The target cells' adhesion and proliferation are detected by impedance. Impedance was collected every 15 minutes and imaging every 1 hour. After the tumor cells have adhered for 12 hours, collect the effector cells (mainly T cells) amplified in 8.2. According to the effect-to-target ratio of 5:1, add the prepared effector cell suspension to each well containing target cells (50ul). tumor cells).
  • Antibodies can regulate the binding of ligands to their receptors and subsequent signals by binding to ligands.
  • Single-domain antibodies have unique advantages in molecular modification and fusion protein construction due to their simple structure (containing only one peptide chain to perform antibody-related functions) and the ease of engineering.
  • the ligand is IL-2 (SEQ ID NO: 6) or an IL-2 variant (SEQ ID NO: 7), and the ligand binding protein is anti-IL-2 Single domain antibody (SEQ ID NO:10-13).
  • a single domain antibody that is anti-IL-2 and binds to its CD25 epitope can antagonize the binding of IL-2 to CD25.
  • VHH/IL-2 fusion protein as described in the present invention, we can obtain a product that does not bind to CD25 (or does not bind to CD25). Binds to high-affinity IL-2 receptors) while retaining a biased form of IL-2 that binds/activates intermediate-affinity IL-2 receptors.
  • Anti-IL-2-VHH-02, SEQ ID NO:11), 506(9)-F4 (corresponding to Anti-IL-2-VHH-03 in the present invention, SEQ ID NO:12) and 514(11) -G5 (corresponding to Anti-IL-2-VHH-04 in the present invention, SEQ ID NO: 13) was used in the experiment of this embodiment.
  • Anti-IL-2-VHH-01-Fc, Anti-IL-2-VHH-02-Fc, Anti-IL-2-VHH-03-Fc, Anti-IL-2-VHH- 04-Fc can combine well with IL-2, but the control Fc-Isotype cannot combine with IL-2: Anti-IL-2-VHH-01, Anti-IL-2-VHH-02, Anti-IL -2-VHH-03 and Anti-IL-2-VHH-04 are single domain antibodies that specifically bind IL-2.
  • Anti-IL-2-VHH-01-Fc, Anti-IL-2-VHH-02-Fc and Anti-IL-2-VHH-03-Fc can antagonize CD25 and IL-2 very well interaction, while Anti-IL-2-VHH-04-Fc cannot antagonize the interaction between CD25 and IL-2, indicating: Anti-IL-2-VHH-01, Anti-IL-2-VHH-02, Anti -IL-2-VHH-03 is a single domain antibody that is anti-IL-2 and recognizes its CD25-binding epitope. It can block the interaction and signal transduction of IL-2 and CD25; Anti-IL-2-VHH-04 It can only bind IL-2 and does not have the function of blocking the interaction between IL-2 and CD25.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Table 3.
  • the CD25-His recombinant protein was coated on the ELISA plate at an amount of 400ng/well, kept at room temperature for 2 hours, and blocked with 5% skimmed milk powder at room temperature. After blocking, wash 3 times with washing buffer, then add 100ul Ab-VHH/IL-2-C1, Ab-VHH/IL-2-C2, Ab-VHH/IL-2-C3 or Ab-VHH/IL to each well.
  • -2-C4 gradient dilution series (3.125nM to 500nM) was reacted at room temperature for 2 hours and washed 3 times with washing buffer.
  • Goat anti-Human-IgG(HPR) The antibody reacted at room temperature for 1 hour and washed 3 times with washing buffer.
  • TMB chromogenic solution (solaibao, PR1200-500ML) was added, and the reaction was terminated by 1M H2SO4. Use a microplate reader (Tecan, SPARK10M) to read the absorbance value at 460 nm.
  • GraphPadPrism9 was used for data processing and graph analysis to obtain wild-type Ab-VHH/IL-2-C1, Ab-VHH/IL-2-C2, Ab-VHH/IL-2-C3 and Ab-VHH/IL-2.
  • the binding curve of C4 and CD25 is shown in Figure 15. It was found from the experimental results:
  • the Ab-VHH/IL-2-C4 fusion protein formed by the Anti-IL-2-VHH-04 single domain antibody that does not block the interaction between IL-2 and CD25 retains IL-2 (original ligand) Function of binding to CD25;
  • control IGG protein does not bind to CD122, further demonstrating that Ab-VHH/IL-2-C1, Ab-VHH/IL-2-C2, Ab-VHH/IL-2-C3 and Ab-VHH/IL-2-C4 bind CD122 binding is a specific response to the VHH/IL-2 fusion protein.
  • IGG-Isotype cannot activate STAT5 phosphorylation, further indicating that Ab-VHH/IL-2-C1, Ab-VHH/IL-2-C2, Ab-VHH/IL-2-C3 and Ab-VHH/IL-2-
  • the activation of NK92 by C4 is a response specifically triggered by the VHH/IL-2 fusion protein.
  • the ligand is IL-15 (SEQ ID NO: 14), and the ligand-binding protein is the extracellular domain fragment of IL15RA ( SEQ ID NO:15-17).
  • the extracellular domain of IL15RA is a protein that naturally antagonizes the binding of IL-15 to IL15RA.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Table 4.
  • Wild-type Human IL-15 can combine well with IL15RA.
  • Ab-IL15RA/IL-15-Complex fusion protein promotes the activation of NK92 cells
  • IGG-Isotype cannot activate the phosphorylation of STAT5, further indicating that the activation of NK92 by Ab-IL15RA/IL-15-C1, Ab-IL15RA/IL-15-C2 and Ab-IL15RA/IL-15-C3 is IL15RA/IL- 15. Fusion protein specifically stimulates reactions.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Table 5 and Table 6.
  • the plasmid containing the first polypeptide chain and the plasmid containing the second polypeptide chain were co-transfected into EXPi293F cells, And according to the "Expi293F mammalian cell expression protein" process described in 2.2, Ab-IL15RA/IL-15-Complex11 to 14 were expressed in Expi293F cells.
  • the expression vector has a His tag, and Ni-NTAbeads (nickel column) is further used to purify the protein supernatant expressed by Expi293F.
  • the fusion protein may include an Fc region, and the Fc region may be connected to the C-terminal or N-terminal of the first polypeptide chain, or may be connected to the C-terminal or N-terminal of the second polypeptide chain.
  • the Ab-IL15RA/IL-15-Complex12 fusion protein we further designed the Ab-IL15RA/IL-15-Complex fusion protein connected to the Fc region at different positions.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Tables 7-10.
  • the plasmid containing the first polypeptide chain and the plasmid containing the second polypeptide chain were co-transfected into EXPi293F cells, and According to the "Expi293F mammalian cell expression protein" process described in 2.2, Ab-IL15RA/IL-15-Complex21 to 24 were expressed in Expi293F cells and further purified with ProteinAbeads.
  • Wild-type Human IL-15 can bind to IL15RA very well; Ab-IL15RA/IL-15-Complex13-14, Ab-IL15RA/IL-15-Complex21-24 fusion protein has no obvious binding to IL15RA, suggesting that Ab -IL15RA/IL-15-Complex13-14 and Ab-IL15RA/IL-15-Complex21-24 fusion proteins have significantly reduced binding/activation of IL15RA (or IL15R ⁇ trimer) receptors compared with wild-type IL-15. ability;
  • Wild-type Human IL-15 and IL15R ⁇ basically have no detectable binding signals to each other in the ELISA binding experiment.
  • the binding of Ab-IL15RA/IL-15-Complex13-14 and Ab-IL15RA/IL-15-Complex21-24 fusion proteins to IL15R ⁇ (CD122) is significantly enhanced, probably The complex structure formed by IL-15 and IL15RA enhances its affinity with IL15R ⁇ (CD122).
  • the control IGG protein does not bind to IL15R ⁇ (CD122), further indicating that the binding of Ab-IL15RA/IL-15-Complex13-14, Ab-IL15RA/IL-15-Complex21-24 fusion protein and IL15R ⁇ (CD122) is IL15RA/IL-15 fusion protein-specific response.
  • the ligand is IL-2 (SEQ ID NO: 6) or an IL-2 variant (SEQ ID NO: 7), and the ligand-binding protein is the extracellular domain of CD122 (UniProtID: P14784 amino acids 27-239), forming a CD122/IL-2 fusion protein (Ab-CD122/IL-2-Complex) through the first polypeptide chain and the second polypeptide chain. Because killer T cells, NK cells, etc.
  • the Ab-CD122/IL-2-Complex has It has the potential to specifically activate regulatory T cells without activating the function of killer T or NK cells, thereby promoting the body to restore immune balance, and has a potentially important role in the treatment of autoimmune diseases.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Table 11 and Table 12.
  • the plasmid containing the first polypeptide chain and the plasmid containing the second polypeptide chain were co-transfected into EXPi293F cells, And according to the "Expi293F mammalian cell expression protein" process described in 2.2, Ab-CD122/IL-2-Complex1 to 4 were expressed in Expi293F cells.
  • the expression vector has a His tag, and Ni-NTAbeads (nickel column) is further used to purify the protein supernatant expressed by Expi293F.
  • the ligand is a cytokine (including interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemokine, growth factor, etc.), protein hormone molecule, Ligands on the cell membrane or their variants, modifications, and truncations.
  • the ligand-binding protein is a receptor for the ligand or a variant of the receptor.
  • the variants include: mutants, transformations, truncations of the receptor, as well as the extracellular domain of the receptor and the receptor. Mutants, modifications, and truncations of the receptor extracellular domain.
  • the nucleotide sequences capable of expressing the first polypeptide chain and the second polypeptide chain are cloned into the vector (Ab -Vector) to obtain the corresponding expression plasmid.
  • the specific sequences and combinations of the first polypeptide chain and the second polypeptide are shown in Table 13.
  • the plasmid containing the first polypeptide chain and the plasmid containing the second polypeptide chain were co-transfected into EXPi293F cells, and according to 2.2 According to the "Expi293F mammalian cell expression protein" process, the above-mentioned Ab-Complex fusion protein is expressed in Expi293F cells.
  • the expression vector has a His tag, and Ni-NTAbeads (nickel column) is further used to purify the protein supernatant expressed by Expi293F.
  • This embodiment discloses a kit, which includes a container containing the Ab-CD25/IL-2-Complex fusion protein of Example 2 or the Ab-VHH/IL-2-Complex fusion of Example 3. protein, or the Ab-IL15RA/IL-15-Complex fusion protein of Example 4, or the Ab-CD122/IL-2-Complex fusion protein of Example 5, or the Ab-IL4/IL4RA-Complex, Ab of Example 6 -EGFR/Anti-EGFR-VHH-Complex ⁇ Ab-PD1/PD-L1-Complex, Ab-IL2/Anti-IL2-VHH-Complex fusion protein.
  • Using the kit of this embodiment can promote the activation of NK cells or promote immune cells to kill tumor cells or improve autoimmune diseases, etc., so as to promote the treatment of related diseases.
  • the present invention can be applied to activate NK cells or promote immune cells to kill tumor cells or improve autoimmune diseases to promote the treatment of related diseases and promote the body to restore immune balance, and can be used in clinical medicine. It has industrial practicality.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种融合蛋白，该融合蛋白包含两条多肽链，通过将配体和配体结合蛋白分别连接到抗体的重链恒定结构域CH1和轻链恒定结构域CL，形成融合蛋白。经第一多肽链和第二多肽链形成抗体样CD25/IL-2融合蛋白(Ab-CD25/IL-2-Complex)，其具有消除或降低的对高亲和力IL-2受体(IL-2Rαβγ)的亲和力，并保留/增强对中等亲和力IL-2受体(IL-2Rβγ)的亲和力。VHH/IL-2融合蛋白(Ab-VHH/IL-2-Complex)和IL15RA/IL-15融合蛋白(Ab-IL15RA/IL-15-Complex)。以及该融合蛋白的缀合物、药物组合物、核酸分子、表达载体、宿主细胞、生产方法、试剂盒，及其用途。

Description

一种融合蛋白、制备方法及其应用 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及一种含配体蛋白的融合蛋白以及应用。本发明还涉及该融合蛋白的衍生物、缀合物、药物组合、核酸分子、表达载体和宿主细胞以及该融合蛋白的制备方法。
背景技术
配体(Ligand)是一类能通过和其受体蛋白相互结合并传递信号的分子。本发明所述的配体指的是蛋白类的配体，包括：细胞因子、细胞膜上的配体、蛋白类激素等。
细胞因子(cytokine，CK)是一类非常重要的、游离的配体，是细胞产生的低分子量可溶性蛋白质，按照其功能可分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。细胞因子通过结合细胞表面相应的细胞因子受体，介导细胞内信号转导而发挥多种生物学功能，包括免疫调节、血细胞生成、细胞生长以及损伤组织修复等。因此基于细胞因子的药物是药物开发中的重要领域。然而许多天然的细胞因子并不适合作为药物。因为一种细胞因子通常可结合多种表达在不同细胞表面的多种受体，传递着不同的信号，另外同一种受体也可以结合不同的细胞因子，这导致细胞因子的作用具有多效性、重叠性、拮抗性、协同性等多种生理特性。临床治疗中需要特定激活细胞因子参与的一个特异信号，而不激活不必要的信号，进一步执行预期功能，而达到治疗效果好而毒副作用小的目的。细胞因子受体绝大多数是跨膜蛋白，由胞外域、跨膜区和胞浆区组成。
例如，以白细胞介素2为例，白细胞介素2(Interleukin-2，IL-2)也称作T细胞生长因子(TCGF)，是一种15.5kDa球状糖蛋白，它具有133个氨基酸的长度。IL-2的结构由四个反平行的、两亲性的α螺旋和一些链接序列(loop)组成(Smith，Science240，1169-76(1988)；Bazan，Science257，410-413(1992))。IL-2主要来源于活化的CD4+T细胞、活化的CD8+T细胞、自然杀伤(NK)细胞、树突状细胞和巨噬细胞，可通过与细胞表面的IL-2受体结合来调节其作用。
IL-2受体是由三个亚基组成的复合体，三个亚基分别为IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。这三个亚基的表达以及他们与IL-2的亲和力各不相同。其中IL-2Rβ亚基和IL-2Rγ亚基形成的异源二聚IL-2受体主要由细胞毒性CD8+T细胞和NK细胞表达，与IL-2中等亲和力结合，称为中等亲和力IL-2受体(IL-2Rβγ)。由IL-2Rα亚基、IL-2Rβ亚基和IL-2Rγ亚基形成的异源三聚IL-2受体主要表达在调节性T细胞(Treg)(Byman，0.和Sprent.J.Nat.Rev.Immunol.12，180-190(2012))，并与IL-2高亲和性结合(亲和力比二聚体受体高约100倍)，称为高亲和力IL-2受体(IL-2Rαβγ)。另外发现一些内皮细胞表面也有IL-2的受体α(CD25)亚基的表达。IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ时，两个受体亚基形成异源二聚体，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转录和表达；IL-2Rα并非信号所必需，但可以促进IL-2与IL-2Rβ和IL-2Rγ的结合。IL-2Rγ在所有免疫细胞中均有表达；IL-2Rβ在CD8+T细胞、NK细胞、调节性T细胞中均有表达，而且在T细胞被激活后还会提升表达水平；IL-2Rα在调节性T细胞持续高表达，在被激活的CD8+T细胞中会有短暂表达，随即下调表达水平。由于静息状态的效应T细胞和NK细胞在细胞表面上没有IL-2Rα，故对于IL-2相对不敏感。而Treg细胞在体内一贯表达最高水平的IL-2Rα，因此，正常情况下IL-2会优先剌激Treg细胞增殖。
IL-2在体内具有扩充淋巴细胞群体和提高这些细胞的效应器的功能，尤其是对CD8+T细胞和NK细胞的增殖和活化赋予了IL-2的抗肿瘤能力。IL-2是历史上第一个用于肿瘤免疫治疗的细胞因子，抗肿瘤作用已得到临床证实。高剂量IL-2治疗已经批准用于具有转移性肾细胞癌和恶性黑素瘤的患者。经过多年的临床应用，人们对于IL-2的认识更加深入，IL-2的临床应用也带来了诸多问题。
首先与IL-2免疫疗法有关的一项担忧是由重组人IL-2治疗产生的副作用。接受高剂量IL-2治疗的患者发生血管(或毛细血管)渗漏综合征(VLS)，血管通透性的一种病理性升高，导致多个器官中的流体外渗(引起例如肺和皮肤水肿和肝细胞损伤)和血管内流体消减(引起血压下降和心率补偿性升高)。除了IL-2停药以外，VLS没有治疗。研究发现，发生VLS可能与IL-2与内皮细胞表达的IL-2RA结合有关(Kriegetal.，ProcNatAcadSciUSA107，11906-11(2010))。其次，由于IL-2与高表达在抑制性调节T细胞(Treg)的高亲和力IL-2受体结合，而维持外周Treg细胞的数量和活性(MaloyandPowrie，NatureImmunol6，1171-72(2005))。Treg细胞遏制效应T细胞破坏它们的靶物；或是经由细胞-细胞接触抑制T细胞辅助和激活；或是经由释放免疫遏制性细胞因子，诸如IL-10或TGF-β，消减IL-2诱导的抗肿瘤免疫力(Imaietal.，CancerSci98，416-23(2007))。另外，由于IL-2的分子量较小，其在体内的半衰期短，需要持续给药才能维持体内高浓度的IL-2。
抑制IL-2与高亲和力IL2受体(IL-2Rαβγ)的结合，保留或增强与中等亲和力IL2受体(IL-2Rβγ)的结合，并延长其半衰期是克服IL-2在肿瘤免疫治疗中问题的关键。
再比如IL-15是IL-2家族的另一具有抗肿瘤活性的成员，其受体由三个受体亚基组成：IL-15受体α(IL15RA或IL-15Rα)、IL-2受体β(IL-2Rβ，也称IL-15Rβ或CD122)和γc(也称CD132)。IL-15与IL-2结构十分相似，同属于螺旋型细胞因子家族。IL-15的异三聚体受体与IL-2受体共用IL-2Rβ/IL-15Rβ(CD122)和共同γc链(CD132)。
IL-15Rα作为IL-15受体复合物的独特组分，主要在单核细胞和树突状细胞上表达。与其他γc家族细胞因子不同的是，IL-15作为细胞因子首先与IL-15Rα表达细胞结合，然后，IL-15/IL-15Rα复合物递呈给激活的T细胞或NK细胞上的IL-2β/15Rβ和γc。这使得IL-15的活性受到限制。IL-15突变体(IL-15N72D)和IL-15RαSu/Fc的二聚体蛋白，已被证明在小鼠模型中具有优异的抗肿瘤活性。但仍然存在清除率高，激活外周免疫细胞而产生毒性等缺点。临床使用中，需要具有可以直接结合IL-15Rβγ受体、并可以直接激活T细胞/NK细胞、同时避免刺激调节性T细胞(Treg)的偏向型IL-15。
技术问题
现在已经有多种方法被开发来克服这些与IL-2免疫疗法有关的问题。例如，通过IL-2突变体来改变IL-2对不同受体的亲和特异性。如Merck公司的突变体(R38W，F42K，WO2008003473A2)，降低与α受体亚基的相互作用，以其达到效应T细胞活化以增强功效；而Roche的IL-2突变体(F42A，Y45A和L72G，US2016/0208017A1)，其与α受体不结合，但可以正常结合β和γ受体亚基复合物，并可以发挥效应，目前正在临床中。还有一些方法通过开发IL-2的抗体封闭IL-2与IL-2Rα的结合，而保留与IL-2Rβγ的结合。但目前这些开发大部分都处于研究 阶段，临床上仍需要具有偏向性激活IL-2Rβγ的IL-2或衍生物用于肿瘤免疫治疗。
因此，开发出能够偏向性激活中等亲和力IL2受体(IL-2Rβγ)的长效型细胞因子或蛋白，进而更好地作为临床药物，特别是用于肿瘤免疫治疗，十分必要。鉴于细胞因子等蛋白类配体在治疗应用中普遍存在功能多样性和活性受限的问题，亟需发展功能偏向性配体的构建方法，以便于解决临床应用中的问题。
技术解决方案
本发明的目的在于构建一种包括配体和其结合蛋白的融合蛋白，该融合蛋白包含两条多肽链，将配体、配体结合蛋白分别连接到抗体的重链恒定结构域CH1和轻链恒定结构域CL上得到融合蛋白，与原配体蛋白相比：削弱/消除了原配体蛋白在某些方面的功能，保留/增强了其他特定方面的功能，进而发挥特异性功能，可以用于临床药物。
第一方面，本发明提供一种融合蛋白，该融合蛋白充分利用配体结合蛋白潜在的可以通过和配体的结合进而调控其功能的特性(如：受体作为天然存在的可以拮抗配体和其自身相互作用的拮抗剂；或靶向目标配体的抗体或其抗原结合蛋白，具有潜在的阻断该配体和其受体的相互作用或潜在的激活该配体的功能等)，通过将配体和其结合蛋白构建于融合蛋白中，从而使配体结合蛋白达到调控配体功能的作用，进而发挥配体特异性的功能。
具体的，该融合蛋白包含第一多肽和第二多肽，这两条肽链基于抗体的重链恒定结构域CH1和轻链恒定结构域CL进行改造，通过将配体和配体结合蛋白分别连接到CH1和CL上，形成融合蛋白；
在一些具体的实施例中，融合蛋白的第一多肽、第二多肽从N端到C端分别具有如下所示的多肽结构式：
第一多肽：A-L1-C1       (式I)
第二多肽：B-L2-C2       (式II)
在其它一些具体的实施例中，融合蛋白的第一多肽、第二多肽从N端到C端分别具有如下所示的多肽结构式：
第一多肽：C1-L1-A       (式Ⅲ)
第二多肽：C2-L2-B  (式Ⅳ)
上述式中：A为配体，B为可以与A相互作用的配体结合蛋白；也可以是A为配体结合蛋白，B为可以与A相互作用的配体；所述配体包括野生型配体或其变体；所述的变体包括该配体的突变体、改造体和截断体，以及该配体的胞外域或该配体胞外域的突变体、改造体、截断体；所述的配体结合蛋白包括野生型配体结合蛋白或其变体；所述的变体包括该配体结合蛋白的突变体、改造体和截断体，以及该配体结合蛋白的胞外域或该配体结合蛋白胞外域的突变体、改造体、截断体；
C1为抗体轻链恒定结构域CL，C2为抗体重链恒定结构域CH1；且C1和C2之间形成有二硫键；从而使所述两条肽链具有异二聚体的形式；在一些实施方案中，所述CL是人源抗体的CL(包括κ轻链(kappa light chain)的恒定区(IGKC)和λ轻链(lambda light chain)的恒定区(IGLC))或者具有与人CL至少80％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列相同性的氨基酸序列，所述CH1是人源抗体的CH1或者具有与人CH1至少80％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列相同性的氨基酸序列；在一些具体实施方案中，所述CL是人源IGKC，其氨基酸序列示于SEQ ID NO:1；所述CH1是人源CH1，其氨基酸序列示于SEQ ID NO:2。
L1、L2为各自独立的为接头元件(在一些实施例中，也可以无接头元件L1、L2)，可选的接头元件包括但不限于如下序列：
1)GGGGS；
2)GSGGGGS；
3)GGGGSGGGS(SEQIDNO:3)；
4)GSGGGSGGGGSGGGGS(SEQIDNO:4)；
5)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
6)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
7)GGGSGGGSGGGSGGGSGGGS；
8)GGSGGSGGSGGS；
9)(GGGGS)n，其中，n为正整数(例如2、3、4、5或6等)；
10)其他本领域人员常用的接头序列。
在一些具体的实施例中，所述的融合蛋白还包括抗体的Fc区，所述Fc区连接于第一多肽或第二多肽；进一步增加融合蛋白的分子量，进而延长其在体内半衰期。Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。在一些实施方案中，可以在野生型的Fc序列上引入突变用于改变Fc介导的相关活性。所述突变包括但不限于：a).改变Fc介导的CDC活性的突变；b).改变Fc介导的ADCC活性的突变；或c).改变FcRn介导的体内半衰期的突变。此类突变描述于下列文献中：Leonard GPresta，Current Opinionin Immunology 2008，20:460–470；Esohe E.Idusogieetal.，J Immunol 2000，164:4178-4184；RAPHAELA.CLYNE Setal.，Nature Medicine，2000，Volume6，Number 4:443-446；Pau l R.Hintonetal.，J Immunol，2006，176:346-356。
在一些实施方案中，Fc序列上可以引入突变，从而使得突变的Fc更容易形成同二聚体或者异二聚体。如Ridgway，Prestaetal.1996以及Carter2001中提到的利用Fc接触界面氨基酸侧链基团空间作用的knob-hole模型，使得不同Fc突变之间更容易形成异二聚体；再比如如CN102558355或者CN103388013A中，通过改变Fc接触界面氨基酸所带的电荷，进而改变Fc接触界面之间的离子相互作用力，使得不同的Fc突变对之间更容易形成异二聚体(CN102558355A)，亦或是具有相同突变的Fc之间更容易形成同二聚体(CN103388013A)。
在一些具体的实施方案中，免疫球蛋白Fc区优选是人免疫球蛋白Fc区，更优选是人IgG1的Fc区。在一些具体实施方案中，所述免疫球蛋白Fc区的氨基酸序列示于SEQ ID NO:5。
在一些实施例中，所述的Fc区连接在第一多肽的C端，使第一多肽形成多肽结构式A-L1-C1-L3-Fc或C1-L1-A-L3-Fc。
在一些实施例中，所述的Fc区连接在第二多肽的C端，使第二多肽形成多肽结构式B-L2-C2-L3-Fc或C2-L2-B-L3-Fc。
在一些实施例中，所述的Fc区连接在第一多肽的N端，使第一多肽形成多肽结构式Fc-L3-A-L1-C1或Fc-L3-C1-L1-A。
在一些实施例中，或所述的Fc区连接在第二多肽的N端，使第二多肽形成多肽结构式Fc-L3-B-L2-C2或Fc-L3-C2-L2-B。
在一些实施例中，所述的融合蛋白还可以形成类免疫球蛋白样的四聚体结构，其包含两条第一多肽链和两条第二多肽链。
上述式中，L3为独立的为接头元件(在一些实施例中，也可以无接头元件L3)，可选的接头元件包括但不限于如下序列：
1)GGGGS；
2)GSGGGGS；
3)GGGGSGGGS(SEQIDNO:3)；
4)GSGGGSGGGGSGGGGS(SEQIDNO:4)；
5)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
6)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
7)GGGSGGGSGGGSGGGSGGGS；
8)GGSGGSGGSGGS；
9)(GGGGS)n，其中，n为正整数(例如2、3、4、5或6等)；
10)其他本领域人员常用的接头序列。
在一些具体的实施方案中，还包括该融合蛋白的衍生物。
进一步的，在一些实施方式中，所述的配体是细胞因子(包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等)、蛋白类激素分子、细胞膜上的配体或它们的变体、改造体、截断体。所述的配体结合蛋白是该配体的受体或其受体的变体，所述的变体包括：该受体的突变体、改造体、截断体，以及该受体胞外域和该受体胞外域的突变体、改造体、截断体。受体作为天然存在的可以拮抗配体和其自身相互作用的拮抗剂，是能够调控配体功能的重要的潜在分子。通过将受体作为天然拮抗细胞因子的蛋白(如CD25是天然拮抗IL-2与CD25结合的蛋白、CD122是天然拮抗IL-2与CD122结合的蛋白等)，将细胞因子和受体构建于融合蛋白中，从而使配体结合蛋白达到调控配体功能的作用，进而发挥配体特异性的功能。
在一些实施方式中，所述的配体结合蛋白是该配体的抗体或其抗原结合片段；通过抗体或其抗原结合蛋白，阻断该配体和其受体的相互作用或潜在的激活该配体的功能等，进而发挥配体特异性的功能。单域抗体和单链抗体是保留原抗体对抗原特异性的最小功能单位，且都是以单肽链形式发挥其功能。优选地所述的抗体或其抗原结合片段是单域抗体或单链抗体。单域抗体和单链抗体因其结构简单(仅含一条肽链即可发挥抗体相关功能)便于进行工程改造的特性，在分子改造和融合蛋白构建中，具有独特的优势。
单域抗体(sdAb，single domain antibody)是指包含了抗体中单个可变域的片段。和完整的抗体一样，它可以选择性的和特定抗原结合。与完整抗体的150－160kDa的质量相比，单域抗体小得多，大约只有12－15kDa。第一个单域抗体是由骆驼科动物中发现的重链抗体改造而来的(Hamers-Casterman C，et.al(1993)6428:446-448)。驼类以及软骨鱼(鲨鱼)体内存在天然的缺失轻链的重链抗体(Heavy Chain Antibody,HcAb)，重链抗体作为天然缺失轻链的抗体分子，在基因工程改造中相对于传统IgG抗体分子具有更大的优势。从驼类的重链抗体中人造工程制作出来的重链可变区，称为VHH单域抗体(Variable domain of heavy chain of heavy chain antibody)；软骨鱼(鲨鱼)也有重链抗体(IgNAR，免疫球蛋白新抗原感受器Immunoglobulin new antigen receptor的缩写)，从该类抗体也可以制作出称为“V-NAR区段”的单域抗体(Variable domain of Ig new antigen receptor)。单域抗体具有与原重链 抗体相当的结构稳定性以及与抗原的结合活性，是目前已知的可结合目标抗原的最小单位，其分子量只有15KD，仅有传统抗体的十分之一，因此也被称为纳米抗体(nanobody)。和普通抗体相比，单域抗体分子量小，结构简单，易于进行基因改造，体积小，抗原特异性好，组织穿透力强，稳定性高，在疾病的诊断及治疗方面有广阔的应用前景。单域抗体作为已知的可结合目标抗原的最小单位，因其分子量小、结构简单，是作为配体结合蛋白用于构建本发明所述的融合蛋白的重要选择之一。
单链抗体(Single-chain antibody variable fragment，scFv)，是由抗体重链可变区(heavy chain variable domain，VH)和轻链可变区(light chain variable domain，VL)通过一个10-25个氨基酸组成的柔性短肽(linker)连接而成，是一种重组抗体形式(约27kDa)。本质上，scFv是一种融合蛋白，并保留了原始免疫球蛋白对抗原的特异性。scFv较小的分子尺寸，带来了强大的肿瘤内穿透力、在血液中快速降解、人体内负反馈小等优势，这也为scFv的临床应用奠定了基础。类似地，单链抗体因其只有一条肽链、结构简单的特性，也是作为配体结合蛋白用于构建本发明所述的融合蛋白的重要选择之一。
在一些具体的实施方式中，所述的配体是IL-2，所述的配体结合蛋白是CD25的胞外域，经第一多肽链和第二多肽链形成CD25/IL-2融合蛋白(Ab-CD25/IL-2-Complex)；该融合蛋白或其衍生物具有以下特征中的至少一个：
(a)与野生型IL-2相比，分子量更大，体内半衰期更长；
(b)与野生型IL-2相比，很大程度降低与CD25的结合或与CD25不结合；
(c)与野生型IL-2相比，能够保留或增强与CD122的结合；
(d)能够有效激活中等亲和力IL-2受体(IL-2Rβγ)介导的信号；
(f)促进NK细胞的活化；
(g)促进免疫细胞杀伤肿瘤细胞。
在一些具体的实施方式中，所述的配体是IL-2，所述的配体结合蛋白是CD122的胞外域，经第一多肽链和第二多肽链形成CD122/IL-2融合蛋白(Ab-CD122/IL-2-Complex)。因为杀伤性T细胞、NK细胞等主要表达CD122/CD132二聚体受体，调节性T细胞(Treg)主要表达含CD25的三聚体受体，因此该Ab-CD122/IL-2-Complex具有潜在的特异性激活调节性T细胞，而不激活杀伤性T或NK细胞的功能，从而达到促进机体恢复免疫平衡的作用，具有潜在的用于治疗自身免疫性疾病的重要作用。该融合蛋白或其衍生物具有以下特征中的至少一个：
(a)与野生型IL-2相比，分子量更大，体内半衰期更长；
(b)与野生型IL-2相比，很大程度降低与CD122的结合或与CD122不结合；
(c)具有潜在的特异性激活调节性T细胞，而不激活杀伤性T或NK细胞，从而达到促进机体恢复免疫平衡的作用，是潜在的用于治疗自身免疫性疾病的药物。
在一些实施方式中，所述的配体是IL-2，所述的配体结合蛋白是抗IL-2的VHH，经第一多肽链和第二多肽链形成VHH/IL-2融合蛋白(Ab-VHH/IL-2-Complex)；该融合蛋白或其衍生物具有以下特征中的至少一个：
(a)与野生型IL-2相比，分子量更大，体内半衰期更长；
(b)与野生型IL-2相比，很大程度降低与CD25的结合或与CD25不结合；
(c)与野生型IL-2相比，能够保留或增强与CD122的结合；
(d)能够有效激活中等亲和力IL-2受体(IL-2Rβγ)介导的信号；
(f)促进NK细胞的活化。
在一些实施方式中，所述的配体是IL-15，所述的配体结合蛋白是IL15RA的胞外域，经第一多肽链和第二多肽链形成IL15RA/IL-15融合蛋白(Ab-IL15RA/IL-15-Complex)；该融合蛋白或其衍生物具有以下特征中的至少一 个：
(a)与野生型IL-15相比，分子量更大，体内半衰期更长；
(b)与野生型IL-15相比，很大程度降低与IL15RA的结合或与IL15RA不结合；
(c)与野生型IL-15相比，能够保留或增强与IL-15Rβ(CD122)的结合；
(d)能够有效激活IL-15二聚体受体(IL-15Rβγ)介导的信号；
(f)促进NK细胞的活化；
(g)促进免疫细胞杀伤肿瘤细胞。
第二方面，本发明提供一种缀合物，包括上述的融合蛋白或其衍生物，所述融合蛋白或其衍生物直接或通过接头元件间接地与其他模块连接。
一些实施例中，所述的其他模块包括抗原结合模块、细胞毒素、放射性同位素、生物活性蛋白质、可检测标记物、药物、毒素、细胞因子、酶金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、中任一种或其组合。
更优选地，所述抗原结合模块是抗体或抗原结合片段；最优选地，所述抗体或抗原结合片段靶向肿瘤细胞上或肿瘤细胞环境中呈现的抗原。
一些实施方式中，所述抗原结合模块靶向肿瘤细胞上呈现的或肿瘤细胞环境中的抗原。一些实施方式中，所述抗原结合模块靶向功能细胞(例如CD8+T细胞、NK细胞、CIK细胞、TIL细胞、巨噬细胞、DC细胞等)上的抗原。
一些实施方式中，融合蛋白连接于至少一个其他模块。一些实施方式中，融合蛋白和其他模块形成融合蛋白，即融合蛋白与其他模块共享肽键。一些实施方式中，融合蛋白连接于至少一个其他模块，例如第一和第二其他模块。一些实施方式中，其他模块为抗原结合模块。一些实施方式中，融合蛋白与第一抗原结合模块共享氨基或羧基端肽键，且第二抗原结合模块与以下共享氨基或羧基端肽键：i)融合蛋白或ii)第一抗原结合模块。一些具体的实施方式中，融合蛋白与所述第一其他模块共享羧基端肽键，而与所述第二其他模块共享氨基末端肽键。一些实施方式中，所述其他模块是抗原结合模块。所述抗原结合模块可以是抗体或抗原结合片段，包括但不限于免疫球蛋白分子(例如IgG(例如IgG1)类免疫球蛋白分子)、抗体或其抗原结合片段。一些具体实施方式中，抗体或抗原结合片段选自包含抗体重链可变区和抗体轻链可变区的多肽复合物、Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体、多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)、受体结合区和相互作用蛋白结合区。在融合蛋白连接于超过一个抗原结合模块例如第一和第二抗原结合模块的情况下，每个抗原结合模块可以独立地选自各种形式的抗体和抗原结合片段，例如，第一抗原结合模块可以是纳米抗体分子，而第二抗原结合模块可以是scFv分子，或第一和第二抗原结合模块中的每一个都是纳米抗体分子，或第一和第二抗原结合模块中的每一个是Fab分子。一些实施方式中，在融合蛋白连接于超过一个抗原结合模块例如第一和第二抗原结合模块的情况下，能独立地选择每个抗原结合模块所针对的抗原，例如，所述第一和所述第二抗原结合模块针对不同的抗原或针对同一抗原。
一些实施方式中，所述抗原结合模块结合的抗原可以选自下组：生腱蛋白C的A1域(TNCA1)、生腱蛋白C的A2域(TNCA2)、纤连蛋白的外域(ExtraDomainB)(EDB)、癌胚抗原(CEA)和黑素瘤有关的硫酸软骨素蛋 白聚糖(MCSP)。一些实施方式中，肿瘤抗原包括但不限于MAGE、MART-1/Melan-A、gp100、二肽基肽酶IV(DPPIV)、腺苷脱氨酶结合蛋白(ADAbp)、亲环素(cyclophilin)b、结肠直肠有关的抗原(CRC)-C017-1A/GA733、癌胚抗原(CEA)及其免疫原性表位CAP-1和CAP-2、etv6、aml1、前列腺特异性抗原(PSA)及其免疫原性表位PSA-1、PSA-2和PSA-3、前列腺特异性膜抗原(PSMA)、T细胞受体/CD3-zeta链、肿瘤抗原的MAGE家族(例如MAGE-A1、MAGE-A2、MAGE-A3、MAGE-A4、MAGE-A5、MAGE-A6、MAGE-A7、MAGE-A8、MAGE-A9、MAGE-A10、MAGE-A11、MAGE-A12、MAGE-Xp2(MAGE-B2)、MAGE-Xp3(MAGE-B3)、MAGE-Xp4(MAGEB4)、MAGE-C1、MAGE-C2、MAGE-C3、MAGE-C4、MAGE-C5)、肿瘤抗原的GAGE家族(例如GAGE-1、GAGE-2、GAGE-3、GAGE-4，GAGE-5，GAGE-6，GAGE-7，GAGE-8，GAGE-9)、BAGE、RAGE、LAGE-1、NAG、GnT-V、MUM-1、CDK4、酪氨酸酶、p53、MUC家族、HER2/neu、p21ras、RCAS1、α-胎蛋白、E-钙粘蛋白、α-连环蛋白(catenin)、β-连环蛋白和γ-连环蛋白、p120ctn、gp100Pmel117、PRAME、NY-ESO-1、cdc27、腺瘤样结肠息肉蛋白(adenomatouspolyposiscoliprotein，APC)、胞衬蛋白(fodrin)、连接蛋白(Connexin)37、Ig独特型、p15、gp75、GM2和GD2神经节苷脂、病毒产物如人乳头状瘤病毒蛋白、肿瘤抗原的Smad家族、lmp-1、P1A、EBV编码的核抗原(EBNA)-1、脑糖原磷酸化酶、SSX-1、SSX-2(HOM-MEL-40)、SSX-1、SSX-4、SSX-5、SCP-1和CT-7、以及c-erbB-2。一些实施方式中，病毒抗原的非限制性例子包括流感病毒血凝素、Epstein-Barr病毒LMP-1、丙肝病毒E2糖蛋白、HIVgp160和HIVgp120。一些实施方式中，ECM抗原的非限制性例子包括多配体聚糖(syndecan)、类肝素酶(heparanase)、整联蛋白、骨桥蛋白(osteopontin)、link、钙粘蛋白、层粘连蛋白、EGF型层粘连蛋白、凝集素、纤连蛋白、notch、生腱蛋白和matrixin。
第三方面，本发明公开了一种药物组合物，包括上所述的融合蛋白或其衍生物或上述的缀合物以及药学上可接受的稀释剂、载体或助剂。所述药物组合物可以为冻干制剂或可注射溶液。
第四方面，本发明提供了一种编码上述融合蛋白或其衍生物的核酸分子。所述的核酸分子包含两个多核苷酸模块，第一多核苷酸编码如本发明第一方面所述的第一多肽链，第二多核苷酸编码如本发明第一方面所述的第二多肽链。本发明的核酸可为RNA、DNA或cDNA。
第五方面，本发明提供含有上述融合蛋白或其衍生物的核酸序列的表达载体。所述载体可以是真核表达载体、原核表达载体、病毒载体。
在一些实施方案中，所述的表达载体是病毒载体，可以产生具有生理功能的病毒，如常见的一些溶瘤病毒：单纯疱疹病毒(HSV)、腺病毒、牛痘病毒和呼肠弧病毒等。
第六方面，本发明涉及表达或能够表达一种或多种本发明的融合蛋白和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括但不限于革兰氏阴性细菌菌株(例如大肠杆菌(Escherichiacoli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括但不限于木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物 种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomycescerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomycespombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichiapastoris)及嗜甲醇毕赤酵母(Pichiamethanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括但不限于HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
一些实施方案中，所述的宿主细胞是能够表达本发明的融合蛋白或其衍生物的功能细胞(如：CAR-T、CAR-NK、CD8+T细胞、NK细胞、CIK细胞、TIL细胞、巨噬细胞、DC细胞等)，所述的功能细胞具有以下任一种或多种生理功能：肿瘤杀伤、病原清除、免疫效应等。
第八方面，本发明提供了本发明所述融合蛋白或其衍生物、核酸分子、宿主细胞、免疫缀合物及药物组合物用于制备治疗相关疾病(如增生性疾病、免疫性疾病等)、调节T细胞介导的免疫应答、刺激个体免疫系统的药物中的用途。所述增生性疾病可以是肿瘤或癌(例如转移性肿瘤或癌)，可以是实体瘤(例如转移性肾细胞癌和恶性黑素瘤)。
一些实施方式中，本发明公开的融合蛋白或其衍生物、免疫缀合物、药物组合可用于治疗刺激宿主的免疫系统以获益的疾病情形，特别是期望增强细胞免疫应答的状况，可以包括宿主免疫应答不足或缺陷性的疾病情形。一些实施方式中，施融合蛋白或其衍生物、免疫缀合物的疾病情形包括细胞免疫应答是特异性免疫的关键机制的肿瘤或感染，例如癌症(例如肾细胞癌或黑素瘤)、免疫缺陷(例如HIV阳性患者中、免疫受抑制的患者)、慢性感染等。一些实施方式中，增强细胞免疫应答可以包括以下任一种或多种：免疫功能的一般升高、T细胞功能升高、B细胞功能升高、淋巴细胞功能的恢复、IL-2受体表达提高、T细胞应答性提高、天然杀伤细胞活性或淋巴因子激活的杀伤(LAK)细胞活性升高等。
一些实施方式中，本发明公开的融合蛋白或其衍生物、免疫缀合物、药物组合用于治疗的疾病是增殖性病症，例如癌症。癌症的非限制性例子包括膀胱癌、脑癌、头和颈癌、胰腺癌、肺癌、乳腺癌、卵巢癌、子宫癌、宫颈癌、子宫内膜癌、食道癌、结肠癌、结肠直肠癌、直肠癌、胃癌、前列腺癌、血液癌、皮肤癌、鳞状细胞癌、骨癌和肾癌。其它可使用本公开的融合蛋白或其衍生物治疗的细胞增殖病症包括但不限于位于以下处的新生物：腹部、骨、乳房、消化系统、肝、胰、腹膜、内分泌腺(肾上腺、副甲状腺、垂体、睾丸、卵巢、胸腺、甲状腺)、眼、头和颈、神经系统(中枢和外周)、淋巴系统、骨盆、皮肤、软组织、脾、胸部、和泌尿生殖系统。还包括癌症前期状况或损伤以及癌症转移。在某些实施方式中，癌症选自下组：肾细胞癌、皮肤癌、肺癌、结肠直肠癌、乳腺癌、脑癌、头和颈癌。类似地，其它细胞增殖病症也可用本公开的融合蛋白或其衍生物治疗，包括但不限于：高丙种球蛋白血症(hypergammaglobulinemia)、淋巴增生性病症、病变蛋白血症(paraproteinemias)、紫癜(purpura)、结节病、塞扎里综合征(SezarySyndrome)、Waldenstron's巨球蛋白血症、高歇氏病(Gaucher'sDisease)、组织细胞增多病(histiocytosis)以及任何其它位于上文所列的器官系统中的瘤形成(neoplasia)外的细胞增殖疾病。在另一些实施方式中，所述疾病涉及自身免疫性、移植排斥、外伤后免疫应答和传染性疾病(例如HIV)。
一些实施方式中，提供其中每日至少2次、每日至少1次、每48小时至少1次、每72小时至少一次、每周 至少一次、每2周至少一次、每个月至少一次、每2个月至少一次或每3个月至少一次向受试者施用融合蛋白或其衍生物、免疫缀合物的方法。可通过任何有效途径施用融合蛋白或其衍生物、免疫缀合物。在一些实施方式中，通过肠胃外注射包括皮下注射施用融合蛋白或其衍生物、免疫缀合物。具体实施方式涉及包含药学上可接受的量的融合蛋白或其衍生物、免疫缀合物(例如，治疗有效量)(包括上述的那些试剂)连同一种或多种药学上可接受的稀释剂、载体或赋形剂(例如，等渗注射溶液)的药物组合物。所述药物组合物通常为适于人施用的药物组合物。此外，在一些实施方式中，所述药物组合物包含至少一种另外的预防剂或治疗剂。一些实施方式含有上述药物组合物之一和任选地一种或多种另外的组分的无菌容器。
第九方面，本发明提供了一种试剂盒，所述的试剂盒包含本发明的融合蛋白、其衍生物、缀合物、药物组合物、核酸分子、表达载体或宿主细胞，以及使用说明。试剂盒一般包括表明试剂盒内容物的预期用途的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
第十方面，本发明提供了一种融合蛋白或其衍生物的构建和生产方法，包括在适于表达所述融合蛋白或其衍生物的条件下或使用前述核酸分子，或使用前述表达载体，或使用前述宿主细胞进行表达。
有益效果
本发明融合蛋白具有偏向性配体功能，基于此可以构建功能偏向型配体以克服相关配体分子(如细胞因子)成药过程中的问题。削弱/消除了原配体蛋白在某些方面的功能，保留/增强了其他特定方面的功能，进而发挥特异性功能，可以用于临床药物。
附图说明
图1为本发明所述的Ab-CD25/IL-2-Complex融合蛋白结构示意图1；
图2为本发明所述的Ab-CD25/IL-2-Complex融合蛋白结构示意图2；
图3为本发明所述的Ab-CD25/IL-2-Complex融合蛋白结构示意图3；
图4为Ab-CD25/IL-2-Complex融合蛋白的表达纯化；
图5为Ab-CD25/IL-2-Complex融合蛋白与CD25的结合情况；
图6为Human IL-2、Human IL-2-Fc和CD25的结合情况；
图7为Ab-CD25/IL-2-Complex融合蛋白与CD122的结合情况(柱形图)；
图8为Ab-CD25/IL-2-Complex融合蛋白与CD122的结合情况(曲线图)；
图9为Ab-CD25/IL-2-Complex融合蛋白刺激NK92细胞激活p-STAT5情况；
图10为Ab-CD25/IL-2-Complex融合蛋白促进免疫细胞杀伤肿瘤细胞情况；
图11为Anti-IL-2-VHH-Fc与IL-2结合情况；
图12为Anti-IL-2-VHH-Fc拮抗IL-2与CD25结合情况；
图13为本发明所述的Ab-VHH/IL-2-Complex融合蛋白结构示意图；
图14为Ab-VHH/IL-2-Complex融合蛋白的表达纯化；
图15为Ab-VHH/IL-2-Complex融合蛋白与CD25的结合情况；
图16为Ab-VHH/IL-2-Complex融合蛋白与CD122的结合情况(柱形图)；
图17为Ab-VHH/IL-2-Complex融合蛋白刺激NK92细胞激活p-STAT5情况；
图18为本发明所述的Ab-IL15RA/IL-15-Complex融合蛋白结构示意图；
图19为Ab-IL15RA/IL-15-Complex融合蛋白的表达纯化；
图20为Ab-IL15RA/IL-15-Complex融合蛋白与IL15RA的结合情况；
图21为Ab-IL15RA/IL-15-Complex融合蛋白与IL15Rβ(CD122)的结合情况(柱形图)；
图22为Ab-IL15RA/IL-15-Complex融合蛋白与IL15Rβ(CD122)的结合情况(曲线图)；
图23为Ab-IL15RA/IL-15-Complex融合蛋白刺激NK92细胞激活p-STAT5情况；
图24为Ab-IL15RA/IL-15-Complex融合蛋白促进免疫细胞杀伤肿瘤细胞情况；
图25为Ab-IL15RA/IL-15-Complex12-14融合蛋白的表达情况；
图26为Ab-IL15RA/IL-15-Complex21-24融合蛋白的表达情况；
图27为Ab-IL15RA/IL-15-Complex13-14、21-24融合蛋白与IL15RA的结合情况；
图28为Ab-IL15RA/IL-15-Complex13-14、21-24融合蛋白与IL15Rβ(CD122)的结合情况；
图29为三种Ab-Complex融合蛋白的表达情况。
本发明的最佳实施方式
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步详细的描述。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本发明中的它处另有明确定义，否则本发明使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义，参考例如标准手册，如Sambrook等人，“MolecularCloning:ALaboratoryManual”(第2版)，第1-3卷，ColdSpringHarborLaboratoryPress(1989)；Lewin，“GenesIV”，OxfordUniversityPress，NewYork，(1990)；及Roitt等人，“Immunology”(第2版)，GowerMedicalPublishing，London，NewYork(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解，亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
术语
“配体”(Ligand)是一类能通过和其受体蛋白相互结合并传递信号的分子。本发明所述的配体指的是蛋白类的配体，包括：细胞因子、细胞膜上的配体、蛋白类激素等。本发明所述的配体还涵盖该配体的变体，所述的变体包括该配体的突变体、改造体和截断体，以及该配体的胞外域和该配体胞外域的突变体、改造体、截断体。
“受体”旨在被广义地解释，指能够结合配体并传导信号的蛋白分子。本发明所述的受体还涵盖该受体的变体，所述的变体包括该受体的突变体、改造体和截断体，以及该受体的胞外域和该受体胞外域的突变体、改造体、截断体。
“细胞因子”(cytokine，CK)旨在被广义地解释，是细胞产生的低分子量可溶性蛋白质，按照其功能可分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。细胞因子是一种蛋白多肽， 在细胞信号通路中起重要的作用。该术语涵盖未加工的细胞因子以及源自细胞中的加工的任何形式的细胞因子。该术语还涵盖天然存在的细胞因子变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的人工改造的细胞因子变体、改造体、截断体。
“受体胞外域”旨在被广义地解释，包括上文术语中所述的配体、细胞因子的受体胞外域。该术语涵盖未加工的受体胞外域以及源自细胞中的加工的任何形式的受体胞外域。该术语还涵盖天然存在的受体胞外域的变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的人工改造的受体胞外域的变体、改造体、截断体。
“白介素-2”或“IL-2”指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然的IL-2。该术语涵盖未加工的IL-2以及源自细胞中的加工的任何形式的IL-2。该术语还涵盖天然存在的IL-2变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的IL-2变体、改造体、截断体。例示性野生型人IL-2的氨基酸序列如SEQ ID NO：6所示。未加工的人IL-2额外包含N端20个氨基酸的信号肽(参见UniProt条目号：P60568)，所述信号肽在成熟的IL-2分子中是缺乏的。本发明中，“白介素-2”、“白介素2”、“白细胞介素-2”、“白细胞介素2”“IL2”和“IL-2”可以互换使用。
“CD25”或“IL-2受体的α亚基”指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然CD25，包括“全长”的未加工的CD25以及源自细胞中的加工的任何形式的CD25，还包括天然存在的CD25变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的CD25变体、改造体、截断体。在某些实施方案中，CD25是人CD25(参见UniProt条目号：P01589)，其胞外域片段例示性序列如SEQ ID NO：8-9所示。本发明所述的“CD25”与“IL-2Rα”以及“IL-2受体的α亚基”可以互换使用。
“白介素-15”或“IL-15”指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然的IL-15。该术语涵盖未加工的IL-15以及源自细胞中的加工的任何形式的IL-15。该术语还涵盖天然存在的IL-15变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的IL-15变体、改造体、截断体。例示性野生型人IL-15的氨基酸序列如SEQ ID NO：14所示。未加工的人IL-15额外包含N端的信号肽(参见UniProt条目号：P40933)，所述信号肽在成熟的IL-15分子中是缺乏的。本发明中，“白介素-15”、“白介素215、“白细胞介素-15”、“白细胞介素15”、“IL15”和“IL-15”可以互换使用。
“IL15RA”或“IL-15受体的α亚基”指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然IL15RA，包括“全长”的未加工的IL15RA以及源自细胞中的加工的任何形式的IL15RA，还包括天然存在的IL15RA变体，例如剪接变体或等位变体。该术语还涵盖具有类似功能的IL15RA变体、改造体、截断体。在某些实施方案中，IL15RA是人IL15RA(参见UniProt条目号：Q13261)，其胞外域片段例示性序列如SEQ ID NO：15-17所示。本发明所述的“IL15RA”与“IL-15Rα”以及“IL-15受体的α亚基”可以互换使用。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述，是本领域中公知且达成一致的标准。
“衍生物”旨在被广义地解释，包括任意目标蛋白相关的产品。包括但不限于人和非人的目标蛋白同系物、 片段或截短体、融合蛋白(如与信号肽融合或其他活性、非活性成份融合，活性成份例如抗体或其抗原结合片段)、修饰形式(如PEG化、糖基化、白蛋白缀合/融合、Fc缀和/融合、羟乙基化等)、和保守修饰的蛋白等。
“免疫缀合物”是一种特定的缀合物，其包含至少一个融合蛋白或其衍生物和至少一个抗原结合模块。在某些实施方式中，所述免疫缀合物包含至少一个融合蛋白或其衍生物和至少两个抗原结合模块。依照本发明的具体的免疫缀合物基本由通过一种或多种接头序列连接的一个融合蛋白或其衍生物和抗原结合模块组成。可以通过多种相互作用并以多种构造将抗原结合模块与融合蛋白或其衍生物连接。
“免疫球蛋白”(Immunoglobulin，Ig)又称抗体(Antibody，Ab)，是一种主要由浆细胞分泌，被免疫系统用来鉴别与中和外来物质如细菌、病毒等病原体的大型Y形蛋白质，常规的免疫球蛋白由4条肽链组成，包括轻链(L链)和重链(H链)。轻链与重链之间通过二硫键(—S—S—)相连接，重链和轻链上分别具有可变区(V区)和恒定区(C区)，其中重链和轻链的V区分别称为VH和VL，重链和轻链的C区分别称为CH(CH1、CH2和CH3等)和CL。“抗体”在本文中以最广义使用，并且涵盖各种抗体结构，只要它们展现出期望的抗原结合活性，所述抗体包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗原结合片段。抗体可以包括鼠源抗体、人抗体、骆驼抗体、鲨鱼抗体、人源化抗体、嵌合抗体、重链抗体、纳米抗体、单域抗体等。例示性的，抗体可以是免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，单链Fv(scFv)，纳米抗体(即VHH)，VH/VL结构域。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗原结合片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(singlechainantibody)或单链Fv(scFv)。
“单域抗体”旨在被广义地解释，指包含了抗体中单个可变域的片段。既包括来自驼类和软骨鱼等生物体内的重链抗体的可变区，也包括来自人工改造的抗体重链\轻链的可变区，如由人工改造的小鼠产生的重链抗体的可变区。
“单链抗体”旨在被广义地解释，包括由抗体重链可变区(heavy chain variable domain，VH)和轻链可变区(light chain variable domain，VL)通过柔性短肽(linker)连接而成的重组抗体(约27kDa)，以及其它可以发挥抗体功能、单肽链形式的抗体。
在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。 所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：ComputationalMolecularBiology，Lesk，A.M.，ed.，OxfordUniversityPress，NewYork，1988，Biocomputing:Informatics，and，Genome，Projects，Smith，D.W.，ed.，AcademicPress，NewYork，1993；ComputerAnalysisofSequenceData，PartI，Griffin，A.M.，andGriffin，H.G.，eds.，HumanaPress，NewJersey，1994；SequenceAnalysisinMolecularBiology，vonHeinje，G.，AcademicPress，1987和SequenceAnalysisPrimer，Gribskov，M.andDevereux，J.，eds.，MStocktonPress，NewYork，1991。
“载体”是指可以在其中插入多核苷酸的核酸媒介物。当载体允许插入其中的多核苷酸编码的蛋白质的表达时，该载体称为表达载体。该载体可以通过转化、转导或转染入宿主细胞而使携带的遗传物质元件在宿主细胞中表达。载体是本领域技术人员所熟知的，包括但不限于质粒，噬菌体，粘粒，人工染色体如酵母人工染色体(YAC)，细菌人工染色体(BAC)或P1衍生人工染色体(PAC)，噬菌体如λ噬菌体或M13噬菌体和动物病毒。可用作载体的病毒包括但不限于逆转录病毒(包括慢病毒)，腺病毒，腺相关病毒，疱疹病毒(如单纯疱疹病毒)，痘病毒，杆状病毒，乳头瘤病毒，乳多空病毒(如SV40)。载体可以包含用于控制表达的多个元件，包括但不限于启动子序列，转录起始序列，增强子序列，选择元件和报道基因。另外，载体可以包含复制起点。
“宿主细胞”和“宿主细胞系”可交换使用并指已引入外源核酸的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括初始转化的细胞和自其衍生的后代(不考虑传代数)。后代在核酸内含物上可能与亲本细胞不完全相同，但可以含有突变。本文中包括具有与原始转化细胞中所筛选或选择的功能或生物学活性相同的功能或生物学活性的突变后代。“宿主细胞”包括但不限于原核细胞如大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillussubtilis)，真核细胞如酵母细胞或曲霉属(Aspergillus)，昆虫细胞如S2果蝇细胞或Sf9，以及哺乳动物细胞如成纤维细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞。
下面采用相关具体实施例对本发明做进一步说明。
实施例一
实验材料准备
表1中提供了实施例1-4中使用的相关实验材料的信息。
表1实验材料
实施例2
Ab-CD25/IL-2-Complex融合蛋白的制备及功能测试
如本发明第一方面所述的融合蛋白，在本具体实施方案中，所述的配体是IL-2(SEQ ID NO:6)或IL-2变体(SEQ ID NO:7)，所述的配体结合蛋白是CD25的胞外域片段(SEQ ID NO:8-9)。CD25胞外域是天然拮抗IL-2与CD25结合的蛋白，我们希望通过构建CD25/IL-2如本发明所述的融合蛋白，从而获得偏向型IL-2：降低与CD25的结合或与CD25不结合，保留结合/激活中等亲和力IL-2受体。
2.1 Ab-CD25/IL-2-Complex融合蛋白的设计
根据本发明第一方面所述的多肽结构式，根据“式I”第一多肽和“式II”第二多肽所设计的Ab-CD25/IL-2-Complex融合蛋白如图1A和B所示；根据“式III”第一多肽和“式IV”第二多肽所设计的Ab-CD25/IL-2-Complex融合蛋白如图2A和B所示。
图1和图2的融合蛋白不含Fc区，进一步地我们设计了如图3所示含Fc区的Ab-CD25/IL-2-Complex融合蛋白。
由于含Fc的Ab-CD25/IL-2-Complex融合蛋白能够形成免疫球蛋白样的四聚体复合体，具有更高的分子量，有利于延长目标蛋白在体内的半衰期，我们进一步根据图3的结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽链和第二多肽链具体的序列和组合如表2所示。
表2.各Ab-CD25/IL-2-Complex的两条多肽链序列
2.2 Expi293F哺乳动物细胞表达Ab-CD25/IL-2-Complex融合蛋白
将2.1中构建好的质粒，根据表2中第一多肽链和第二多肽链的配对关系，将含有第一多肽链的质粒和含有第二多肽链的质粒共同转染到EXPi293F细胞中，转染流程如下：Expi293F细胞于37℃，8％CO2培养箱中135rpm摇晃培养到4至5×10^6cells/ml，将细胞用新鲜培养液稀释到3×10^6cells/ml的浓度，30ml/瓶。取1.5mlOpti-Medium+30ug质粒预混5分钟(A液)，1.5mlOpti-Medium+60ulPEI(2ug/ul)(B液)预混5分钟，将A液及B液混合并于室温中静置20分钟(每孔剂量)，将上述液体滴入培养瓶中，轻摇混匀，并将细胞放回至37℃，8％CO2培养箱中135rpm继续培养。转染24小时后，补加D-Glucose至终浓度4.5g/L，补加VPA至终浓度3mM。转染5天后，2000rcf，5分钟离心收集细胞上清培养液。
将上清液与ProteinAbeads旋转孵育2h，然后将液体和珠子全部转移至空柱中，用WashingBuffer洗掉杂蛋白，用ElutionBuffer洗脱蛋白质，洗脱后的蛋白在PBS中透析，透析后的蛋白采用0.22uM的滤膜过滤，用BCA的方法测定蛋白浓度，并将蛋白质跑SDS-PAGE胶，经考马斯亮蓝染色后，观察蛋白的表达情况，如图4所示。如图4可知Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2、Ab-CD25/IL-2-C3均可正常表达。
2.3检测Ab-CD25/IL-2-Complex融合蛋白与CD25结合情况
按400ng/孔的量在ELISA板包被CD25重组蛋白，室温2h，5％脱脂奶粉室温封闭。封闭结束后用washing buffer清洗3次，之后每孔加入100ul Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2或Ab-CD25/IL-2-C3的梯度稀释系列(0.625nM至1000nM)，室温反应2h，washing buffer清洗3次。加入Alpaca-VHH-Fc-Anti-IL-2抗体室温反应1h，washing buffer清洗3次。加入Rabbit-anti-VHH(HRP)(Genscript，A01861-200)室温孵育1h，孵育结束后，加入TMB显色液(索莱宝，PR1200-500ML)，并通过1MH2SO4终止反应。使用酶标仪(Tecan，SPARK10M)读取460nm处的吸光值。采用GraphPadPrism9进行数据处理和作图分析，获得野生型Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3与CD25的结合曲线，如图5所示。同时检查了Human IL-2-Fc和CD25的结合情况，如图6所示。
由实验结果发现：
(1)野生型Human IL-2可以很好的和CD25结合。
(2)Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3在使用浓度达到1000nM的情况下和CD25都没有明显结合，提示Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3很可能没办法结合/激活高亲和力IL-2受体。
(3)融合Fc的IL-2蛋白(Human IL-2-Fc)与CD25的结合能力和野生型Human IL-2差不多，说明Fc不会显著影响IL-2和CD25的结合情况，进一步说明Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3与CD25极其显著削弱的结合反应是CD25/IL-2融合蛋白特异的效果。
2.4检测Ab-CD25/IL-2-Complex融合蛋白与CD122结合情况
2.4.1Human IL-2和Ab-CD25/IL-2-Complex融合蛋白进行Biotin偶联
将500μg相关蛋白溶解于500μlPBS中，使其终浓度为1mg/ml。加入5μl、10mg/ml的NHS-biotin溶液混合均匀(NHS用DMSO溶解)。室温孵育30min。加入50μl100mM的甘氨酸溶液，室温孵育10min，混合均匀终止反应。采用PBS对偶联后的蛋白进行透析。透析后，采用0.22uM滤膜过滤，采用BCA测量蛋白浓度，冻存于-80度备用。
2.4.2ELISA检测Ab-CD25/IL-2-Complex融合蛋白与CD122结合情况
按400ng/孔的量在ELISA板中包被CD122重组蛋白，然后分别加入等摩尔量偶联Biotin后的阴性对照IGG-Isotype、Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2或Ab-CD25/IL-2-C3，采用HRP-Streptavidin检测各蛋白和CD122的结合情况，如图7所示。
由实验结果可知，野生型Human IL-2和CD122有较弱的相互作用。与野生型Human IL-2相比，Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3与CD122的结合显著增强，很可能是IL-2和CD25形成的复合结构，增强了其和CD122的亲和力。对照IGG蛋白与CD122不结合，进一步说明Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3与CD122的结合是CD25/IL-2融合蛋白特异的反应。
为了进一步确认Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3与CD122的结合能力，进一步通过Ab-CD25/IL-2-Complex融合蛋白的梯度稀释系列(0.625nM至1000nM)结合实验检测三个融合蛋白和CD122 的结合曲线，如图8所示。从图8中可以看出Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3可以很好地和CD122结合，提示该融合蛋白保留结合/激活中等亲和力IL-2受体的能力。
2.5 Ab-CD25/IL-2-Complex融合蛋白促进NK92细胞的激活
NK92细胞(ATCC，CRL-2407)采用天津灏洋生物的TBDNK92KIT培养基进行传代培养。
刺激前，先采用无IL2培养基，培养NK92细胞2-3天。按所需的样本量，将无IL-2培养的NK92细胞接种于24孔板中。采用PBS(阴性对照)、Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3和IGG-Isotype(阴性对照)分别对上述IL-2饥饿处理的NK92细胞进行处理。处理时间：20min，60min；处理浓度：15.625nM；刺激后1000rcf，5min，收集细胞沉淀。用RIPAlysisbuffer裂解细胞，加入5×Loadingbuffer，westernblot检测P-STAT5和Actin，如图9所示。
由实验结果可知：和HumanIL-2类似，Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3都可以很好地激活NK92细胞中STAT5的磷酸化，提示该融合蛋白具有激活NK细胞的功能。IGG-Isotype不能激活STAT5的磷酸化，进一步说明Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2和Ab-CD25/IL-2-C3对NK92的激活是CD25/IL-2融合蛋白特异激发的反应。
2.6 Ab-CD25/IL-2-Complex融合蛋白促进免疫细胞杀伤肿瘤细胞
2.6.1人外周血单个核细胞(PBMC)的提取
将新鲜血液用PBS按照1:1进行稀释轻轻上下吹打。将Ficoll试剂上下颠倒5-10次充分震荡混匀，将Ficoll试剂分装到离心管中待用。将稀释后的血液贴壁加入含Ficoll的离心管中，将离心管转移至离心机中，配平，室温400g离心30min，设置升速1降速0。用1mL移液器轻轻将上层血清吸出。离心结束后分4层，由下到上分别为，红细胞，Ficoll溶液，白膜层，PBS。将枪头插进白膜层中小心吸取白膜层(即淋巴细胞层)转移至新的15ml离心管中。尽量避免吸取到下层或上层的细胞，避免造成污染。加入10mlPBS至上述转移出来的淋巴细胞中，轻轻吹打混匀，100×g，10min离心。弃上清，加入1ml裂红液，3min裂红。加入清洗液终止裂红，300×g，5min离心。加入清洗液补至体积到10ml清洗，100×g，8min离心清洗细胞2次。弃上清，收集细胞沉淀备用。
2.6.2免疫细胞的扩增和活化
将上述分离的PBMC加入24孔细胞培养皿中，细胞培养皿预先涂有10μg/mL抗CD3(novoprotein，GMP-A018)和抗CD28(novoprotein,GMP-A063)并在4℃下保持过夜，抗CD3和抗CD28抗体可以促进T细胞的活化和增殖。PBMC是在含有10％胎牛血清的RPMI 1640中培养，含1:100青霉素-链霉素。将细胞分成不同份，按终浓度15.625nM的用量分别加入：CD25-Fc(对照)、IGG-Isotype(对照)、Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2或Ab-CD25/IL-2-C3。细胞隔天半数传代，等量添加新鲜培养基，并添加相应的刺激物：CD25-Fc(对照)、IGG-Isotype(对照)、Human IL-2、Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2或Ab-CD25/IL-2-C3，将增殖和活化好的免疫细胞用于后续的肿瘤杀伤实验。
2.6.3RTCA esight检测免疫细胞对肿瘤细胞的杀伤能力
步骤如下：在E-Plate 96孔培养板中加入50μL1640完全培养基，37度平衡1个小时，检测阻抗基线。接 种肿瘤细胞(人肺鳞癌细胞H226)数量5000个/孔，将孔板室温水平放置30分钟，使细胞均匀沉降到孔板底部，通过阻抗检测靶细胞贴壁增殖。阻抗每15分钟采集一次，成像每1个小时采集一次。待肿瘤细胞贴壁12h后，收集8.2中扩增好的效应细胞(主要是T细胞)，按效靶比为5:1，将配制好的效应细胞悬液以每孔50ul加入含靶细胞(肿瘤细胞)的孔中。将加好的孔板放回xCELLigence RTCA eSight(安捷伦)中对应的位置，静置平衡30min。然后开始数据采集，实时监测效应细胞杀伤靶细胞的过程，阻抗15min测一次，图像1小时采集一次。结果如图10所示：与IGG-Isotype相比，Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2、Ab-CD25/IL-2-C3同HumanIL-2类似都可以显著促进免疫细胞杀伤H226肿瘤细胞，说明Ab-CD25/IL-2-C1、Ab-CD25/IL-2-C2与Ab-CD25/IL-2-C3可以有效促进免疫细胞杀伤肿瘤，并且这个活性是CD25/IL-2融合蛋白特异性的反应。
实施例3
Ab-VHH/IL-2-Complex融合蛋白的制备及功能测试
抗体可以通过和配体的结合，调控配体和其受体的结合以及后续的信号。单域抗体因其结构简单(仅含一条肽链即可发挥抗体相关功能)便于进行工程改造的特性，在分子改造和融合蛋白构建中，具有独特的优势。在本具体实施方案中，所述的配体是IL-2(SEQ ID NO:6)或IL-2变体(SEQ ID NO:7)，所述的配体结合蛋白是抗IL-2的单域抗体(SEQ ID NO:10-13)。抗IL-2且结合其CD25表位的单域抗体能够拮抗IL-2与CD25的结合，我们希望通过构建VHH/IL-2如本发明所述的融合蛋白，从而获得不结合CD25(或不结合高等亲和力IL-2受体)而保留结合/激活中等亲和力IL-2受体的偏向型IL-2。
3.1抗IL-2-单域抗体确定和功能验证
在本申请人之前申请的专利“白细胞介素2结合分子、其衍生物、试剂盒及其生产方法和用途”(申请号：202110528965.8)中，筛获得了一系列抗IL-2的单域抗体。我们进一步筛选了其中的四个抗体514(18)-G6(对应本发明中的Anti-IL-2-VHH-01，SEQ ID NO:10)、514(18)-D2(对应本发明中的Anti-IL-2-VHH-02，SEQ ID NO:11)、506(9)-F4(对应本发明中的Anti-IL-2-VHH-03，SEQ ID NO:12)和514(11)-G5(对应本发明中的Anti-IL-2-VHH-04，SEQ ID NO:13)用于本实施例的实验。
我们首先将表达上述四个单域抗体的核苷酸序列克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达、带humanIGG1-Fc标签的载体(LV082-AbV-Human-IGG1-Fc-1GS)中，获得表达Anti-IL-2-VHH-Fc的4个表达质粒，进一步根据如2.2所述的“Expi293F哺乳动物细胞表达蛋白”的流程制备4种Anti-IL-2-VHH-Fc蛋白。进一步地，利用ELISA实验检测了这些Anti-IL-2-VHH-Fc蛋白与IL-2的结合情况(图11)，及拮抗CD25和IL-2结合的情况(图12)。
如图11所示，Anti-IL-2-VHH-01-Fc、Anti-IL-2-VHH-02-Fc、Anti-IL-2-VHH-03-Fc、Anti-IL-2-VHH-04-Fc都可以很好的和IL-2结合，而对照Fc-Isotype不能和IL-2结合说明：Anti-IL-2-VHH-01、Anti-IL-2-VHH-02、Anti-IL-2-VHH-03和Anti-IL-2-VHH-04是能够特异性结合IL-2的单域抗体。
进一步地，我们检测这几个Anti-IL-2-VHH-Fc蛋白拮抗CD25和IL-2(His-tag)结合的情况。实验流程如下：在96孔ELISA板中加入100ul含200ngCD25-Fc蛋白，室温包被2h。封闭和洗涤后，每孔分别加入100ul含 100ngIL-2(His-tag)和抗IL-2-VHH-Fc蛋白的梯度稀释系列(1ng/ml至10^5ng/ml)，室温反应2h。洗涤后，加入Anti-His Tag Antibody(HRP)室温孵育1h。之后加入TMB底物，用1M H2SO4终止反应。使用酶标仪(Tecan，SPARK10M)读取460nm处的吸光值。采用GraphPadPrism9进行数据处理和作图分析，获得Anti-IL-2-VHH-01-Fc、Anti-IL-2-VHH-02-Fc、Anti-IL-2-VHH-03-Fc、Anti-IL-4-VHH-01-Fc对CD25/IL-2阻断曲线，如图12所示。由实验结果可知，Anti-IL-2-VHH-01-Fc、Anti-IL-2-VHH-02-Fc和Anti-IL-2-VHH-03-Fc可以很好的拮抗CD25和IL-2的相互作用，而Anti-IL-2-VHH-04-Fc不能拮抗CD25和IL-2的相互作用，说明：Anti-IL-2-VHH-01、Anti-IL-2-VHH-02、Anti-IL-2-VHH-03是抗IL-2且识别其CD25结合表位的单域抗体，可以阻断IL-2和CD25的相互作用及信号转导；Anti-IL-2-VHH-04仅可以结合IL-2，不具有阻断IL-2和CD25相互作用的功能。
3.2Ab-VHH/IL-2-Complex融合蛋白的设计
如图13所示，我们设计了含Fc区的Ab-VHH/IL-2-Complex融合蛋白。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表3所示。
表3.各Ab-VHH/IL-2-Complex的两条多肽链序列
3.3 Expi293F哺乳动物细胞表达Ab-VHH/IL-2-Complex融合蛋白
类似地，根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，我们表达并获得了各Ab-VHH/IL-2-Complex融合蛋白，经SDS-PAGE-考马斯亮蓝染色，检测表达情况如图14所示：Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4均可正常表达。
3.4检测Ab-VHH/IL-2-Complex融合蛋白与CD25结合情况
按400ng/孔的量在ELISA板包被CD25-His重组蛋白，室温2h，5％脱脂奶粉室温封闭。封闭结束后用washing buffer清洗3次，之后每孔加入100ul Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3或Ab-VHH/IL-2-C4的梯度稀释系列(3.125nM至500nM)室温反应2h，washing buffer清洗3次。加入Goat anti-Human-IgG(HPR) 抗体室温反应1h，washing buffer清洗3次。加入TMB显色液(索莱宝，PR1200-500ML)，并通过1M H2SO4终止反应。使用酶标仪(Tecan，SPARK10M)读取460nm处的吸光值。采用GraphPadPrism9进行数据处理和作图分析，获得野生型Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4与CD25的结合曲线，如图15所示。由实验结果发现：
(1)不具有阻断IL-2和CD25相互作用的Anti-IL-2-VHH-04单域抗体形成的Ab-VHH/IL-2-C4融合蛋白保留了IL-2(原配体)和CD25结合的功能；
(2)具有阻断IL-2和CD25相互作用的Anti-IL-2-VHH-01、Anti-IL-2-VHH-02和Anti-IL-2-VHH-03单域抗体形成的Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2和Ab-VHH/IL-2-C3在使用浓度达到500nM的情况下和CD25都没有明显结合，提示Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2和Ab-VHH/IL-2-C3很可能没办法结合/激活高亲和力IL-2受体。
3.5检测Ab-VHH/IL-2-Complex融合蛋白与CD122结合情况
类似地，根据2.4所述的“检测融合蛋白与CD122结合”的实验流程，我们首先将Ab-VHH/IL-2-Complex以及阴性对照IGG-Isotype进行biotin偶联，然后通过ELISA检测了Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4与CD122的结合情况。结果如图16所示，Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4均保留了IL-2(原配体)和CD122结合的能力。对照IGG蛋白与CD122不结合，进一步说明Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4与CD122的结合是VHH/IL-2融合蛋白特异的反应。
3.6 Ab-VHH/IL-2-Complex融合蛋白促进NK92细胞的激活
类似地，根据2.5所述的“融合蛋白促进NK92细胞的激活”的实验流程，我们检测了Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4刺激NK92激活STAT5磷酸化的情况。结果如图17所示，Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4都可以很好地激活NK92细胞中STAT5的磷酸化，提示该融合蛋白具有激活NK细胞的功能。IGG-Isotype不能激活STAT5的磷酸化，进一步说明Ab-VHH/IL-2-C1、Ab-VHH/IL-2-C2、Ab-VHH/IL-2-C3和Ab-VHH/IL-2-C4对NK92的激活是VHH/IL-2融合蛋白特异激发的反应。
实施例4
Ab-IL15RA/IL-15-Complex融合蛋白的制备及功能测试
如本发明第一方面所述的融合蛋白，在本具体实施方案中，所述的配体是IL-15(SEQ ID NO:14)，所述的配体结合蛋白是IL15RA的胞外域片段(SEQ ID NO:15-17)。IL15RA胞外域是天然拮抗IL-15与IL15RA结合的蛋白，我们希望通过构建IL15RA/IL-15如本发明所述的融合蛋白，从而获得不结合IL15RA受体而直接结合/激活IL-15Rβγ受体的偏向型IL-15。
4.1 Ab-IL15RA/IL-15-Complex融合蛋白的设计
如图18所示，我们设计了含Fc区的Ab-IL15RA/IL-15-Complex融合蛋白。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表4所示。
表4.各Ab-IL15RA/IL-15-Complex的两条多肽链序列
4.2 Expi293F哺乳动物细胞表达Ab-IL15RA/IL-15-Complex融合蛋白
类似地，根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，我们表达并获得了各Ab-IL15RA/IL-15-Complex融合蛋白，经SDS-PAGE-考马斯亮蓝染色，检测各融合蛋白的表达情况，如图19所示：Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3均可正常表达(*号标记的迁移条带推测可能是糖基化修饰的条带)。
4.3检测Ab-IL15RA/IL-15-Complex融合蛋白与IL15RA结合情况
根据2.4.1所述的“融合蛋白进行Biotin偶联”实验流程，我们对Ab-IL15RA/IL-15-Complex融合蛋白和HumanIL-15进行biotin偶联，并进一步利用这些biotin偶联蛋白的梯度稀释系列(0.625nM至500nM)检测Ab-IL15RA/IL-15-Complex融合蛋白与IL15RA结合曲线(实验流程参考2.4.2)，如图20所示。
由实验结果发现：
(1)野生型Human IL-15可以很好的和IL15RA结合。
(2)Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3在使用浓度达到500nM的情况下和IL15RA都没有明显结合，提示Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3与野生型IL-15相比，具有显著降低结合/激活IL15RA(或IL15Rαβγ三聚体)受体的能力。
4.4检测Ab-IL15RA/IL-15-Complex融合蛋白与IL15Rβ(CD122)结合情况
类似地，根据2.4所述的“检测融合蛋白与CD122结合”的实验流程，我们首先将Ab-IL15RA/IL-15-Complex、HumanIL-15和阴性对照IGG-Isotype进行biotin偶联，然后通过ELISA检测了HumanIL-15、Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2、Ab-IL15RA/IL-15-C3和IGG-Isotype分别与IL15Rβ(CD122)的结合情况。结果如图21所示，野生型Human IL-15和IL15Rβ(CD122)在ELISA结合实验中基本检测不到相互结合的信号。与野生型Human IL-15相比，Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3与IL15Rβ(CD122)的 结合非常显著地增强，很可能是IL-15和IL15RA形成的复合结构，增强了其和IL15Rβ(CD122)的亲和力。对照IGG蛋白与IL15Rβ(CD122)不结合，进一步说明Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3与IL15Rβ(CD122)的结合是IL15RA/IL-15融合蛋白特异的反应。
为了进一步确认Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3与IL15Rβ(CD122)的结合能力，进一步通过Ab-IL15RA/IL-15-Complex融合蛋白的梯度稀释系列(0.625nM至500nM)结合实验检测三个融合蛋白和IL15Rβ(CD122)的结合曲线，结果如图22所示：Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3可以很好地和IL15Rβ(CD122)结合，提示该融合蛋白保留/增强了结合/激活IL15Rβ(或者IL15Rβγ二聚体)受体的能力。
4.5 Ab-IL15RA/IL-15-Complex融合蛋白促进NK92细胞的激活
类似地，根据2.5所述的“融合蛋白促进NK92细胞的激活”的实验流程，我们检测了Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3刺激NK92激活STAT5磷酸化的情况。结果如图23所示，Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3都可以很好地激活NK92细胞中STAT5的磷酸化，提示该融合蛋白具有激活NK细胞的功能。IGG-Isotype不能激活STAT5的磷酸化，进一步说明Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3对NK92的激活是IL15RA/IL-15融合蛋白特异激发的反应。
4.6 Ab-IL15RA/IL-15-Complex融合蛋白促进免疫细胞杀伤肿瘤细胞
类似地，根据2.6所述的“融合蛋白促进免疫细胞杀伤肿瘤细胞”的实验流程，我们检测了Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3对免疫细胞杀伤肿瘤细胞(HT226，人肺鳞癌细胞)的影响。
结果如图24所示：与IGG-Isotype相比，Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3同Human-IL-15类似都可以显著促进免疫细胞杀伤H226肿瘤细胞，说明Ab-IL15RA/IL-15-C1、Ab-IL15RA/IL-15-C2和Ab-IL15RA/IL-15-C3可以有效促进免疫细胞杀伤肿瘤，并且这个活性是IL15RA/IL-15融合蛋白特异性的反应。
4.7不含Fc区的Ab-IL15RA/IL-15-Complex融合蛋白的设计及制备
参考图1和图2所示的结构示意图，将IL-2更换为IL-15，将CD25的胞外域更换为IL-15RA的胞外域，我们设计了不含Fc区的Ab-IL15RA/IL-15-Complex融合蛋白。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表5和表6所示。
表5.不含Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列(式I&式II组合)

表6.不含Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列(式III&式IV组合)
进一步，根据表5和表6中第一多肽链和第二多肽链的配对关系，将含有第一多肽链的质粒和含有第二多肽链的质粒共同转染到EXPi293F细胞中，并根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，将Ab-IL15RA/IL-15-Complex11至14在Expi293F细胞中进行表达。表达载体上带有His标签，进一步采用Ni-NTAbeads(镍柱)对Expi293F表达的蛋白上清进行纯化。
我们将表达的Ab-IL15RA/IL-15-Complex12至14融合蛋白，经SDS-PAGE、考马斯亮蓝染色，检测这些融合蛋白的表达情况，如图25所示为Ab-IL15RA/IL-15-Complex12至14的表达情况。
4.7在不同位置连接Fc区的Ab-IL15RA/IL-15-Complex的制备及功能测试
如本发明第一方面所述，融合蛋白可以包含Fc区，且Fc区可以连接于第一多肽链的C端或者N端，也可以连接于第二多肽链的C端或者N端。在Ab-IL15RA/IL-15-Complex12融合蛋白的基础上，我们进一步设计了在不同位置连接Fc区的Ab-IL15RA/IL-15-Complex融合蛋白。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表7-10所示。
表7.第一多肽链C端连接Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列
表8.第二多肽链C端连接Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列

表9.第一多肽链N端连接Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列
表10.第二多肽链N端连接Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列
进一步，根据表7-10中第一多肽链和第二多肽链的配对关系，将含有第一多肽链的质粒和含有第二多肽链的质粒共同转染到EXPi293F细胞中，并根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，将Ab-IL15RA/IL-15-Complex21至24在Expi293F细胞中进行表达，并进一步用ProteinAbeads进行纯化。
我们将表达的Ab-IL15RA/IL-15-Complex21至24融合蛋白，经SDS-PAGE、考马斯亮蓝染色，检测这些融合蛋白的表达情况，如图26所示：Ab-IL15RA/IL-15-Complex21至24均可正常表达。
4.8检测Ab-IL15RA/IL-15-Complex13-14、21-24融合蛋白与IL15RA和CD122的结合情况
根据2.4.1所述的“融合蛋白进行Biotin偶联”实验流程，我们对Ab-IL15RA/IL-15-Complex13至14、Ab-IL15RA/IL-15-Complex21至24融合蛋白、HumanIL-15和阴性对照IGG-Isotype进行biotin偶联，并进一步利用等摩尔量(125nM)的这些biotin偶联蛋白分别与IL15RA和CD122进行ELISA结合实验检测(实验流程参考2.4.2)，如图27-28所示。
由实验结果发现：
(1)野生型Human IL-15可以很好的和IL15RA结合；Ab-IL15RA/IL-15-Complex13-14、Ab-IL15RA/IL-15-Complex21-24融合蛋白和IL15RA没有明显结合，提示Ab-IL15RA/IL-15-Complex13-14、Ab-IL15RA/IL-15-Complex21-24融合蛋白与野生型IL-15相比，具有显著降低结合/激活IL15RA(或IL15Rαβγ三聚体)受体的能力；
(2)野生型Human IL-15和IL15Rβ(CD122)在ELISA结合实验中基本检测不到相互结合的信号。与野生型Human IL-15相比，Ab-IL15RA/IL-15-Complex13-14、Ab-IL15RA/IL-15-Complex21-24融合蛋白与IL15Rβ(CD122)的结合非常显著地增强，很可能是IL-15和IL15RA形成的复合结构，增强了其和IL15Rβ(CD122)的亲和力。对照IGG蛋白与IL15Rβ(CD122)不结合，进一步说明Ab-IL15RA/IL-15-Complex13-14、Ab-IL15RA/IL-15-Complex21-24融合蛋白与IL15Rβ(CD122)的结合是 IL15RA/IL-15融合蛋白特异的反应。
实施例5
Ab-CD122/IL-2-Complex融合蛋白的设计及制备
在一些具体的实施方式中，所述的配体是IL-2(SEQ ID NO:6)或IL-2变体(SEQ ID NO:7)，所述的配体结合蛋白是CD122的胞外域(UniProtID：P14784第27-239位氨基酸)，经第一多肽链和第二多肽链形成CD122/IL-2融合蛋白(Ab-CD122/IL-2-Complex)。因为杀伤性T细胞、NK细胞等主要表达CD122/CD132二聚体受体，调节性T细胞(Treg)主要表达含CD25的三聚体受体，因此该Ab-CD122/IL-2-Complex具有潜在的特异性激活调节性T细胞，而不激活杀伤性T或NK细胞的功能，从而达到促进机体恢复免疫平衡的作用，具有潜在的用于治疗自身免疫性疾病的重要作用。
参考图1和图2所示的结构示意图，将CD25的胞外域更换为CD122的胞外域，我们设计了不含Fc区的Ab-CD122/IL-2-Complex融合蛋白。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表11和表12所示。
表11.不含Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列(式I&式II组合)
表12.不含Fc区的Ab-IL15RA/IL-15-Complex的两条多肽链序列(式III&式IV组合)
进一步，根据表11和表12中第一多肽链和第二多肽链的配对关系，将含有第一多肽链的质粒和含有第二多肽链的质粒共同转染到EXPi293F细胞中，并根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，将Ab-CD122/IL-2-Complex1至4在Expi293F细胞中进行表达。表达载体上带有His标签，进一步采用Ni-NTAbeads(镍柱)对Expi293F表达的蛋白上清进行纯化。
实施例6
多种Ab-Complex融合蛋白的设计及制备
根据本发明第一方面所述，所述配体是是细胞因子(包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等)、蛋白类激素分子、细胞膜上的配体或它们的变体、改造体、截断体。所述的配体结合蛋白是该配体的受体或其受体的变体，所述的变体包括：该受体的突变体、改造体、截断体，以及该受体胞外域和该受体胞外域的突变体、改造体、截断体。
参考图1的结构示意图，我们选取了以下配体/配体结合蛋白对，设计了不含Fc区的多种Ab-Complex融合蛋白，具体的有：Ab-IL4/IL4RA-Complex、Ab-EGFR/Anti-EGFR-VHH-Complex、Ab-PD1/PD-L1-Complex、Ab-IL2/Anti-IL2-VHH-Complex。
根据本发明第一方面所述的多肽结构式，将可以表达第一多肽链和第二多肽链的核苷酸序列分别克隆到厦门柏慈生物科技有限公司用于Expi293F细胞表达的载体(Ab-Vector)中，获得相对应的表达质粒。第一多肽肽链和第二多肽具体的序列和组合如表13所示。
表13.多种Ab-Complex融合蛋白的两条多肽链序列(式I&式II组合)
进一步，根据表13中第一多肽链和第二多肽链的配对关系，将含有第一多肽链的质粒和含有第二多肽链的质粒共同转染到EXPi293F细胞中，并根据2.2所述的“Expi293F哺乳动物细胞表达蛋白”流程，将上述Ab-Complex融合蛋白在Expi293F细胞中进行表达。表达载体上带有His标签，进一步采用Ni-NTAbeads(镍柱)对Expi293F表达的蛋白上清进行纯化。
我们将上述Ab-Complex融合蛋白，经SDS-PAGE、考马斯亮蓝染色，检测各融合蛋白的表达情况，如图29所示为这些Ab-Complex融合蛋白的表达情况。
实施例7
本实施例公开了一种试剂盒，包含容器，所述容器内设有实施例2的Ab-CD25/IL-2-Complex融合蛋白，或实施例3的Ab-VHH/IL-2-Complex融合蛋白，或实施例4的Ab-IL15RA/IL-15-Complex融合蛋白，或实施例5的Ab-CD122/IL-2-Complex融合蛋白，或实施例6的Ab-IL4/IL4RA-Complex、Ab-EGFR/Anti-EGFR-VHH-Complex、 Ab-PD1/PD-L1-Complex、Ab-IL2/Anti-IL2-VHH-Complex融合蛋白。采用本实施例试剂盒，可促进NK细胞的活化或促进免疫细胞杀伤肿瘤细胞或改善自身免疫性疾病等，以促进相关疾病的治疗。
工业实用性
本发明可以应用于NK细胞的活化或促进免疫细胞杀伤肿瘤细胞或改善自身免疫性疾病等，以促进相关疾病的治疗，促进机体恢复免疫平衡的作用，可以用于临床药物。具有工业实用性。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。

Claims (18)

  1. 一种融合蛋白，其特征在于：包含第一多肽和第二多肽，
    所述的第一多肽、第二多肽从N端到C端分别具有如下所示的多肽结构式：
    第一多肽：A-L1-C1       (式I)
    第二多肽：B-L2-C2        (式II)
    或所述的第一多肽、第二多肽从N端到C端分别具有如下所示的多肽结构式：
    第一多肽：C1-L1-A       (式Ⅲ)
    第二多肽：C2-L2-B  (式Ⅳ)
    其中，
    A为配体，B为可以与A相互作用的配体结合蛋白；或A为配体结合蛋白，B为可以与A相互作用的配体；所述配体包括野生型配体或其变体；所述的变体包括该配体的突变体、改造体和截断体，以及该配体的胞外域和该配体胞外域的突变体、改造体、截断体；所述的配体结合蛋白包括野生型配体结合蛋白或其变体；所述的变体包括该配体结合蛋白的突变体、改造体和截断体，以及该配体结合蛋白的胞外域和该配体结合蛋白胞外域的突变体、改造体、截断体；
    C1为抗体轻链恒定结构域CL，C2为抗体重链恒定结构域CH1；所述CL包含人源抗体的CL或者具有与人源抗体的CL至少80％的序列相同性的氨基酸序列；所述CH1包含人源抗体的CH1或者具有与人源抗体的CH1至少80％的序列相同性的氨基酸序列；
    L1、L2各自独立的为接头元件或无。
  2. 如权利要求1的融合蛋白，其特征在于：所述融合蛋白还包括抗体Fc区，所述Fc区连接于第一多肽或第二多肽；所述的Fc区连接在第一多肽的C端，使第一多肽形成多肽结构式A-L1-C1-L3-Fc或C1-L1-A-L3-Fc；或所述的Fc区连接在第二多肽的C端，使第二多肽形成多肽结构式B-L2-C2-L3-Fc或C2-L2-B-L3-Fc；或所述的Fc区连接在第一多肽的N端，使第一多肽形成多肽结构式Fc-L3-A-L1-C1或Fc-L3-C1-L1-A；或所述的Fc区连接在第二多肽的N端，使第二多肽形成多肽结构式Fc-L3-B-L2-C2或Fc-L3-C2-L2-B，其中，
    抗体的Fc区包括人源IgG1、IgG2、IgG3、IgG4、IgA、IgD、IgM、IgE的Fc段或其变体、改造体；L3为接头元件或无。
  3. 如权利要求1或2所述的融合蛋白，其特征在于：所述的配体为细胞因子、蛋白类激素分子、细胞膜上的配体或它们的变体、改造体、截断体；所述的配体结合蛋白是该配体的受体或其受体的变体、或该配体的抗体或其抗原结合片段；所述的变体包括：该受体的突变体、改造体、截断体，以及该受体胞外域和该受体胞外域的突变体、改造体、截断体。
  4. 如权利要求3的融合蛋白，其特征在于：所述的细胞因子包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子。
  5. 如权利要求3的融合蛋白，其特征在于：所述的抗体或其抗原结合片段是单域抗体或单链抗体。
  6. 如权利要求3所述的融合蛋白，其特征在于：
    所述的配体是IL-2，所述的配体结合蛋白是CD25的胞外域；
    所述的配体是IL-2，所述的配体结合蛋白是CD122的胞外域；
    或所述的配体是IL-2，所述的配体结合蛋白是抗IL-2的VHH；
    或所述的配体是IL-15，所述的配体结合蛋白是IL15RA。
  7. 如权利要求1～6所述的融合蛋白，其特征在于：所述的融合蛋白选自下组中任意一组：
    1)所述的配体包含SEQIDNO：6-7任一多肽序列或与SEQIDNO：6-7任一多肽序列至少80％序列相同性的氨基酸序列，且所述的配体结合蛋白包含SEQIDNO：8-13任一多肽序列或与SEQIDNO：8-13任一多肽序列至少80％序列相同性的氨基酸序列；
    2)所述的配体包含SEQIDNO：14或与SEQIDNO：14至少80％序列相同性的氨基酸序列，且所述的配体结合蛋白包含SEQIDNO：15-17任一多肽序列或与SEQIDNO：15-17任一多肽序列至少80％序列相同性的氨基酸序列；
    3)所述的多肽结构式式I中的A包含SEQIDNO：6-7任一多肽序列或与SEQIDNO：6-7任一多肽序列至少80％序列相同性的氨基酸序列，且式II中的B包含SEQIDNO：8-13任一多肽序列或与SEQIDNO：8-13任一多肽序列至少80％序列相同性的氨基酸序列；
    4)所述的多肽结构式式I中的A包含SEQIDNO：14或与SEQIDNO：14至少80％序列相同性的氨基酸序列，且式II中的B包含SEQIDNO：15-17任一多肽序列或与SEQIDNO：15-17任一多肽序列至少80％序列相同性的氨基酸序列。
  8. 如权利要求1～7所述的融合蛋白，其特征在于：所述的融合蛋白选自下组中任意一组：
    1)所述的第一多肽链包含SEQIDNO：18-19任一多肽序列或与SEQIDNO：18-19任一多肽序列至少80％序列相同性的氨基酸序列，且第二多肽链包含SEQIDNO：20-21任一多肽序列或与SEQIDNO：20-21任一多肽序列至少80％序列相同性的氨基酸序列；
    2)所述的第一多肽链包含SEQIDNO：22或与SEQIDNO：22至少80％序列相同性的氨基酸序列，且第二多肽链包含SEQIDNO：23-25任一多肽序列或与SEQIDNO：23-25任一多肽序列至少80％序列相同性的氨基酸序列。
  9. 缀合物，其特征在于：包括权利要求1～8任一项所述的融合蛋白或其衍生物，所述融合蛋白或其衍生物直接或通过接头元件间接地与其他模块连接。
  10. 如权利要求9所述的缀合物，其特征在于：所述的其他模块包括抗原结合模块、细胞毒素、放射性同位素、生物活性蛋白质、可检测标记物、药物、毒素、细胞因子、酶金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、中任一种或其组合；更优选地，所述抗原结合模块是抗体或抗 原结合片段；最优选地，所述抗体或抗原结合片段靶向肿瘤细胞上或肿瘤细胞环境中呈现的抗原，或免疫细胞上的抗原。
  11. 药物组合物，其特征在于：包括权利要求1～8任一项所述的融合蛋白或其衍生物或权利要求9-10任一项所述的缀合物以及药学上可接受的稀释剂、载体或助剂。
  12. 核酸分子，其特征在于：其编码权利要求1～8任一项所述的融合蛋白或权利要求9-10所述的缀合物。
  13. 表达载体，其特征在于：包含权利要求12所述的核酸分子。
  14. 宿主细胞，其包含权利要求13所述的表达载体，或权利要求12所述的核酸分子，或表达权利要求1～8任一项所述的融合蛋白或其衍生物或权利要求9-10中的缀合物。
  15. 权利要求1～8任一项所述的融合蛋白或其衍生物、权利要求9-10中的缀合物、权利要求11的药物组合物、权利要求12的核酸分子、权利要求13的表达载体或权利要求14的宿主细胞用于制备治疗增生性疾病或免疫性疾病、调节T细胞介导的免疫应答、刺激个体免疫系统的药物中的用途。
  16. 如权利要求15所述的用途，其特征在于：所述增生性疾病是肿瘤或癌。
  17. 试剂盒，其特征在于：包含权利要求1～8任一项所述的融合蛋白或其衍生物、权利要求9-10中的缀合物、权利要求11的药物组合物、权利要求12的核酸分子、权利要求13的表达载体或权利要求14的宿主细胞。
  18. 一种制备融合蛋白的方法，其特征在于：在适于表达所述的融合蛋白的条件下使用权利要求12的核酸分子，或使用权利要求13的表达载体，或使用权利要求14的宿主细胞进行表达。
PCT/CN2023/079786 2022-03-10 2023-03-06 一种融合蛋白、制备方法及其应用 WO2023169349A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202380014988.4A CN118339194A (zh) 2022-03-10 2023-03-06 一种融合蛋白、制备方法及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210233916.6 2022-03-10
CN202210233916.6A CN115141283A (zh) 2022-03-10 2022-03-10 一种融合蛋白、制备方法及其应用

Publications (1)

Publication Number Publication Date
WO2023169349A1 true WO2023169349A1 (zh) 2023-09-14

Family

ID=83404950

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/079786 WO2023169349A1 (zh) 2022-03-10 2023-03-06 一种融合蛋白、制备方法及其应用

Country Status (2)

Country Link
CN (2) CN115141283A (zh)
WO (1) WO2023169349A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115141283A (zh) * 2022-03-10 2022-10-04 厦门柏慈生物科技有限公司 一种融合蛋白、制备方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130089516A1 (en) * 2010-04-02 2013-04-11 University Of Rochester Protease activated cytokines
CN107759697A (zh) * 2016-08-19 2018-03-06 安源生物科技(上海)有限公司 制备融合蛋白的方法
US20190070264A1 (en) * 2014-12-19 2019-03-07 Jiangsu Hengrui Medicine Co., Ltd. Interleukin 15 protein complex and use thereof
US20200040052A1 (en) * 2018-05-14 2020-02-06 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides and methods of use thereof
CN112154153A (zh) * 2018-03-28 2020-12-29 百时美施贵宝公司 白介素-2/白介素-2受体α融合蛋白以及使用方法
WO2021054867A1 (ru) * 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Иммуноцитокин, включающий гетеро димерный белковый комплекс на основе il-15 и il-15rα
CN115141283A (zh) * 2022-03-10 2022-10-04 厦门柏慈生物科技有限公司 一种融合蛋白、制备方法及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130089516A1 (en) * 2010-04-02 2013-04-11 University Of Rochester Protease activated cytokines
US20190070264A1 (en) * 2014-12-19 2019-03-07 Jiangsu Hengrui Medicine Co., Ltd. Interleukin 15 protein complex and use thereof
CN107759697A (zh) * 2016-08-19 2018-03-06 安源生物科技(上海)有限公司 制备融合蛋白的方法
CN112154153A (zh) * 2018-03-28 2020-12-29 百时美施贵宝公司 白介素-2/白介素-2受体α融合蛋白以及使用方法
US20200040052A1 (en) * 2018-05-14 2020-02-06 Werewolf Therapeutics, Inc. Activatable interleukin-2 polypeptides and methods of use thereof
WO2021054867A1 (ru) * 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Иммуноцитокин, включающий гетеро димерный белковый комплекс на основе il-15 и il-15rα
CN115141283A (zh) * 2022-03-10 2022-10-04 厦门柏慈生物科技有限公司 一种融合蛋白、制备方法及其应用

Also Published As

Publication number Publication date
CN115141283A (zh) 2022-10-04
CN118339194A (zh) 2024-07-12

Similar Documents

Publication Publication Date Title
CN113383013B (zh) 一种人白细胞介素2变体或其衍生物
EP3371311B1 (en) Bi-functional chimeric proteins and uses thereof
US11649270B2 (en) T-cell receptor recognizing SSX2 antigen
US11912770B2 (en) Blocking type PD-L1 single-domain camel antibody and application thereof
JP7121496B2 (ja) 癌治療で使用するためのペグ化インターロイキン－１０
CN111647068A (zh) 一种人白细胞介素2变体或其衍生物
JP2020530299A (ja) Ｃｄ８結合物質
JP2020530298A (ja) Ｐｄ−１およびｐｄ−ｌ１結合物質
JP2020506727A (ja) 標的化キメラタンパク質及びその使用
CN113480668A (zh) 抗b7h3嵌合抗原受体及其应用
CN102573922A (zh) 靶向性免疫缀合物
WO2023169349A1 (zh) 一种融合蛋白、制备方法及其应用
CN110198953A (zh) 针对ny-eso的高亲和力tcr
EP3889183A1 (en) A protein complex comprising an immunocytokine
CA3149010A1 (en) T cell receptor for identifying ssx2 antigen short peptide
WO2023046156A1 (en) Il-2 variants and fusion proteins thereof
CN115057919A (zh) 一种人白细胞介素2变体及其用途
TW202237632A (zh) ｐＨ依賴性突變型介白素－２多肽
CN116178572A (zh) 构建嵌合蛋白的嵌合区域、构建方法、嵌合蛋白及其应用
JP2023552093A (ja) ガンマデルタｔ細胞調節剤を同定する方法
CN114478789A (zh) 抗pd-l1与ox40双特异性抗体及其用途
EP4357357A1 (en) Human interleukin-2 variant and use thereof
RU2799437C2 (ru) Вариант человеческого интерлейкина-2 или его производное
CN110016074B (zh) Mage-a3人源化t细胞受体
CN114375303A (zh) Magea10特异性t细胞受体及其用途

Legal Events

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

Ref document number: 23765927

Country of ref document: EP

Kind code of ref document: A1