WO2023083379A1 - 一种以白介素15为活性成份的融合蛋白构建体及其应用 - Google Patents

一种以白介素15为活性成份的融合蛋白构建体及其应用 Download PDF

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WO2023083379A1
WO2023083379A1 PCT/CN2022/132019 CN2022132019W WO2023083379A1 WO 2023083379 A1 WO2023083379 A1 WO 2023083379A1 CN 2022132019 W CN2022132019 W CN 2022132019W WO 2023083379 A1 WO2023083379 A1 WO 2023083379A1
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fusion protein
fab
seq
homodimer
terminal
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傅阳心
彭华
申姣
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Institute of Biophysics of CAS
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Priority to EP22892171.4A priority patent/EP4435011A4/en
Priority to JP2024529257A priority patent/JP2024539432A/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—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
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the invention belongs to the field of medicine and biology, and specifically relates to a fusion protein construct with interleukin-15 as an active ingredient and its application.
  • PD-1/PD-L1 blocking antibody With the rapid development of immune checkpoint therapy, PD-1/PD-L1 blocking antibody has become one of the important methods in clinical treatment.
  • the PD-1/PD-L1 immune blocking antibody relieves the tumor's inhibition of tumor infiltrating lymphocytes (tumor infiltrating lymphocytes, TILs), and regains its ability to recognize and kill tumors.
  • TILs tumor infiltrating lymphocytes
  • TILs tumor infiltrating lymphocytes
  • TILs tumor infiltrating lymphocytes
  • TILs tumor infiltrating lymphocytes
  • IL-2 is a cytokine required for the expansion of T cells, and it is also one of the earliest immunotherapy drugs approved by the US FDA (Food and Drug Administration, FDA) for malignant melanoma and kidney cancer.
  • FDA Food and Drug Administration
  • IL-2 has not been widely used clinically for the following reasons: (1) short half-life; (2) activation of immunosuppressive regulatory T cells (Regulatory T cells, Treg); (3) activation of effector T cells Cells will be cleared through activation induced cell death (AICD); (4) Activation of vascular endothelial cells will cause severe toxic side effects.
  • IL-15 is another important T cell growth factor and a pleiotropic cytokine that maintains and activates innate and adaptive immune responses.
  • IL-15 can promote the activation, proliferation and survival of CD8+ T cells; activate and maintain memory T cells.
  • IL-15 can also promote NK and NKT cell activation and proliferation.
  • IL-15 can also promote the activation, proliferation and differentiation of dendritic cells in an autocrine form, and promote the expression of MHC-II and CD80/CD86, and improve the ability of dendritic cells to cross-present antigens.
  • IL-15 has the following advantages in tumor therapy: (1) IL-15 can avoid activation-induced cell death, and induce the production and steady-state proliferation of long-lived memory T cells. (2) IL-15 does not expand Treg cells. (3) IL-2 acts on vascular endothelial cells to cause systemic toxicity, while IL-15 does not cause this side effect. Therefore, IL-15 is currently a potential cytokine for tumor immunotherapy.
  • IL-15 The main reasons for the limited clinical application of IL-15 are the short half-life and low therapeutic effect.
  • IgG1 Fc domain ALT-803
  • the combination of ALT-803 does not have great advantages.
  • an increase in the number of systemic circulating NK and CD8+ T cells was detected in all patients, which indicates that the toxic side effects of IL-15 are very obvious at therapeutic doses.
  • many preclinical studies use the method of mutating IL-15 to reduce the binding of IL-15 to its receptor.
  • IL-15 Although it can reduce the binding of IL-15 to peripheral NK cells, it will also affect the anti-tumor activity of IL-15. And the artificially modified IL-15 is also more likely to induce anti-IL-15 antibodies in the human body, thereby further reducing the therapeutic effect of IL-15.
  • PD-1 itself has great advantages. Compared with other tissues in the whole body, PD-1 has a higher expression in tumors. More importantly, CD8+ T cells in the tumor express high levels of PD-1 molecules, which allows the ⁇ PD-1 antibody to directly act on this group of killer T cells. At the same time, the ⁇ PD-1 blocking antibody can also block the combination of PD-1 and PD-L1, relieve the inhibition of T cells, and further enhance the anti-tumor ability of T cells.
  • IL-15 can promote the proliferation of CD8+ T cells, which may enhance the killing ability of T cells during PD-1/PD-L1 antibody blocking therapy.
  • the concept of PD-1/PD-L1 antibody and cytokine fusion protein is very common now, but most fusion protein forms still produce obvious toxic side effects. The reason is that the receptors of cytokines are widely distributed, such as IL-15, whose receptors are expressed on both T cells and NK cells, and have a high affinity with the receptors (Kd ⁇ 10 -9 M).
  • the affinity between the cytokine and the receptor is likely to be greater than the affinity between the antibody and the antigen, and cannot produce an effective tumor targeting effect, so it will still bring obvious effects during systemic injection. toxic side effect.
  • the advantage of the fusion protein constructed in this patent is that it uses the steric hindrance effect to adjust the length of the connecting fragment 1 between hIgG globulin and IL-15, and hinders the binding of IL-15 to its receptor IL-2/15R ⁇ (CD122).
  • the fusion protein-targeted drug reduces the combination with NK cells expressing IL-15 receptors during peripheral circulation, thereby reducing peripheral toxic and side effects.
  • the fusion protein specifically binds to CD8+ T cells with high expression of PD-1 through the ⁇ PD-1 antibody, and at the same time uses the high affinity of the ⁇ PD-1 antibody to assist IL-15 and CD8+ T cells The binding of the upper receptors can specifically activate this group of CD8+ T cells.
  • ⁇ PD-1 antibody blocks the inhibition of PD-1/PD-L1 on T cells, it further promotes the proliferation of T cells and enhances the killing ability of T cells through the signal of IL-15, and eliminates peripheral toxic side effects. , to enhance the anti-tumor effect of the fusion protein-targeted drug.
  • the present invention firstly relates to a fusion protein, and the fusion protein comprises the following block
  • the first structural unit the alpha subunit sushi domain of the interleukin 15 (IL-15) receptor;
  • connecting fragment 1 connects the second and third structural units
  • the connecting fragment 1 connects the first and third structural units.
  • the amino acid sequence of the connecting fragment 1 is an integer multiple repetition of GGGGS, represented by (G 4 S) n .
  • the integer n is any integer from 1 to 7; more preferably, the integer n is 1-5; most preferably, the integer n is 3.
  • fusion protein may further comprise the following blocks:
  • the fourth structural unit further connected to the N-terminal of the fusion protein (the N-terminal of the third structural unit): the Fab block of the therapeutic antibody;
  • the therapeutic antibodies include but are not limited to: anti-PD1/PD-L1 antibody, Her2 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-RANKL antibody, anti-VEGFR antibody, anti-EGFR antibody;
  • the Fab block of the therapeutic antibody is anti-PD-1 Fab (Fab fragment of PD1 antibody).
  • the anti-PD-1 Fab includes a heavy chain (variable region + constant region) and a light chain (variable region + constant region), wherein the heavy chain is located at the N-terminal of the fusion protein;
  • the anti-PD-1 Fab is an anti-PD-1 Fab of mouse or human origin;
  • the light chain amino acid sequence of the mouse or human anti-PD-1 Fab is shown in Seq ID No.4, Seq ID No.18, and Seq ID No.19; the anti-PD
  • the heavy chain amino acid sequence of -1 Fab is shown in Seq ID No.5, Seq ID No.20, Seq ID No.21.
  • the IL-15 is murine or human IL-15, the amino acid sequences of which are shown in Seq ID No.1 and Seq ID No.15 respectively;
  • the amino acid sequence of the ⁇ subunit sushi domain of the murine or human IL-15 receptor is shown in Seq ID No.3 and Seq ID No.17;
  • the amino acid sequence of connecting fragment 2 is shown in Seq ID No.6.
  • the present invention also relates to a homodimer composed of the fusion protein, preferably, monomers of the homodimer are connected to each other through dimerization of a third structural unit.
  • the homodimer is:
  • the sequence of the monomeric fusion protein from N-terminal to C-terminal is: human IgG1-Fc, connecting fragment 1 (G 4 S), sushi domain of ⁇ subunit of IL-15 receptor, connecting fragment 2, IL-15; preferred Its amino acid sequence structure is shown in SEQ ID No.7;
  • the sequence of the monomeric fusion protein from N-terminal to C-terminal is: human IgG1-Fc, connecting fragment 1 (G 4 S), IL-15, connecting fragment 2, ⁇ subunit sushi domain of IL-15 receptor; preferred Its amino acid sequence structure is shown in SEQ ID No.8;
  • the sequence is: anti-PD-1 Fab, human IgG1-Fc, connecting fragment 1 (G 4 S), sushi domain of the ⁇ subunit of IL-15 receptor, connecting fragment 2.
  • IL-15 preferably, its amino acid sequence structure is shown in SEQ ID No.9;
  • the order is: anti-PD-1 Fab, human IgG1-Fc, connecting fragment 1 (G 4 S), IL-15, connecting fragment 2, IL-15 receptor ⁇ Subunit sushi domain; preferably, its amino acid sequence structure is shown in SEQ ID No.10;
  • composition of monomers from N-terminal to C-terminal is as follows: fusion protein linked by human IgG1-Fc, connecting fragment 1 ((G 4 S) 3 ), human or mouse IL-15, connecting fragment 2,
  • the ⁇ subunit sushi domain of IL-15 receptor preferably, its amino acid sequence structure is shown in SEQ ID No.11;
  • the constituent monomers are as follows from N-terminal to C-terminal: anti-PD-1 Fab, human IgG1-Fc, connecting fragment 1 ((G 4 S) 3 ), IL-15, connecting fragment 2, IL-15 receptor
  • anti-PD-1 Fab anti-PD-1 Fab
  • human IgG1-Fc connecting fragment 1 ((G 4 S) 3 )
  • IL-15 connecting fragment 2
  • IL-15 receptor The ⁇ subunit sushi domain of the body; preferably, its amino acid sequence structure is shown in SEQ ID No.12;
  • Homodimer 7 (Fc-(G 4 S) 5 -IL-15-R ⁇ ):
  • composition of monomers from N-terminal to C-terminal is as follows: fusion protein linked by human IgG1-Fc, connecting fragment 1 ((G 4 S) 5 ), IL-15, connecting fragment 2, IL-15 receptor ⁇ subunit sushi domain; preferably, its amino acid sequence structure is shown in SEQ ID No.13;
  • Homodimer 8 (anti-PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ ):
  • the constituent monomers are as follows from N-terminal to C-terminal: anti-PD-1 Fab, human IgG1-Fc, connecting fragment 1 ((G 4 S) 5 ), IL-15, connecting fragment 2, IL-15 receptor
  • anti-PD-1 Fab anti-PD-1 Fab
  • human IgG1-Fc connecting fragment 1 ((G 4 S) 5 )
  • IL-15 connecting fragment 2
  • IL-15 receptor The ⁇ subunit sushi domain of the body; preferably, its amino acid sequence structure is shown in SEQ ID No.14;
  • the present invention also relates to nucleotide fragments encoding said fusion protein.
  • the present invention also relates to the application of the fusion protein and the fusion protein dimer in the preparation of medicine; preferably, the medicine is an antitumor medicine, most preferably, the medicine is anti-B-cell lymphoma, anti-neoplastic Drugs for colorectal cancer, against melanoma, or against lung cancer.
  • the present invention also relates to a preparation method of the fusion protein, the preparation method comprising the following steps:
  • an expression vector comprising the gene encoding the fusion protein, preferably, the expression vector is a pEE12.4 expression vector;
  • IL-15 fusion proteins Two forms of IL-15 fusion proteins were designed. Their structures are similar, and the positions of IL-15 and the sushi domain of the ⁇ subunit of IL-15 receptor are exchanged at the C-terminus of Fc respectively. The biological activities of the two IL-15 fusion proteins differ by approximately 10,000-fold.
  • connecting fragment 1 A variety of connecting fragments (connecting fragment 1) were designed, at the position where the C-terminus of Fc is connected to the sushi domain of the alpha subunit of IL-15/IL-15 receptor.
  • the basic unit of connecting fragment 1 is five amino acids GGGGS, and the length of connecting fragment 1 will directly affect the biological activity of IL-15.
  • fusion protein refers to the protein product obtained by linking the coding regions of two or more genes by gene recombination methods, chemical methods or other appropriate methods, and expressing gene recombination under the control of the same regulatory sequence.
  • the first block polypeptide at the N-terminal of the fusion protein connects the C-terminus of the polypeptide to the N-terminus of the next block (or connecting fragment) polypeptide, and so on; therefore, the N-terminus of the polypeptide at the N-terminal block of the fusion protein is a fusion
  • the N-terminal of the protein, the C-terminal of the polypeptide located in the C-terminal block of the fusion protein is the C-terminal of the fusion protein.
  • IL-15 wild type or wild type IL-15 refers to human IL-15 or non-human mammal IL-15 or non-mammal IL-15 of natural origin; IL-15 polypeptide.
  • IL-15R ⁇ subunit may be IL-15R ⁇ or a functional fragment thereof of any species, such as human IL-15R ⁇ or non-human mammalian IL-15R ⁇ or non-mammalian IL-15R ⁇ .
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc.
  • human IL-15R ⁇ preferably human interleukin 15 receptor alpha ectodomain fragment.
  • the term "Sushi domain” defines at least one sushi-containing polypeptide
  • the extracellular region of IL-15R ⁇ or a fragment thereof contains an IL-15R ⁇ sushi domain, the extracellular region of IL-15R ⁇ contains a domain called a sushi domain (Wei et al. 2001, J. Immunol. 167: 277-282).
  • the sushi domain of IL-15R ⁇ has a ⁇ -sheet conformation, which is encoded by exon 2 of IL-15R ⁇ , starts at the cysteine residue (C1) encoded by the first exon 2, and ends at the Four exon 2 encoded cysteine residues (C4).
  • the “sushi domain of the IL-15R ⁇ subunit” can be defined as starting at the first cysteine residue after the signal peptide (C1 ), and terminate at the fourth cysteine residue (C4) after the signal peptide, both residues C1 and C4 can be included in the sushi domain sequence.
  • Fc is an abbreviation for the Fc region of an immunoglobulin, which refers to the constant region of the immunoglobulin chain, especially the carboxy-terminal or a part of the constant region of the heavy chain of the immunoglobulin, which has no antigen-binding activity and is the site of cell interaction.
  • the "Fc” of the present invention may be any Fc or its variants, derived from human or non-human mammals.
  • an Fc may comprise two or more domains of heavy chains CH1, CH2, CH3, CH4 in combination with an immunoglobulin hinge region.
  • Fc can be derived from different species, preferably human immunoglobulins.
  • immunoglobulins can be divided into different classes, and there are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.
  • An "Fc region” preferably includes at least one immunoglobulin hinge region, and the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the initial amino acid position of the hinge region can be changed.
  • the Fc comprises an Fc with increased antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) activity due to interaction with the Fc receptor (such as CD16a, CD16b, CD32a, CD16b, CD64 and C1q protein) binding affinity enhancement or weakening.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • mutations are included in the Fc domain to reduce ADCC activity or CDC activity.
  • Fc mutations include, but are not limited to, in the Fc domain (1) mutations of N297, such as but not limited to N297A, N297G; (2) mutations of L234, such as L234A, L234G, and/or mutations of L235, such as L235A or L235G; (3) a mutation of P329, such as P329G; or (4) a mutation of D265, such as D265A; or a combination of substitutions at any or all of these positions.
  • Fc mutations include mutations that increase serum half-life.
  • the Fc has the following substitutions: T250Q, or M428L, or a T250Q/M428L double mutation in CH3 (Hinton et al., J Biol Chem. 279(8):6213-6, 2004). In other embodiments, the Fc has the M252Y/S254T/T256E triple mutation (Dall'Acqua WF et al., J Immunol 169(9):5171-80, 2002).
  • Fc has N434A mutation (Petkova SB et al., International Immunology 18(12):1759-1769, 2006.) or M428L/N434S double mutation, or M428/N434A double mutation (Zalevsky J et al ., Nat Biotechnol. 28(2):157–159, 2010).
  • the Fc region has been modified to increase its serum half-life.
  • the modification that increases serum half-life is M428L.
  • antibody refers to an immunoglobulin molecule that specifically binds to a target antigen or has immunoreactivity, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates) and Antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • Fab fragment refers to an antibody digested with papain to generate two identical antigen-binding fragments, each Fab fragment containing the variable region of the heavy chain and the variable region of the light chain, as well as the constant region of the light chain and the region of the heavy chain.
  • Fab fragment herein refers to a light chain fragment comprising the VL region and the constant region (CL) of the light chain, and an antibody fragment comprising the VH region and the first constant region (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 region of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residue in the constant region of the heavy chain carries a free thiol group.
  • F(ab')2 is an antibody fragment with two antigen-combining sites (two Fab fragments) and a part of the Fc region produced by pepsin treatment of an intact antibody.
  • fusion protein refers to the protein product obtained by linking the coding regions of two or more genes by gene recombination methods, chemical methods or other appropriate methods, and expressing the gene recombination under the control of the same regulatory sequence.
  • the coding regions of two or more genes may be fused at one or several positions by sequences encoding peptide linkers or connecting peptides.
  • fusion protein of the present invention further includes antibody/Fc fusion protein constructs/complexes, or compositions of antibody/Fc fusion protein constructs/complexes formed in a non-covalent manner.
  • linking fragment 1/2" refers to the peptide used to link IL-15 with another protein molecule or protein fragment in the present invention to ensure correct folding and stability of the protein.
  • Said another molecule includes without limitation IL-15R ⁇ , Fc, Fc variants, antibodies and the like.
  • PD1 antibody and ⁇ PD-1 refer to antibodies against programmed death protein 1 (PD1).
  • exemplary antibodies include, but are not limited to, those listed in US Pat. Specific embodiments of such antibodies include BGB-A317, nivolumab (Bristol-Myers Squibb), labrolizumab (Merck), and pembrolizumab (Merck).
  • Figure 1 Schematic diagram of the structure of the fusion protein
  • FIG. 1A schematic diagram of the structure of Fc-G 4 S-IL-15-R ⁇ fusion protein
  • FIG. 1C schematic diagram of the structure of Fc-G 4 S-R ⁇ -IL-15 fusion protein
  • FIG. 1D schematic diagram of the structure of ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 fusion protein.
  • Fig. 2 Comparison of biological activities of several fusion proteins with different linker fragment 1.
  • Fig. 3 Comparison of the receptor-binding abilities of several fusion proteins connected with Fragment 1.
  • Fig. 5 The results of intraperitoneal administration of Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-R ⁇ -IL-15 to treat mouse MC38 tumor model.
  • FIG. 6 Body weight changes of MC38 tumor model in mice treated with intraperitoneal administration of Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-R ⁇ -IL-15.
  • FIG. 7 Survival curve of mouse MC38 tumor model treated with intraperitoneal administration of Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-R ⁇ -IL-15.
  • FIG. 8 Peripheral blood lymphocyte data diagram of Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-R ⁇ -IL-15 intraperitoneally administered to treat mouse MC38 tumor model, the vertical axis in the figure is "# /ul refers to the number of cells per ul of blood".
  • Fig. 9 The results of intraperitoneal administration of ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-IL-15-R ⁇ in the mouse MC38 tumor model.
  • Fig. 10 Results of intraperitoneal administration of ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and ⁇ PD-1 Fab combined with Fc-G 4 S-IL-15-R ⁇ to treat mouse MC38 tumor model.
  • Fig. 11 Results of intraperitoneal administration of ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 in the mouse MC38 tumor model.
  • FIG. 12 Body weight changes of mouse MC38 tumor model treated with ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 administered intraperitoneally.
  • FIG. 13 Data graph of peripheral blood lymphocytes of mouse MC38 tumor model treated by intraperitoneal administration of ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15.
  • Fig. 14 Results of intraperitoneal administration of ⁇ PD-1-IL-15 fusion proteins with different linker fragment 1 and Fc fragments to treat mouse MC38 small tumor model on the seventh day.
  • Figure 15 Changes in the body weight of the mouse MC38 small tumor model on the seventh day after intraperitoneal administration of ⁇ PD-1-IL-15 fusion proteins with different linker fragments 1 and Fc fragments.
  • Fig. 16 Results of intraperitoneal administration of ⁇ PD-1-IL-15 fusion proteins with different linker fragments 1 and Fc fragments to treat mouse MC38 large tumor model on the 14th day.
  • FIG. 17 Results of intraperitoneal administration of ⁇ PD-1-IL-15 fusion proteins with different linker fragments 1 to treat the large tumor model of mouse MC38 on the 14th day.
  • FIG. 18 Changes in body weight of the MC38 large tumor model in mice treated with intraperitoneal administration of ⁇ PD-1-IL-15 fusion proteins with different linker fragments 1 on the 14th day.
  • FIG. 19 Data graph of peripheral blood lymphocytes of MC38 large tumor model of mice treated with intraperitoneal administration of different ⁇ PD-1-IL-15 fusion proteins of fragment 1.
  • Fig. 20 is a graph showing the results of intratumoral administration of ⁇ PD-1 Fab-Fc-(G 4 S) 3 -IL-15-R ⁇ to treat the mouse B16 tumor model.
  • Figure 21 Systemic administration of anti-human PD-1 Fab-Fc-G 4 S-human IL-15-R ⁇ and anti-human PD-1 Fab-Fc-(G 4 S) 3 -human IL-15-R ⁇ Diagram of the results of drug treatment of CD34 + humanized mouse A549 lung cancer tumor model.
  • Figure 22 ⁇ PD-1 Fab-Fc-(G 4 S) 3 -R ⁇ -IL-15, Fc-G 4 S-R ⁇ -IL-15, ⁇ PD-1 Fab-Fc-(G 4 S) 3 -IL - 15-R ⁇ and Fc-(G 4 S) 3 -IL-15-R ⁇ fusion protein SDS-PAGE electrophoresis results.
  • Embodiment 1 design and construction of fusion protein
  • Fc-G 4 S-IL-15-IL-15R ⁇ sushi hereinafter named Fc-G 4 S-IL-15-R ⁇
  • Fc-G 4 S-IL-15-R ⁇ Fc-G 4 S-IL-15-IL-15R ⁇ sushi-IL-15
  • Figure 1A, C wherein, IL-15R ⁇ sushi is the sushi domain of the IL-15 receptor ⁇ subunit, and its amino acid sequence is shown in Seq ID No.3, 17, IL-15R ⁇ sushi -The amino acid sequence of the connecting fragment 2 between 15R ⁇ sushi and IL-15 is shown in Seq ID No.6; the amino acid sequence of the connecting fragment 1 between IL-15R ⁇ sushi or IL-15 and Fc is an integer multiple repetition of GGGGS, with (G 4 S) n represents; the amino acid sequence of Fc-G 4 S-IL-15-R ⁇ is shown in Seq ID No.8; the amino acid sequence of Fc-G 4 S-R ⁇ -IL-15 is shown in Seq ID No.
  • connection fragment 1 of Fc-(G 4 S) 3 -IL-15-R ⁇ and Fc-(G 4 S) 5 -IL-15-R ⁇ is three times and five times that of GGGGS respectively;
  • Fc-(G 4 S) The amino acid sequence of 3 -IL-15-R ⁇ is shown in Seq ID No.11; the amino acid sequence of Fc-(G 4 S) 5 -IL-15-R ⁇ is shown in Seq ID No.13;
  • ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ and ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 are shown in Figure 1B, D; among them, the amino acid sequence of anti-PD-1 Fab is light The chain is shown in Seq ID No.4, 18, 19; the heavy chain of anti-PD-1 Fab amino acid sequence is shown in Seq ID No.5, 20, 21; ⁇ PD-1Fab Fc-G 4 S-IL-15- The amino acid sequence of R ⁇ is shown in Seq ID No.10; the amino acid sequence of ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 is shown in Seq ID No.9;
  • the connecting fragment 1 of ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ and ⁇ PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ is three times and five times that of GGGGS, respectively ;
  • the amino acid sequence of ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ is shown in Seq ID No.12; the amino acid sequence of ⁇ PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ As shown in Seq ID No.14;
  • the above four forms of fusion protein genes are constructed in eukaryotic expression vectors, and are transiently expressed by a single plasmid or multiple plasmids in 293F cells, and the collected cell supernatant is purified by protein A.
  • the purified protein was quantified by ELISA and Nanodrop. The purity was detected by SDS-PAGE (4 ⁇ g loading for each sample).
  • the pEE12.4-IgG ⁇ -hIgG1 Fc plasmid comes from our laboratory and contains the signal peptide of mouse IgG ⁇ and the constant region sequence of human IgG1. All the genes involved in this application (IL-15, IL-15 R ⁇ and ⁇ PD-1 Fab) were synthesized by a third-party commercial synthesis company, and then ligated by restriction enzymes or homologous recombination into the pEE12.4 expression vector. Plasmids were extracted with Tiangen’s plasmid extraction kit and stored at -20°C.
  • the plasmid/PEI mixture was added to the cell suspension, and placed in an incubator at 37°C, 8% CO 2 , 85rpm;
  • valproic acid a cell proliferation inhibitor
  • Sample preparation filter the collected cell supernatant with a 0.22 ⁇ m filter membrane to remove cell debris, and add a final concentration of 0.05% NaN 3 ;
  • the protein concentration and purity were determined by SDS-PAGE electrophoresis, NanoDrop2000 and ELISA.
  • the protein was aliquoted and stored at -80°C.
  • the aliquoted protein should be thawed slowly at 4°C, and freezing and thawing should not be repeated.
  • lymphocyte proliferation-promoting test (CCK8 test) are as follows:
  • CTLL2 cells were cultured with 1640 complete medium containing 100 U/ml commercialized recombinant IL2 cytokine;
  • Fc-G 4 S-IL-15-R ⁇ and Fc-G 4 S-R ⁇ -IL-15 are about 10,000 times different, and the biological activity of Fc-G 4 S-R ⁇ -IL-15 Significantly increased, indicating that the biological activity of the two forms of IL-15 fusion protein is related to the relative position of IL-15 and R ⁇ ;
  • 5 ⁇ 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice.
  • 15 ⁇ g Fc-G 4 S-IL-15-R ⁇ or Fc-G 4 S-R ⁇ -IL-15 was administered intraperitoneally for 3 times with an interval of 2 days.
  • the control group was administered in the same way PBS.
  • the tumor was significantly controlled by intraperitoneal administration of Fc-G 4 S-R ⁇ -IL-15, but the body weight decreased significantly, 40% of the mice died, and CD8+T cells in peripheral blood lymphocytes , B220+ cells, NK cells, NKT cell expansion. It shows that Fc-G 4 S-R ⁇ -IL-15 has a significant anti-tumor effect, but the toxic and side effects are obvious.
  • 5 ⁇ 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice.
  • intraperitoneally administer ⁇ PD-1 Fab 15 ⁇ g+Fc-G 4 S-IL-15-R ⁇ 15ug or ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ (equimolar mass administration: 30 ⁇ g), administered 3 times with an interval of 2 days, and the control group was given PBS in the same way.
  • 5 ⁇ 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice.
  • the control group was given PBS in the same way.
  • ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 With intraperitoneal administration of ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15, the tumor was well controlled, but the weight loss was obvious, and CD8+T cells and B220+ cells in peripheral blood lymphocytes, NK cells, NKT cell expansion. It shows that ⁇ PD-1 Fab Fc-G 4 S-R ⁇ -IL-15 has a significant anti-tumor effect, but the toxicity and side effects are obvious.
  • 5 ⁇ 10 5 MC38 tumor cells were subcutaneously inoculated into the lower left side of C57 mice.
  • the tumor grows to 40mm 3 , ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ , ⁇ PD-1 Fab mFc-G 4 S-IL-15-R ⁇ , ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ , ⁇ PD-1 Fab mFc-(G 4 S) 3 -IL-15-R ⁇ and aEGFR Fab Fc-G 4 S-IL-15-R ⁇ , administered 3 times at intervals
  • the control group was given PBS in the same way.
  • 5 ⁇ 10 5 MC38 cells were subcutaneously inoculated into the lower left side of C57 mice.
  • the tumor grew to 100mm 3 , ⁇ PD-1 Fab Fc-G 4 S-IL-15-R ⁇ , ⁇ PD-1 Fab mFc-G 4 S-IL-15-R ⁇ , ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ , ⁇ PD-1 Fab mFc-(G 4 S) 3 -IL-15-R ⁇ and aEGFR Fab Fc-(G 4 S) 3 -IL-15-R ⁇ , administered for 3 times, with an interval of 2 days, the control group was given PBS in the same way.
  • mice 5 ⁇ 10 5 MC38 cells were subcutaneously inoculated into the lower left side of C57 mice.
  • ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ , ⁇ PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ were administered intraperitoneally, and Three times with an interval of 2 days, the control group was given PBS in the same way.
  • ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ and ⁇ PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ were administered intraperitoneally, and the tumors were significantly improved. Good control, and no significant difference. It shows that when the connecting fragment 1 is (G 4 S) 5 , compared with (G 4 S) 3 , the fusion protein does not show a better anti-tumor effect.
  • mice After intraperitoneal administration of ⁇ PD-1 Fab Fc-(G 4 S) 5 -IL-15-R ⁇ , the mice had a slight weight loss, and peripheral blood lymphocytes expanded significantly. It shows that when the connecting fragment 1 is (G 4 S) 5 , the fusion protein exhibits peripheral toxic side effects.
  • the tumor can be controlled by intratumoral administration of ⁇ PD-1 Fab Fc-(G 4 S) 3 -IL-15-R ⁇ . It shows that the fusion protein has a certain anti-tumor effect on B16.
  • the tumor can be controlled. It shows that the fusion protein has an anti-tumor effect on human-derived A549 lung cancer tumors on humanized mice.

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Abstract

本发明涉及一种以白介素15为活性成份的融合蛋白构建体及其应用，具体的，所述的融合蛋白构建体包括： (1)第一结构单元：白介素15(IL-15)受体的α亚基sushi结构域； (2)第二结构单元：IL-15； (3)第三结构单元：抗体Fc或突变的Fc片段；以及关键的连接片段1；连接片段1的氨基酸序列为GGGGS的整数倍重复。

Description

一种以白介素15为活性成份的融合蛋白构建体及其应用 技术领域
本发明属于医药生物领域，具体而言，涉及一种以白介素15为活性成份的融合蛋白构建体及其应用。
背景技术
随着免疫检查点疗法的迅速发展，PD-1/PD-L1阻断性抗体成为临床治疗中重要的方法之一。PD-1/PD-L1免疫阻断抗体解除了肿瘤对肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TILs)的抑制，重新发挥对肿瘤的识别杀伤功能。然而，在治疗后期，由于过度活化，T细胞逐渐进入耗竭状态，丧失功能。临床肿瘤患者对PD-1/PD-L1治疗的响应率普遍较低，且面临耐受或复发的风险。因此，如何解决PD-1/PD-L1临床应用上的瓶颈成为当前免疫治疗过程中最重要的问题。
由于具有扩增T淋巴细胞的能力，T细胞生长因子成为免疫治疗的重要靶点。其中，IL-2是T细胞扩增所需的细胞因子，也是美国FDA(Food and Drug Administration,FDA)最早批准用于恶性黑色素瘤和肾癌的免疫治疗药物之一。然而，IL-2一直未被广泛应用于临床，有以下几方面原因：(1)半衰期短；(2)激活免疫抑制性调节T细胞(Regulatory T cells,Treg)；(3)激活的效应T细胞会通过活化诱导的细胞死亡(Activation induced cell death,AICD)被清除；(4)激活血管内皮细胞而产生严重毒副作用。IL-15是另一个重要的T细胞生长因子，也是一个多效性的细胞因子，可维持和活化天然免疫和适应性免疫反应。IL-15可促进CD8+T细胞活化、增殖以及存活；活化和维持记忆性T细胞。IL-15还可促进NK和NKT细胞活化和增殖。此外，IL-15还可通过自分泌的形式促进树突状细胞的活化、增殖和分化，并促进MHC-II和CD80/CD86的表达，提高树突状细胞交叉提呈抗原的能力。IL-15相较于IL-2在肿瘤治疗方面具有以下优势：(1)IL-15可以避免活化诱导的细胞死亡，并诱导长寿命的记忆性T细胞的产生和稳态增殖。(2)IL-15不会扩增Treg细胞。(3)IL-2作用于血管内皮细胞从而引起系统性毒性，而IL-15不会导致此副作用。因此，IL-15是目前很有潜力的肿瘤免疫治疗细胞因子。
IL-15的临床应用受到限制的主要原因是较短的半衰期以及较低的治疗效果。有些研究者将IL-15突变体与IL-15Rα连接，并且与IgG1 Fc domain融合(ALT-803)用以延长半衰期。然而相比免疫检查点阻断治疗，ALT-803的联合并没有很大优势。与此同时，所有病人体内都检测到系统循环的NK和CD8+T细胞数量的增加，这表明了在治疗剂量下，IL-15带来的毒副作用十分明显。目前很多临床前研究采用突变IL-15的方法来降低IL-15与其受体的结合，虽然能够减少IL-15与外周NK细胞的结合，但同时会影响IL-15的抗肿瘤活性。并且人为改造的IL-15也更容易在人体内诱导产生抗IL-15抗体，从而进一步降低IL-15的治疗效果。
PD-1作为热门的靶点，自身具有很大的优势。相比全身其他组织，PD-1在肿瘤内具有较高的表达。更重要的是，肿瘤内的CD8+T细胞表达高水平的PD-1分子，这就使得αPD-1抗体可以直接作用于这群杀伤性T细胞。同时，αPD-1阻断性抗体还可以阻断PD-1与PD-L1的结合，解除对T细胞的抑制，进一步提升T细胞的抗肿瘤能力。
正如前文所述，PD-1/PD-L1抗体阻断治疗的效果并不显著，原因可能是T细胞的逐渐耗竭。而IL-15作为T细胞激动剂，可以促进CD8+T细胞的增殖，从而可能提升PD-1/PD-L1抗体阻断治疗过程中T细胞的杀伤能力。现在PD-1/PD-L1抗体与细胞因子融合蛋白的构想十分常见，但大部分融合蛋白形式仍会产生明显的毒副作用。究其原因，细胞因子的受体分布广泛，例如IL-15，其受体在T细胞、NK细胞上均有表达，且与受体的亲和力很高(Kd～10 -9M)。这就导致在抗体-细胞因子融合蛋白中，细胞因子与受体的亲和力很可能大于抗体与抗原的亲和力，无法产生有效的肿瘤靶向效果，故而在系统性注射过程中仍会带来明显的毒副作用。
本专利构建的融合蛋白优势在于，利用空间位阻效应，调整hIgG球蛋白与IL-15之间连接片段1的长度，阻碍IL-15与其受体IL-2/15Rβ(CD122)的结合。该融合蛋白靶向型药物在外周循环时，减少与表达IL-15受体的NK细胞结合，从而降低了外周的毒副作用。而在肿瘤微环境中，该融合蛋白通过αPD-1抗体特异性地结合到PD-1高表达的CD8+T细胞上，同时利用αPD-1抗体的高亲和力辅助IL-15与CD8+T细胞上受体的结合，从而特异性地激活这群CD8+T细胞。在αPD-1抗体阻断PD-1/PD-L1对T细胞的抑制的同时，通过IL-15的信号进一步促进T细胞的增殖、增强T细胞的杀伤能力，在消除外周毒副作用的情况下，提升该融合蛋白靶向型药物抗肿瘤的效果。
发明内容
本发明首先涉及一种融合蛋白，所述的融合蛋白包含如下嵌段
(1)第一结构单元：白介素15(IL-15)受体的α亚基sushi结构域；
(2)第二结构单元：IL-15；
(3)位于融合蛋白N端的第三结构单元：抗体Fc或突变的Fc片段；
(4)连接所述第一、第二结构单元的连接片段2；
当所述的融合蛋白的C端是第一结构单元时，连接片段1连接所述第二、第三结构单元；
当所述的融合蛋白的C端是第二结构单元时，连接片段1连接所述第一、第三结构单元。
所述连接片段1的氨基酸序列为GGGGS的整数倍重复，以(G 4S) n表示，优选的，所述的整数n为1～7的任意整数；更优选的，所述的整数n为1～5；最优选的，所述的整数n为3。
进一步的，所述的融合蛋白还可以进一步包含如下嵌段：
(5)进一步连接于所述融合蛋白N端(第三结构单元N端)的第四结构单元：治疗型抗体的Fab嵌段；
所述的治疗型抗体包括但不限于：抗PD1/PD-L1抗体、Her2抗体、抗CD20抗体、抗CD19抗体、抗RANKL抗体、抗VEGFR抗体、抗EGFR抗体；
优选的，所述的治疗型抗体的Fab嵌段为anti-PD-1 Fab(PD1抗体的Fab片段)。
所述的anti-PD-1 Fab包括重链(可变区+恒定区)和轻链(可变区+恒定区)，其中重链位于所述的融合蛋白N端；
优选的，所述的anti-PD-1 Fab为鼠源或人源的anti-PD-1 Fab；
更优选的，所述的鼠源或人源anti-PD-1 Fab的轻链氨基酸序列如Seq ID No.4、Seq ID No.18、Seq ID No.19所示；所述的anti-PD-1 Fab的重链氨基酸序列如Seq ID No.5、Seq ID No.20、Seq ID No.21所示。
Seq ID No.4：
Figure PCTCN2022132019-appb-000001
Seq ID No.18：
Figure PCTCN2022132019-appb-000002
Seq ID No.19：
Figure PCTCN2022132019-appb-000003
Figure PCTCN2022132019-appb-000004
Seq ID No.5：
Figure PCTCN2022132019-appb-000005
Seq ID No.20：
Figure PCTCN2022132019-appb-000006
Seq ID No.21：
Figure PCTCN2022132019-appb-000007
所述的IL-15为鼠源或人源的IL-15，其氨基酸序列分别如Seq ID No.1、Seq ID No.15所示；
Seq ID No.1：
Figure PCTCN2022132019-appb-000008
Seq ID No.15：
Figure PCTCN2022132019-appb-000009
所述的鼠源或人源IL-15受体的α亚基sushi结构域的氨基酸序列如Seq ID No.3、Seq ID No.17所示；
Seq ID No.3：
Figure PCTCN2022132019-appb-000010
Seq ID No.17：
Figure PCTCN2022132019-appb-000011
所述的Fc或突变的Fc片段氨基酸序列分别如Seq ID No.2、Seq ID No.16所示；
Seq ID No.2：
Figure PCTCN2022132019-appb-000012
Seq ID No.16：
Figure PCTCN2022132019-appb-000013
连接片段2的氨基酸序列如Seq ID No.6所示。
Seq ID No.6：
Figure PCTCN2022132019-appb-000014
本发明还涉及由所述融合蛋白构成的同源二聚体，优选的，所述的同源二聚体的单体之间通过第三结构单元的二聚化相互连接构成。
更优选的，所述的同源二聚体为：
(1)同源二聚体1(Fc-G 4S-Rα-IL-15)：
单体融合蛋白自N端到C端依次为：人源IgG1-Fc、连接片段1(G 4S)、IL-15受体的α亚基sushi结构域、连接片段2、IL-15；优选的，其氨基酸序列结构如SEQ ID No.7所示；
SEQ ID No.7：
Figure PCTCN2022132019-appb-000015
(2)同源二聚体2(Fc-G 4S-IL-15-Rα)：
单体融合蛋白自N端到C端依次为：人源IgG1-Fc、连接片段1(G 4S)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.8所示；
SEQ ID No.8：
Figure PCTCN2022132019-appb-000016
(3)同源二聚体3(anti-PD-1 Fab Fc-G 4S-Rα-IL-15)：
单体融合蛋白自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1(G 4S)、IL-15受体的α亚基sushi结构域、连接片段2、IL-15；优选的，其氨基酸序列结构如SEQ ID No.9所示；
SEQ ID No.9：
Figure PCTCN2022132019-appb-000017
(4)同源二聚体4(anti-PD-1 Fab Fc-G 4S-IL-15-Rα)：
单体融合蛋白自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1(G 4S)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.10所示；
SEQ ID No.10：
Figure PCTCN2022132019-appb-000018
(5)同源二聚体5(Fc-(G 4S) 3-IL-15-Rα)：
组成的单体自N端到C端依次为：人源IgG1-Fc连接成的融合蛋白、连接片段1((G 4S) 3)、人源或鼠源的IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.11所示；
SEQ ID No.11：
Figure PCTCN2022132019-appb-000019
(6)同源二聚体6(anti-PD-1 Fab Fc-(G 4S) 3-IL-15-Rα)：
组成的单体自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1((G 4S) 3)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.12所示；
SEQ ID No.12：
Figure PCTCN2022132019-appb-000020
Figure PCTCN2022132019-appb-000021
(7)同源二聚体7(Fc-(G 4S) 5-IL-15-Rα)：
组成的单体自N端到C端依次为：人源IgG1-Fc连接成的融合蛋白、连接片段1((G 4S) 5)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.13所示；
SEQ ID No.13：
Figure PCTCN2022132019-appb-000022
(8)同源二聚体8(anti-PD-1 Fab Fc-(G 4S) 5-IL-15-Rα)：
组成的单体自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1((G 4S) 5)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，其氨基酸序列结构如SEQ ID No.14所示；
SEQ ID No.14：
Figure PCTCN2022132019-appb-000023
本发明还涉及编码所述融合蛋白的核苷酸片段。
本发明还涉及所述融合蛋白、融合蛋白二聚体在制备药物中的应用；优选的，所述的药物为抗肿瘤药物，最优选的，所述药物为抗B细胞性淋巴瘤、抗结直肠癌、抗黑色素瘤或抗肺癌的药物。
本发明还涉及所述的融合蛋白的制备方法，所述的制备方法包括如下步骤：
(1)构建包含所述编码所述融合蛋白的编码基因的表达载体，优选的，所述的表达载体是pEE12.4表达载体；
(2)通过瞬时转染宿主细胞的方法构建包含所述表达载体的宿主细胞，优选的，所述的宿主细胞是293F细胞；
(3)培养所述宿主细胞并收集细胞上清；
(4)通过Protein A的亲和层析柱纯化蛋白纯化所述融合蛋白。
本发明的有益效果包括：
1、设计了两种形式的IL-15融合蛋白，它们的结构是相似的，分别在Fc的C端交换IL-15和IL-15受体的α亚基sushi结构域的位置。两种IL-15融合蛋白的生物学活性相差约10000倍。
2、设计了多种连接片段(连接片段1)，在Fc的C端与IL-15/IL-15受体的α亚基sushi结构域相连的位置。连接片段1的基本单元为五个氨基酸GGGGS，连接片段1的长短会直接影响IL-15的生物学活性。
2、在MC38小肿瘤模型(接种肿瘤细胞7天后，肿瘤大小约30mm 3)中，系统性注射αPD-1 Fab Fc-G 4S-IL-15-Rα融合蛋白后，可以完全清除肿瘤；在MC38大肿瘤模型(接种肿瘤细胞14天后，肿瘤大小约100mm 3)中，系统性注射αPD-1 Fab Fc-G 4S-IL-15-Rα融合蛋白后，可以起到明显的控制作用。
3、系统性注射αPD-1 Fab Fc-G 4S-IL-15-Rα融合蛋白，在3倍治疗剂量时，该融合蛋白靶向性药物治疗组小鼠没有任何体重的下降。
4、在CD34 +人源化小鼠的A549肺癌肿瘤模型中，系统性注射anti-human PD-1 Fab Fc-G 4S-human IL-15-Rα或anti-human PD-1 Fab Fc-(G 4S) 3-human IL-15-Rα融合蛋白后，可以起到明显的控制作用。
【术语解释】
术语“融合蛋白”是指，通过基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物，如未额外说明，融合蛋白N端的第一嵌段多肽以该多肽的C端和下一嵌段(或连接片段)多肽的N端相连，依次类推；因此，位于融合蛋白N端嵌段的多肽的N端为融合蛋白的N端，位于融合蛋白C端嵌段的多肽的C端为融合蛋白的C端。
术语“IL-15野生型”或“野生型IL-15”是指，天然来源的人IL-15或非人哺乳动物IL-15或非哺乳动物IL-15；也可以指代本领域已经通用的IL-15多肽。
术语“IL-15Rα亚基”可以是任何物种的IL-15Rα或者其功能性片段，如人IL-15Rα或非人哺乳动物IL-15Rα或非哺乳动物IL-15Rα。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的IL-15Rα；优选人的白介素15受体α胞外域片段。
术语“Sushi结构域”：定义至少一个含sushi多肽的IL-15Rα或其片段的胞外区含有IL-15Rαsushi结构域，IL-15Rα的胞外区含有被称为sushi结构域的结构域(Wei et al.2001，J.Immunol.167：277-282)。IL-15Rα的sushi结构域具有β折叠构象，其是由IL-15Rα的外显子2编码的，开始于第一外显子2编码的半胱氨酸残基(C1)，并终止于第四外显子2编码的半胱氨酸残基(C4)。当考虑标准N-末端至C-末端方向的IL-15Rα蛋白序列时，“IL-15Rα亚基的sushi结构域”能够被定义为开始于信号肽之后的第一半胱氨酸残基(C1)，并终止于信号肽之后的第四半胱氨酸残基(C4)，残基C1和C4均可包括在sushi结构域序列中。
术语“Fc”是免疫球蛋白Fc区的简称，指免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。本发明所述“Fc”可以是任何Fc或其变体，来源于人或非人哺乳动物。例如，Fc可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。Fc可以来源于不同的种属，优选人的免疫球蛋白。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2。“Fc区”优选包括至少一个免疫球蛋白绞链区，以及IgG的CH2和CH3结构域。更优选包括IgG1的一个CH2结构域，一个CH3结构域和一个免疫球蛋白绞链区，铰链区起始氨基酸位置可以变动。在一些实施方式中，Fc包含具有增加的抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)或补体依赖性细胞毒性(CDC)活性的Fc，其是由于与Fc受体(诸如CD16a、CD16b、CD32a、CD16b、CD64和C1q蛋白) 的结合亲和力增强或减弱所致。在另一些实施方式中，Fc结构域中包含突变，以减小ADCC活性或CDC活性。这些可包括但不限于在Fc结构域中(1)N297的突变，诸如但不限于N297A、N297G；(2)L234的突变，诸如L234A、L234G，和/或L235的突变，诸如L235A或L235G；(3)P329的突变，诸如P329G；或(4)D265的突变，诸如D265A；或者在任何或所有这些位置处的替换的组合。在另一些实施方式中，Fc突变(所有编号均为Kabat编号系统的Eu索引)包括增加血清半衰期的突变。在一种实施方式中，Fc具有以下替换：在CH3中的T250Q、或M428L、或T250Q/M428L双突变(Hinton et al.,J Biol Chem.279(8):6213-6,2004)。在另一些实施方式中，Fc具有M252Y/S254T/T256E三重突变(Dall’Acqua WF et al.,J Immunol169(9):5171-80,2002)。在另一些实施方式中，Fc具有N434A突变(Petkova SB et al.,International Immunology 18(12):1759–1769,2006.)或M428L/N434S双突变、或M428/N434A双突变(Zalevsky J et al.,Nat Biotechnol.28(2):157–159,2010)。在另一些实施方式中，Fc区已经被修改以增加其血清半衰期。在一些实施方式中，增加血清半衰期的修改为M428L。
术语“抗体”(Antibody，Ab)是指，与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。
术语“Fab”片段时指，抗体经木瓜蛋白酶消化生成两个同一的抗原结合片段，每个Fab片段含有重链可变区和轻链可变区，还有轻链的恒定区和重链的第一恒定区(CH1)。如此，本文术语“Fab片段”指包含轻链的VL区和恒定区(CL)的轻链片段，和重链的VH区和第一恒定区(CH1)的抗体片段。Fab’片段因在重链CH1区的羧基末端增加少数残基而与Fab片段不同，包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其重链恒定区的半胱氨酸残基携带游离硫醇基团的Fab’片段。F(ab’)2是完整抗体经胃蛋白酶处理产生的具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的抗体片段。
术语“融合蛋白”(fusion protein)是指，通过基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。本发明的融合蛋白中，两个或多个基因的编码区之间可由编码肽接头或连接肽的序列于一个或数个位置融合。本发明术语“融合蛋白”进一步还包括抗体/Fc融合蛋白构建体/复合物，或通过非共价方式形成的抗体/Fc融合蛋白构建体/复合物的组合物。
术语“连接片段1/2”是指，在本发明中用于连接IL-15与另一蛋白分子或蛋白片段，以保证蛋白的正确折叠和稳定性的肽。所述另一分子包括不限于IL-15Rα、Fc、Fc变体、抗体等。
术语“PD1抗体”和“αPD-1”是指针对程序性死亡蛋白1(PD1)的抗体。示例性抗体包括但不限于美国专利7,029,674、7,488,802、7,521,051、8,008,449、8,354,509、8,617,546和8,709,417中列出的抗体。所述抗体的具体实施方案包括BGB-A317、纳武单抗(Bristol-Myers Squibb)、labrolizumab(Merck)和派姆单抗(Merck)。
附图说明
图1、融合蛋白的结构示意图，
1A，Fc-G 4S-IL-15-Rα融合蛋白结构示意图；
1B，αPD-1 Fab Fc-G 4S-IL-15-Rα融合蛋白结构示意图；
1C，Fc-G 4S-Rα-IL-15融合蛋白结构示意图；
1D，αPD-1 Fab Fc-G 4S-Rα-IL-15融合蛋白结构示意图。
图2、几种不同连接片段1的融合蛋白的生物学活性比较。
图3、几种不同连接片段1的融合蛋白的结合受体能力比较。
图4、Fc-G 4S-IL-15-Rα、Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα及aEGFR Fab Fc-(G 4S) 3-IL-15-Rα融合蛋白与肿瘤内CD8+T细胞的结合能力比较。
图5、Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型结果图。
图6、Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型体重变化。
图7、Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型存活曲线。
图8、Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型外周血淋巴细胞数据图，图中的纵坐标为“#/ul，指每ul血液中的细胞数量”。
图9、αPD-1 Fab Fc-G 4S-IL-15-Rα及Fc-G 4S-IL-15-Rα腹腔给药治疗小鼠MC38肿瘤模型结果图。
图10、αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab联合Fc-G 4S-IL-15-Rα腹腔给药治疗小鼠MC38肿瘤模型结果图。
图11、αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型结果图。
图12、αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型体重变化。
图13、αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab Fc-G 4S-Rα-IL-15腹腔给药治疗小鼠MC38肿瘤模型外周血淋巴细胞数据图。
图14、不同连接片段1及Fc片段的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第七天小肿瘤模型结果图。
图15、不同连接片段1及Fc片段的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第七天小肿瘤模型体重变化。
图16、不同连接片段1及Fc片段的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第十四天大肿瘤模型结果图。
图17、不同连接片段1的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第十四天大肿瘤模型结果图。
图18、不同连接片段1的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第十四天大肿瘤模型体重变化。
图19、不同连接片段1的αPD-1-IL-15融合蛋白腹腔给药治疗小鼠MC38第十四天大肿瘤模型外周血淋巴细胞数据图。
图20、αPD-1 Fab-Fc-(G 4S) 3-IL-15-Rα瘤内给药治疗小鼠B16肿瘤模型结果图。
图21、anti-human PD-1 Fab-Fc-G 4S-human IL-15-Rα及anti-human PD-1 Fab-Fc-(G 4S) 3-human IL-15-Rα系统性给药治疗CD34 +人源化小鼠A549肺癌肿瘤模型结果图。
图22、αPD-1 Fab-Fc-(G 4S) 3-Rα-IL-15、Fc-G 4S-Rα-IL-15、αPD-1 Fab-Fc-(G 4S) 3-IL-15-Rα及Fc-(G 4S) 3-IL-15-Rα融合蛋白SDS-PAGE电泳结果图。
图23、anti-human PD-1 Fab-Fc-G 4S-human IL-15-Rα及anti-human PD-1 Fab-Fc-(G 4S) 3-human IL-15-Rα融合蛋白SDS-PAGE电泳结果图。
具体实施方式
实施例1、融合蛋白的设计构建
1、设计构建以下八种融合蛋白：
(1)Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15融合蛋白：
Fc-G 4S-IL-15-IL-15Rαsushi(以下命名为Fc-G 4S-IL-15-Rα)和Fc-G 4S-IL-15Rαsushi-IL-15(以下命名为Fc-G 4S-Rα-IL-15)结构见图1A，C；其中，IL-15Rαsushi为IL-15受体α亚基的sushi结构域，其氨基酸序列 如Seq ID No.3、17所示，IL-15Rαsushi与IL-15之间的连接片段2的氨基酸序列如Seq ID No.6所示；IL-15Rαsushi或IL-15与Fc之间的连接片段1的氨基酸序列为GGGGS的整数倍重复，以(G 4S) n表示；Fc-G 4S-IL-15-Rα氨基酸序列如Seq ID No.8所示；Fc-G 4S-Rα-IL-15氨基酸序列如Seq ID No.7所示；
(2)在Fc-G 4S-IL-15-Rα的基础上进一步修改连接片段1的Fc-(G 4S) 3-IL-15-Rα及Fc-(G 4S) 5-IL-15-Rα融合蛋白：
Fc-(G 4S) 3-IL-15-Rα及Fc-(G 4S) 5-IL-15-Rα的连接片段1分别为GGGGS的三倍和五倍；Fc-(G 4S) 3-IL-15-Rα氨基酸序列如Seq ID No.11所示；Fc-(G 4S) 5-IL-15-Rα氨基酸序列如Seq ID No.13所示；
(3)在Fc-G 4S-IL-15-Rα及Fc-G 4S-Rα-IL-15的基础上进一步融合anti-PD-1 Fab的αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab Fc-G 4S-Rα-IL-15：
αPD-1 Fab Fc-G 4S-IL-15-Rα及αPD-1 Fab Fc-G 4S-Rα-IL-15的结构见图1B，D；其中，anti-PD-1 Fab氨基酸序列轻链如Seq ID No.4、18、19所示；anti-PD-1 Fab氨基酸序列重链如Seq ID No.5、20、21所示；αPD-1Fab Fc-G 4S-IL-15-Rα氨基酸序列如Seq ID No.10所示；αPD-1 Fab Fc-G 4S-Rα-IL-15氨基酸序列如Seq ID No.9所示；
(4)在αPD-1 Fab Fc-G 4S-IL-15-Rα的基础上进一步修改连接片段1的αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα及αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα融合蛋白：
αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα及αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα的连接片段1分别为GGGGS的三倍和五倍；αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα氨基酸序列如Seq ID No.12所示；αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα氨基酸序列如Seq ID No.14所示；
2、融合蛋白的构建，转染，表达以及回收纯化
以上四种形式的融合蛋白基因构建于真核表达载体中，通过293F细胞单一质粒或者多个质粒进行瞬时表达，收集的细胞上清通过protein A进行纯化。纯化出的蛋白用ELISA和Nanodrop进行定量。通过SDS-PAGE的方法检测其纯度(每个样品4μg上样)。
具体的方法步骤如下：
2.1、质粒的构建
pEE12.4-IgGκ-hIgG1 Fc质粒来自本实验室，包含有小鼠IgGκ的信号肽和人IgG1恒定区序列。本申请涉及的所有基因(IL-15，IL-15 Rα和αPD-1 Fab)都是通过第三方商业合成公司合成，然后通过酶切连接或者同源重组到pEE12.4表达载体上。用天根的质粒大提试剂盒提取质粒，-20℃保存。
2.2、瞬时转染快速无血清表达目的蛋白
(1)293F细胞用SMM293TII培养基，37℃，8％CO 2，135rpm悬浮培养，待细胞密度达到4～4.5×10 6cells/ml，200ml时准备转染；
(2)离心收集细胞。用Freestyle 293培养基洗1次后，用200ml Freestyle 293培养基重悬细胞；
(3)用6ml Freestyle 293培养基稀释360ug质粒，当使用两个质粒共转αPD-1融合蛋白时，αPD-1轻链与重链的比例是2:1(240ug轻链+120ug重链)。用0.22μm的滤膜过滤除菌；
(4)用6ml Freestyle 293培养基稀释720μg Polyethylenimine(PEI)，用0.22μm的滤膜过滤除菌；
(5)将5ml PEI逐滴加到质粒中，同时涡旋震荡，随后静置5～10分钟；
(6)将质粒/PEI混合物加入细胞悬液中，放置在37℃，8％CO 2，85rpm培养箱中培养；
(7)4小时后补加200ml EX-CELLTM 293培养基和2mM L-Glutamine，将转速调至135rpm继续培养；
(8)24小时后加入细胞增殖抑制剂3.8mM valproic acid(VPA)，转染后第6天，收集细胞，8000rpm，4℃离心2小时，收集细胞上清，进行下一步纯化。
2.3、利用Protein A进行目的蛋白纯化
(1)样品准备：将收集好的细胞上清用0.22μm的滤膜过滤，除去细胞碎片，加入终浓度为0.05％NaN 3；
(2)分别用10倍柱体积的双蒸水和PBS冲洗和平衡Protein A层析柱；
(3)利用恒流泵进行上样，流速为10倍柱体积/小时，重复上样；
(4)用10倍柱体积的PBS冲洗柱子，洗去杂蛋白；
(5)使用0.1M Glycine(pH 2.7)进行洗脱，收集洗脱液。将洗脱的蛋白混合后加入适量的neutralization buffer(1M Tris，pH 9.0)进行中和(调节pH值至4左右，观察蛋白溶液，以刚好不产生絮状沉淀为准)；
通过SDS-PAGE电泳，NanoDrop2000以及ELISA确定蛋白浓度和纯度，蛋白分装-80℃保存，分装好的蛋白需在4℃缓慢化冻，不可反复冻融。
αPD-1 Fab-Fc-(G4S)3-Rα-IL-15、Fc-G4S-Rα-IL-15、αPD-1 Fab Fc-(G4S)3-IL-15-Rα及Fc-(G4S)3-IL-15-Rα融合蛋白的SDS-PAGE电泳结果见图22。Anti-human PD-1 Fab Fc-G 4S-human IL-15-Rα及anti-human PD-1Fab Fc-(G 4S) 3-human IL-15-Rα融合蛋白的SDS-PAGE电泳结果见图23。
实施例2、αPD-1-IL-15融合蛋白的生物学功能研究
1、促淋巴细胞生成的功能
促淋巴细胞增殖实验(CCK8试验)步骤如下：
(1)CTLL2细胞用含100U/ml商业化重组IL2细胞因子的1640完全培养基培养；
(2)在实验时，用不含IL2的完全培养基洗2～3遍，稀释细胞至2×10 4/ml；
(3)用不含IL2完全培养基培养稀释样品Fc-G 4S-IL-15-Rα，Fc-G 4S-Rα-IL-15，Fc-(G 4S) 3-IL-15-Rα，αPD-1Fc-(G 4S) 3-IL-15-Rα，αPD-1 Fc-(G 4S) 5-IL-15-Rα。起始浓度为5μg/ml，5倍稀释，做10个稀释梯度；
(4)96孔细胞培养板中加入100μl细胞悬液和100μl样品，用枪头吹打混匀；
(5)培养72小时后，加入20μl CCK8，继续培养3小时后，酶标仪检测450nM和630nM两个波长的OD值。
检测结果如图2所示，表明：
(1)Fc-G 4S-IL-15-Rα与Fc-G 4S-Rα-IL-15的生物学活性相差10000倍左右，Fc-G 4S-Rα-IL-15的生物学活性显著升高， 表明两种形式的IL-15融合蛋白的生物学活性与IL-15及Rα的相对位置有关；
(2)Fc-(G 4S) 3-IL-15-Rα与αPD-1 Fc-(G 4S) 3-IL-15-Rα相比，生物学活性没有改变，表明在PD-1低表达的细胞上，αPD-1抗体的引入不改变IL-15的生物学活性；
(3)αPD-1 Fc-(G 4S) 3-IL-15-Rα与αPD-1 Fc-(G 4S) 5-IL-15-Rα相比，生物学活性依次递增， 说明融合蛋 白型IL-15的生物学活性与连接片段1的长度也具有相关性。
2、IL-15融合蛋白结合能力检测
通过CTLL2报告细胞系检测Fc-G 4S-IL-15-Rα、Fc-G 4S-Rα-IL-15及Fc-(G 4S) 3-IL-15-Rα与IL-15受体结合的能力，结果如图3所示，表明：IL-15与其受体的结合能力与起生物学活性呈正相关，说明Fc-G 4S-IL-15-Rα的低生物学活性是由于与其受体的亲和力低导致的。
通过MC38荷瘤小鼠肿瘤分选的CD8+T细胞检测Fc-G 4S-IL-15-Rα、Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα及aEGFR Fab Fc-(G 4S) 3-IL-15-Rα与CD8+T细胞的结合能力，结果如图4所示，说明PD-1 Fab抗体会介导融合蛋白与CD8+T细胞结合。
3、在小鼠肿瘤模型中检测IL-15及αPD-1-IL-15融合蛋白的肿瘤治疗效果和毒副作用
3.1、MC38肿瘤模型(day 7treatment)
方法1(给药剂量15μg)：
5×10 5个MC38肿瘤细胞皮下接种到C57小鼠左下侧。待肿瘤长至40mm 3，腹腔给药15μg Fc-G 4S-IL-15-Rα或Fc-G 4S-Rα-IL-15，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)，记录小鼠的体重变化和生存曲线。
结果如图5、6、7、8所示，可见，
(1)通过腹腔给药的Fc-G 4S-IL-15-Rα，肿瘤没有得到控制，体重没有改变，小鼠全部存活。说明Fc-G 4S-IL-15-Rα对抗肿瘤没有明显效果，也没有任何毒副作用。
(2)通过腹腔给药的Fc-G 4S-Rα-IL-15，肿瘤得到明显的控制，但是体重下降比较明显，有40％的小鼠死亡，并且外周血淋巴细胞中CD8+T细胞、B220+细胞，NK细胞、NKT细胞扩增。说明Fc-G 4S-Rα-IL-15对抗肿瘤有显著效果，但是毒副作用很明显。
方法2(给药剂量15μg)：
5×10 5个MC38肿瘤细胞皮下接种到C57小鼠左下侧。待肿瘤长至40mm 3，腹腔给药Fc-G 4S-IL-15-Rα(15μg)或αPD-1 Fab Fc-G 4S-IL-15-Rα(等摩尔质量给药：30μg)，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)。
结果如图9所示：可见，通过腹腔给药的Fc-G4S-IL-15-Rα，肿瘤没有得到控制，而腹腔给药的αPD-1 Fab Fc-G 4S-IL-15-Rα治疗组可以显著控制肿瘤生长。说明αPD-1 Fab Fc-G 4S-IL-15-Rα可以通过αPD-1抗体的作用提升治疗效果。
方法3(给药剂量15μg)：
5×10 5个MC38肿瘤细胞皮下接种到C57小鼠左下侧。待肿瘤长至40mm 3，腹腔给药αPD-1 Fab 15μg+Fc-G 4S-IL-15-Rα15ug或αPD-1 Fab Fc-G 4S-IL-15-Rα(等摩尔质量给药：30μg)，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)。
结果如图10所示：可见，通过腹腔给药的αPD-1 Fab+Fc-G 4S-IL-15-Rα，肿瘤没有得到控制，而腹腔给药的αPD-1 Fab Fc-G 4S-IL-15-Rα治疗组可以显著控制肿瘤生长。说明αPD-1 Fab Fc-G 4S-IL-15-Rα的抗肿瘤效果依赖αPD-1 Fab与IL-15融合后的共同作用。
方法4(给药剂量30μg)：
5×10 5个MC38肿瘤细胞皮下接种到C57小鼠左下侧。待肿瘤长至40mm 3，腹腔给药αPD-1 Fab Fc-G 4S-Rα-IL-15或αPD-1 Fab Fc-G 4S-IL-15-Rα，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)，记录小鼠的体重变化。
结果如图11、12、13所示：可见，
(1)通过腹腔给药的αPD-1 Fab Fc-G 4S-IL-15-Rα，肿瘤得到很好控制，体重没有改变，外周血淋巴细胞扩增不明显。说明αPD-1 Fab Fc-G 4S-IL-15-Rα对抗肿瘤有明显效果，且没有任何毒副作用。
(2)通过腹腔给药的αPD-1 Fab Fc-G 4S-Rα-IL-15，肿瘤得到很好控制，但是体重下降比较明显，并且外周血淋巴细胞中CD8+T细胞、B220+细胞，NK细胞、NKT细胞扩增。说明αPD-1 Fab Fc-G 4S-Rα-IL-15对抗肿瘤有显著效果，但是毒副作用很明显。
方法5(给药剂量30μg)：
5×10 5个MC38肿瘤细胞皮下接种到C57小鼠左下侧。待肿瘤长至40mm 3，腹腔给药αPD-1 Fab Fc-G 4S-IL-15-Rα、αPD-1 Fab mFc-G 4S-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab mFc-(G 4S) 3-IL-15-Rα及aEGFR Fab Fc-G 4S-IL-15-Rα，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)，记录小鼠的体重变化。
结果如图14、15所示：可见，
(1)通过腹腔给药的αPD-1 Fab Fc-G 4S-IL-15-Rα、αPD-1 Fab mFc-G 4S-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab mFc-(G 4S) 3-IL-15-Rα，肿瘤都得到很好控制，体重没有明显改变。说明αPD-1  Fab Fc-G 4S-IL-15-Rα、αPD-1 Fab mFc-G 4S-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab mFc-(G 4S) 3-IL-15-Rα对抗肿瘤有明显效果，且没有任何毒副作用。
(2)通过腹腔给药的aEGFR Fab Fc-G 4S-IL-15-Rα，肿瘤没有得到控制，没有体重下降。说明aEGFR抗体无法替代αPD-1抗体在抗肿瘤中发挥的作用。
3.2、MC38肿瘤模型(day 14treatment)
方法1(给药剂量30ug)：
5×10 5MC38细胞皮下接种到C57小鼠左下侧。待肿瘤长至100mm 3，腹腔给药αPD-1 Fab Fc-G 4S-IL-15-Rα、αPD-1 Fab mFc-G 4S-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab mFc-(G 4S) 3-IL-15-Rα及aEGFR Fab Fc-(G 4S) 3-IL-15-Rα，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)。
结果如图16所示：可见，
(1)通过腹腔给药的αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab mFc-(G 4S) 3-IL-15-Rα，肿瘤都得到很好控制。说明当连接片段1为(G 4S) 3时，融合蛋白有更好的抗肿瘤效果。
(2)通过腹腔给药的aEGFR Fab Fc-(G 4S) 3-IL-15-Rα，肿瘤没有得到控制，没有体重下降。说明aEGFR抗体无法替代αPD-1抗体在抗肿瘤中发挥的作用。
(3)通过腹腔给药的αPD-1 Fab Fc-G 4S-IL-15-Rα与αPD-1 Fab mFc-G 4S-IL-15-Rα抗肿瘤效果没有显著差异，说明mFc在抗肿瘤过程中发挥的作用不明显。
方法2(给药剂量30ug)：
5×10 5MC38细胞皮下接种到C57小鼠左下侧。待肿瘤长至100mm 3，腹腔给药αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα，给药3次，间隔2天，对照组采用同样方式给予PBS。测量肿瘤的体积(体积＝长×宽×高/2)，记录小鼠的体重变化。
结果如图17、18、19所示：可见，
(1)通过腹腔给药的αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα、αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα，肿瘤都得到很好控制，且没有明显差别。说明当连接片段1为(G 4S) 5时，相较于(G 4S) 3，融合蛋白没有体现出更好的抗肿瘤效果。
(2)通过腹腔给药的αPD-1 Fab Fc-(G 4S) 5-IL-15-Rα，小鼠有轻微体重下降，外周血淋巴细胞扩增明显。说明当连接片段1为(G 4S) 5时，融合蛋白显现出了外周的毒副作用。
3.3、B16肿瘤模型(day 7treatment)
(1)3×10 5B16细胞皮下接种到C57小鼠左下侧；
(2)待肿瘤长至30mm 3，瘤内给药αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα，给药3次，每次30ug，间隔2天，对照组采用同样方式给予PBS；
(3)测量肿瘤的体积(体积＝长×宽×高/2)。
结果如图20所示：可见，
(1)通过瘤内给药的αPD-1 Fab Fc-(G 4S) 3-IL-15-Rα，肿瘤可以得到控制。说明融合蛋白对B16有一定的抗肿瘤效果。
3.4、CD34 +人源化小鼠A549肺癌肿瘤模型
(1)2×10 6A549细胞皮下接种到CD34 +人源化小鼠左下侧；
(2)待肿瘤长至30mm 3，系统性给药anti-human PD-1 Fab Fc-G 4S-human IL-15-Rα或anti-human PD-1 Fab Fc-(G 4S) 3-human IL-15-Rα，在第10天给药10ug，第17天及第20天给药20ug，对照组采用同样方式给予PBS；
(3)测量肿瘤的体积(体积＝长×宽×高/2)。
结果如图21所示：可见，
(1)通过腹腔给药的anti-human PD-1 Fab Fc-G 4S-human IL-15-Rα或anti-human PD-1 Fab Fc-(G 4S) 3-human IL-15-Rα，肿瘤可以得到控制。说明融合蛋白对人源化小鼠上的人来源A549肺癌肿瘤有抗肿瘤效果。
最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，不用做本发明保护范围的限定。

Claims (10)

  1. 一种融合蛋白，所述的融合蛋白包含如下嵌段
    (1)第一结构单元：白介素15(IL-15)受体的α亚基sushi结构域；
    (2)第二结构单元：IL-15；
    (3)位于融合蛋白N端的第三结构单元：抗体Fc或突变的Fc片段；
    (4)连接所述第一、第二结构单元的连接片段2；
    当所述的融合蛋白的C端是第一结构单元时，连接片段1连接所述第二、第三结构单元；
    当所述的融合蛋白的C端是第二结构单元时，连接片段1连接所述第一、第三结构单元；
    所述连接片段1的氨基酸序列为GGGGS的整数倍重复，以(G 4S) n表示，优选的，所述的整数n为1～7的任意整数；更优选的，所述的整数n为1～5；最优选的，所述的整数n为3。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述的融合蛋白还包含如下嵌段：
    (5)连接于所述第三结构单元N端的第四结构单元：治疗型抗体的Fab嵌段；
    所述的治疗型抗体包括但不限于：抗PD1/PD-L1抗体、Her2抗体、抗CD20抗体、抗CD19抗体、抗RANKL抗体、抗VEGFR抗体、抗EGFR抗体。
    优选的，所述的治疗型抗体的Fab嵌段为anti-PD-1 Fab(PD1抗体的Fab片段)。
  3. 根据权利要求2所述的融合蛋白，其特征在于，
    所述的anti-PD-1 Fab包括重链(可变区+恒定区)和轻链(可变区+恒定区)，其中重链位于所述的融合蛋白N端。
  4. 根据权利要求1-3任一所述的融合蛋白，其特征在于，
    所述的IL-15为鼠源或人源的IL-15，其氨基酸序列分别如Seq ID No.1、Seq ID No.15所示；
    所述的IL-15受体的α亚基sushi结构域为鼠源或人源IL-15受体的α亚基sushi结构域，优选的，所述鼠源或人源IL-15受体的α亚基sushi结构域的氨基酸序列如Seq ID No.3、Seq ID No.17所示；
    所述的Fc或突变的Fc片段氨基酸序列分别如Seq ID No.2、Seq ID No.16所示；
    所述的连接片段2的氨基酸序列如Seq ID No.6所示。
  5. 根据权利要求3所述的融合蛋白，其特征在于，
    所述的anti-PD-1 Fab为鼠源或人源anti-PD-1 Fab，优选的，
    所述的anti-PD-1 Fab的轻链氨基酸序列如Seq ID No.4、Seq ID No.18、Seq ID No.19所示；
    所述的anti-PD-1 Fab的重链氨基酸序列如Seq ID No.5、Seq ID No.20、Seq ID No.21所示。
  6. 由权利要求1-5任一所述的融合蛋白构成的同源二聚体，所述的同源二聚体的单体之间通过第三结构单元的二聚化相互连接构成。
  7. 根据权利要求6所述的同源二聚体，其特征在于，所述的同源二聚体为：
    (1)同源二聚体1(Fc-G 4S-Rα-IL-15)：
    单体融合蛋白自N端到C端依次为：人源IgG1-Fc、连接片段1(G 4S)、IL-15受体的α亚基sushi结构域、连接片段2、IL-15；优选的，同源二聚体1的氨基酸序列结构如SEQ ID No.7所示；
    (2)同源二聚体2(Fc-G 4S-IL-15-Rα)：
    单体融合蛋白自N端到C端依次为：人源IgG1-Fc、连接片段1(G 4S)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域；优选的，同源二聚体2的氨基酸序列结构如SEQ ID No.8所示；
    (3)同源二聚体3(anti-PD-1 Fab Fc-G 4S-Rα-IL-15)：
    单体融合蛋白自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1(G 4S)、IL-15受体的α亚基sushi结构域、连接片段2、IL-15；优选的，同源二聚体3的氨基酸序列结构如SEQ ID No.9所示；
    (4)同源二聚体4(anti-PD-1 Fab Fc-G 4S-IL-15-Rα)：
    单体融合蛋白自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1(G 4S)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域，优选的，同源二聚体4的氨基酸序列结构如SEQ ID No.10所示；
    (5)同源二聚体5(Fc-(G 4S) 3-IL-15-Rα)：
    组成的单体自N端到C端依次为：人源IgG1-Fc连接成的融合蛋白、连接片段1((G 4S) 3)、人源或鼠源的IL-15、连接片段2、IL-15受体的α亚基sushi结构域，优选的，同源二聚体5的氨基酸序列结构如SEQ ID No.11所示；
    (6)同源二聚体6(anti-PD-1 Fab Fc-(G 4S) 3-IL-15-Rα)：
    组成的单体自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1((G 4S) 3)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域，优选的，同源二聚体6的氨基酸序列结构如SEQ ID No.12所示；
    (7)同源二聚体7(Fc-(G 4S) 5-IL-15-Rα)：
    组成的单体自N端到C端依次为：人源IgG1-Fc连接成的融合蛋白、连接片段1((G 4S) 5)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域，优选的，同源二聚体7的氨基酸序列结构如SEQ ID No.13所示；
    (8)同源二聚体8(anti-PD-1 Fab Fc-(G 4S) 5-IL-15-Rα)：
    组成的单体自N端到C端依次为：anti-PD-1 Fab、人源IgG1-Fc、连接片段1((G 4S) 5)、IL-15、连接片段2、IL-15受体的α亚基sushi结构域，优选的，同源二聚体8的氨基酸序列结构如SEQ ID No.14所示。
  8. 编码权利要求1-5任一所述的融合蛋白的核苷酸片段。
  9. 权利要求1-5任一所述的融合蛋白或权利要求6-7任一所述的同源二聚体在制备药物中的应用；优选的，所述的药物为抗肿瘤药物，最优选的，所述药物为抗B细胞性淋巴瘤、抗结直肠癌、抗黑色素瘤或抗肺癌的药物。
  10. 权利要求1-5任一所述的融合蛋白的制备方法，所述的制备方法包括如下步骤：
    (1)构建包含所述编码所述融合蛋白的编码基因的表达载体，优选的，所述的表达载体是pEE12.4表达载体；
    (2)通过瞬时转染宿主细胞的方法构建包含所述表达载体的宿主细胞，优选的，所述的宿主细胞是293F细胞；
    (3)培养所述宿主细胞并收集细胞上清；
    (4)通过Protein A的亲和层析柱纯化蛋白纯化所述融合蛋白。
PCT/CN2022/132019 2021-11-15 2022-11-15 一种以白介素15为活性成份的融合蛋白构建体及其应用 Ceased WO2023083379A1 (zh)

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