WO2017000913A1 - 用于肿瘤靶向治疗的白细胞介素15融合蛋白 - Google Patents

用于肿瘤靶向治疗的白细胞介素15融合蛋白 Download PDF

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WO2017000913A1
WO2017000913A1 PCT/CN2016/088158 CN2016088158W WO2017000913A1 WO 2017000913 A1 WO2017000913 A1 WO 2017000913A1 CN 2016088158 W CN2016088158 W CN 2016088158W WO 2017000913 A1 WO2017000913 A1 WO 2017000913A1
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tumor
fusion protein
sequence
polypeptide
seq
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PCT/CN2016/088158
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French (fr)
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王�忠
李庆
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马卫红
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Priority to AU2016288484A priority Critical patent/AU2016288484B2/en
Priority to KR1020187003435A priority patent/KR102211177B1/ko
Priority to US15/747,029 priority patent/US10611812B2/en
Priority to EP16817277.3A priority patent/EP3318579A4/en
Priority to CA2993891A priority patent/CA2993891C/en
Publication of WO2017000913A1 publication Critical patent/WO2017000913A1/zh
Priority to US16/798,269 priority patent/US11236140B2/en
Priority to US17/588,880 priority patent/US20220332779A1/en

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    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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Definitions

  • the invention relates to the application of the cytokine interleukin-15 in tumor targeted therapy, in particular to the anti-tumor effect of the interleukin-15 fusion protein.
  • Cytokines play an important role in regulating the body's immune system, including anti-tumor immune responses. Some cytokines have been shown to have anti-tumor potential. Interleukin-15 (IL-15) has been extensively studied as a promising anti-tumor drug candidate. IL-15 and IL-2 share the ⁇ and ⁇ chains of the receptor (IL-2/15 ⁇ ), but bind to different ⁇ receptor chains (IL-2R ⁇ /IL-15R ⁇ ). The role of IL-15 is mainly through the trans-presentation model, in which IL-15 binds to IL-15R ⁇ expressed on the surface of antigen-presenting cells, and then binds to the IL-15 ⁇ complex adjacent to the surface of effector cells, ultimately activating effector cells.
  • IL-15 Similar to IL-2, IL-15 promotes proliferation of T cells and natural killer (NK) cells, promotes cytotoxic T cell expansion, and activates NK cells. At the same time, unlike IL-2, IL-15 does not cause activation-induced cell death, nor does it participate in the maintenance of regulatory T cells. Therefore, IL-15 is ranked first in the list of the most promising cancer immunotherapeutics listed by the National Cancer Research Institute.
  • IL-15 still has some shortcomings. First, studies have shown that only high doses of IL-15 can achieve antitumor effects under in vivo conditions. Second, another deficiency of IL-15 is the short half-life. Finally, the role of IL-15 is systemic, not tumor-specific. Cytotoxic T cells or NK cells will be amplified under IL-15 stimulation, but this effect is not limited to the tumor site, but is a systemic distribution. Since extensive activation of the immune system is often fatal, a more desirable therapeutic agent should target the tumor site without affecting normal tissue.
  • the invention provides a tumor targeting fusion protein, which can improve the anti-tumor effect of IL-15 on the one hand, and overcome the short half-life problem of IL-15 on the other hand, and can target the tumor site and target the tumor in a targeted manner. cell.
  • the tumor targeting fusion protein comprises at least (i) an IL-15 polypeptide, an IL-15 polypeptide variant, or a functional fragment thereof, (ii) an IL-15Ra polypeptide, an IL-15Ra polypeptide variant, or a function thereof. a fragment, (iii) an Fc domain, an Fc variant, or a functional fragment thereof, and (iv) an RGD polypeptide or variant thereof.
  • the components of the fusion protein are arranged in the order RGD-Fc-IL-15-IL-15Ra.
  • the Fc domain consists of CH2 and CH3 of human IgG1, which has the sequence shown in SEQ ID NO. 1, or a substitution, deletion or addition of the sequence shown in SEQ ID NO. Or a plurality of amino acid-derived sequences having the same function as the Fc domain.
  • IL-15Ra consists of the IL-15R ⁇ sushi domain and the subsequent 12 amino acids of exon 3 A derivative sequence having the sequence of SEQ ID NO. 2, or the sequence of SEQ ID NO. 2, which has been substituted, deleted or added with one or more amino acids and has the same function as the IL-15RA domain.
  • IL-15 has the sequence set forth in SEQ ID NO. 3, or the sequence shown in SEQ ID NO. 3 is substituted, deleted or added with one or more amino acids and has the same function as the IL-15 domain. Derived sequence.
  • the RGD polypeptide has the sequence set forth in SEQ ID NO. 4, or a derivative sequence in which the sequence set forth in SEQ ID NO. 4 has been substituted, deleted or added with one or more amino acids and has the same function as the RGD polypeptide.
  • the amino acid sequence of the tumor targeting fusion protein is selected from the group consisting of: (a) the sequence set forth in SEQ ID NO. 5; (b) the nucleic acid sequence encoded by SEQ ID NO. An amino acid sequence; (c) an amino acid sequence encoded by the degenerate sequence of the sequence set forth in SEQ ID NO. 6; and (d) a substitution, deletion or addition of one or more amino acids in the sequence set forth in SEQ ID NO. A derivative sequence having the same function as the fusion protein.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a tumor-targeting fusion protein of the invention and a pharmaceutically acceptable additive comprising a carrier, a stabilizer and/or an excipient.
  • Another aspect of the invention provides a pharmaceutical composition comprising the tumor targeting fusion protein of the invention and another anticancer agent.
  • the tumor is an integrin positive tumor, in particular an ⁇ V ⁇ 3 integrin positive tumor, such as melanoma, ovarian cancer, and the like.
  • the tumor is a progressive tumor, an advanced tumor, a tumor with a high tumor burden/burden, or a metastatic tumor.
  • Another aspect of the invention provides a nucleic acid sequence encoding a tumor-targeting fusion protein of the invention, an expression vector comprising the nucleic acid sequence, or a host transformed or transfected with the expression vector.
  • kits comprising the tumor targeting fusion protein of the present invention, a nucleic acid sequence encoding the tumor targeting fusion protein of the present invention, an expression vector comprising the nucleic acid sequence, or a use A host transformed or transfected with an expression vector.
  • the invention provides a tumor targeting fusion protein, which can improve the anti-tumor effect of IL-15 on the one hand, and overcome the short half-life problem of IL-15 on the other hand, and can target the tumor site and target the tumor in a targeted manner. cell.
  • this tumor-targeting protein can be efficiently expressed and purified, and has high antitumor activity, and has the potential to become an immuno-antitumor therapy drug.
  • Figure 1 is a schematic diagram showing the structure of a protein of an embodiment (PFC-1) of a tumor-targeting fusion protein of the present invention.
  • SP signal peptide
  • RGD arginine, glycine-aspartate peptide motif
  • Fc CH2 and CH3 of human IgG1
  • IL-15Ra IL-15R ⁇ sushi domain + subsequent 12 amino acids of exon 3
  • L1 SS
  • L2 G 4 S
  • L4 SG 2 SG 4 SG 3 SG 4 SLQ.
  • Figure 2 shows the fusion protein PFC-1 under non-reducing (NR) or reducing (R) conditions by 10% SDS-PAGE electrophoresis and stained with Coomassie blue.
  • Figure 3 shows the stimulation of the proliferation of mo7e cells by PFC-1 and rhIL-15.
  • the PFC-1 molar concentration is calculated as the molecular weight of the monomer.
  • the experimental results are shown as mean plus or minus standard deviation, and the experimental results represent at least three independent experiments.
  • Figure 4 shows the stimulation of the proliferation of CTLL-2 cells by PFC-1 and rhIL-15.
  • the PFC-1 molar concentration is calculated as the molecular weight of the monomer.
  • the experimental results are shown as mean plus or minus standard deviation, and the experimental results represent at least three independent experiments.
  • Figure 5 shows the in vitro proliferation stimulation of PBMC by the fusion protein PFC-1.
  • PBMC cells were labeled with CFSE and co-cultured with various concentrations of rhIL-15 or PFC-1 for 6 days, and then the degree of cell proliferation was assessed by flow cytometry.
  • Figure 6 shows the binding effect of the fusion protein PFC-1 on HUVEC cell lines by flow cytometry.
  • the experimental results represent at least 3 independent experiments.
  • Figure 7 shows the binding of the fusion protein PFC-1 to SKOV-3 tumor cell lines by flow cytometry.
  • the experimental results represent at least 3 independent experiments.
  • Figure 8 shows the binding of the fusion protein PFC-1 to the LS74T tumor cell line by flow cytometry.
  • the experimental results represent at least 3 independent experiments.
  • Figure 9 shows the results of co-localization of the fusion protein PFC-1 on HUVEC cells and SKOV3 tumor cell models with anti-human CD51/61 ( ⁇ V ⁇ 3 integrin) antibody by laser confocal microscopy.
  • Figure 10 shows the antitumor effect of the fusion protein PFC-1 in mice.
  • Mice with B16F10 mouse melanoma tumors subcutaneously in the back were treated with 5 or 20 ⁇ g of PFC-1 or 200 ul of PBS every 3 days after tumors were grown to a volume of 100 mm 3 .
  • a total of 2 drug injections were received and the tumor volume was measured accordingly.
  • the experimental results were the mean plus or minus standard deviation, with 5 to 8 mice per group.
  • the experimental data were statistically significant by T test, ** represents p ⁇ 0.01.
  • Figure 11 shows the antitumor effect of the fusion protein PFC-1 in mice.
  • Mice with B16F10 mouse melanoma tumors subcutaneously in the back were treated with 10 ⁇ g of PFC-1 for tail vein injection or 200 ul of PBS on the date of development after the tumor grew to 1000 mm 3 volume. A total of 3 drug injections were received and tumor volume was measured daily.
  • the experimental results were the mean plus or minus standard deviation, with 5 to 8 mice per group.
  • the experimental data were statistically significant by T test, ** represents p ⁇ 0.01.
  • Figure 12 shows flow cytometry analysis of the phenotypic changes of CD8+ T cells in vivo in the experimental mice of Figure 10 after receiving PFC-1 treatment.
  • the experimental results were the mean plus or minus standard deviation, with 5 to 8 mice per group.
  • the experimental data were statistically significant by T test, ** for p ⁇ 0.01 and *** for p ⁇ 0.005.
  • Figure 13 shows flow cytometry analysis of NK cell phenotypic changes in vivo after treatment with PFC-1 in the experimental mice of Figure 10.
  • the experimental results were the mean plus or minus standard deviation, with 5 to 8 mice per group.
  • the experimental data were statistically significant by T test, ** for p ⁇ 0.01 and *** for p ⁇ 0.005.
  • Figure 14 shows flow cytometry analysis of the CD44 antigen phenotype changes on CD8+ T cells and NK cells in vivo in the experimental mice of Figure 10 after treatment with PFC-1 to analyze the cell activation ratio.
  • the experimental results were the mean plus or minus standard deviation, with 5 to 8 mice per group.
  • the experimental data were statistically significant by T test, ** for p ⁇ 0.01 and *** for p ⁇ 0.005.
  • Figure 15 shows the results of an anti-tumor nausea migration experiment of the fusion protein PFC-1 in C57BL/6 mice.
  • C57BL/6 mice were injected with 5*10 5 B16F10 melanoma tumor cells via the tail vein on day 0 and received 10 ⁇ g of PFC-1 or 200 ul of equal volume of PBS via tail vein injection on the same day.
  • the mice were euthanized and the lungs were removed and the condition and number of lung tumor sinus were examined under a binocular microscope.
  • the above image is a photograph of a representative mouse lung. Mock, tumor cells were not injected into the lungs of the mice; vehicle, the control group receiving tumor cell injection; PFC-1, receiving the PFC-1 injection group.
  • the experimental results were the mean plus or minus standard deviation, with 5 mice per group. Statistical data were statistically significant by T test.
  • fusion protein protein
  • PFC-1 protein
  • PFC-1 recombinant fusion protein or "fusion molecule” are used interchangeably and refer to a biologically active polypeptide, usually by recombination, chemistry or other suitable Methods Protein or peptide sequences that are covalently linked (ie, fused) together. Fusion proteins can be fused to other peptide or protein sequences at one or more sites via a linker sequence. Alternatively, a linker sequence can be used to assist in the construction of a fusion molecule.
  • the fusion protein may be present as a monomer or a multimer (e.g., a dimer).
  • a "fusion protein” includes at least (i) an IL-15 polypeptide, an IL-15 polypeptide variant, or a functional fragment thereof, (ii) an IL-15Ra polypeptide, an IL-15Ra polypeptide variant, or a function thereof. a fragment, (iii) an Fc domain, an Fc variant, or a functional fragment thereof, and (iv) an RGD polypeptide or variant thereof.
  • component (i) (ii) together constitute an effector module or an effector molecule which is capable of causing activation of effector cells (cytotoxic T cells, NK cells).
  • Component (iii) is used to extend the circulating half-life of IL-15.
  • Component (iv) is a targeting molecule that acts on the receptor molecule expressed on the surface of the tumor cell with high affinity and specificity, thereby enriching other parts of the fusion protein in the tumor foci, thereby killing the tumor cell.
  • the individual components of the fusion protein are arranged in a rational order such that the fusion protein as a whole achieves the intended purpose of the present invention.
  • the order of the components in the fusion protein is the RGD polypeptide-Fc domain-IL-15 polypeptide-IL-15Ra polypeptide.
  • the order of the components in the fusion protein is the RGD polypeptide-Fc domain-IL-15Ra polypeptide-IL-15 polypeptide.
  • the fusion protein The order of the components in the RGD polypeptide-IL-15Ra polypeptide-IL-15 polypeptide-Fc domain.
  • the order of the components in the fusion protein is the RGD polypeptide-IL-15 polypeptide-IL-15Ra polypeptide-Fc domain.
  • Fc domain or “Fc fragment” refers to the "crystallizable fragment” region of an immunoglobulin heavy chain.
  • an Fc domain can interact with another Fc domain to form a dimeric complex.
  • the Fc domain may bind to a protein of a cell surface receptor (Fc receptor) and/or the complement system, or may be modified to attenuate or enhance such binding activity.
  • the Fc domain can be derived from IgG, IgA, IgD, IgM or IgE antibodies and produces immune functions such as opsonization, cell lysis, mast cell degranulation and other Fc receptor dependent processes.
  • the IgG-type immunoglobulin is one of the most abundant proteins in human blood and has a circulating half-life of up to 21 days. It has been reported that the Fc region of IgG binds to the domain of another protein.
  • the prototype fusion protein is a homodimeric protein linked via a cysteine residue in the Fc hinge region of IgG, forming a molecule similar to an IgG molecule without a heavy chain variable region, a CH1 domain, and a light chain.
  • the dimeric nature of a fusion protein comprising an Fc domain may facilitate binding to other molecules (eg, bivalent or bispecific binding). Due to structural homology, Fc fusion proteins show in vivo pharmacokinetic profiles comparable to human IgGs with similar isoforms.
  • the present invention ligates the IL-15/IL-15R ⁇ complex to the Fc portion of the human IgG protein heavy chain.
  • the original immunoglobulin source of native Fc is preferably a human immunoglobulin, preferably IgGl and IgG2.
  • the Fc domain is preferably composed of CH2 and CH3 of human IgG1.
  • Fc variant refers to a molecule or sequence that is modified by a native Fc but still binds to an Fc receptor.
  • Fc variant includes a molecule or sequence that is humanized by a non-human native Fc.
  • certain sites of the native Fc can be removed as certain structural features or biological activities are not required for the fusion molecules of the invention.
  • an "Fc variant” includes a molecule or sequence that lacks one or more native Fc sites or residues.
  • An "Fc domain” includes a molecule or sequence of a native Fc and Fc variant as described above, comprising a monomeric or multimeric form of a molecule, which may be obtained by decomposition of an intact antibody, or by recombinant expression or by other means. get.
  • the arginine-glycine-aspartate (Arg-Gly-Asp, RGD) polypeptide was discovered as a cell adhesion sequence by Pierschbacher and Rouslahti in FN in 1984. They found that the RGD polypeptide enables integrin (intergrin) ⁇ 5 ⁇ 1. It elutes from the affinity column and can adhere to the cells after being fixed to the matrix material. Since then, people have discovered many sugars in the extracellular matrix. Proteins such as LM, collagen, fibrinogen (Fb) and the like all contain highly conserved RGD polypeptides and demonstrate that RGD polypeptides play an important role in mediating the interaction of cells with cells, cells and extracellular matrix proteins.
  • Binding of the RGD polypeptide to cells is also a binding to cell surface integrins. Integrin was discovered in the mid-1990s as a family of cell surface receptors that depend on Ca 2+ . Each integrins consists of two subunits: the alpha subunit and the beta subunit. 18 alpha subunits have been discovered to date. And 8 kinds of ⁇ subunits, which constitute 24 integrin. Integrin receptors that recognize and bind to RGD polypeptides include: ⁇ 3 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ IIb ⁇ 3, ⁇ 5 ⁇ 1, ⁇ v ⁇ 1, ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ v ⁇ 6, ⁇ v ⁇ 8, etc., and particularly have strong selectivity and affinity for ⁇ v ⁇ 3 integrin. ⁇ v ⁇ 3 integrin is highly overexpressed in a variety of tumor cells and tumor-associated angiogenic endothelial cells.
  • the invention combines or fuses a drug with an RGD polypeptide, and uses the RGD polypeptide to guide the fusion of the protein macromolecule drug at the tumor tissue site, and increases the local concentration to enhance the tumor killing effect and limit systemic toxicity.
  • the biological activity of the simple RGD tripeptide is very low, and the 4th amino acid linked to the RGD tripeptide has a great influence on its activity, and the 5th amino acid linked to the RGD tripeptide also plays an important role in the specificity of the peptide binding. .
  • the RGD tripeptide and the GRGD tetrapeptide have no significant response to cell adhesion, but the addition of amino acids at the C-terminus will affect it.
  • Adhesion of cells such as a serine residue after aspartic acid in RGD, will enhance its cell adhesion, and substitution of a left-handed residue with a right-handed residue will result in decreased cell adhesion.
  • the RGD polypeptide sequence is ACDCRGDCFCG, ie, Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly, which comprises an RGD motif at the 5th to 7th amino acid positions.
  • an RGD variant refers to an amino acid sequence having at least one amino acid substitution, deletion or insertion with the RGD polypeptide sequence of the present invention, but is capable of maintaining the integrin receptor binding function of RGD.
  • RGD variants include GRGD, GRGDSPC, GRGDDSY, EPRGDNYR, and the like.
  • the fusion protein of the present invention further comprises a linker sequence between the components, usually a short peptide consisting of 4-20 amino acids. These linker sequences allow for proper positioning of the components to achieve functional activity of the components.
  • an IL-15 polypeptide in a fusion protein of the invention, is covalently linked to an IL-15Ra polypeptide by a linker sequence such that the IL-15 and IL-15a domains are capable of interacting to form a complex.
  • the IL-15 and IL-15Ra domains are positioned such that they interact with immune cells to initiate or inhibit an immune response, or to inhibit or stimulate cell development.
  • the IL-15 or IL-15Ra domain is covalently linked to the immunoglobulin Fc domain by a linker sequence.
  • the linker sequence should allow the Fc domain, IL-15 or IL-15Ra domain to be reasonably positioned to exert the functional activity of each domain.
  • the Fc domain is efficiently positioned to enable formation of a suitable fusion protein complex and to have an extended half-life of the fusion protein complex in vivo.
  • the RGD polypeptide in a fusion protein of the invention, is covalently bound to the Fc domain by a linker sequence.
  • the linker sequence should be such that the RGD polypeptide and Fc domain are reasonably positioned to exert the functional activity of each domain.
  • the RGD polypeptide is efficiently positioned to bind to the integrin molecule on the surface of the tumor cell with high affinity and specificity.
  • the linker sequence comprises from 2 to 20 amino acid sequences, more preferably from 5 to 20 amino acids.
  • the linker sequence is preferably a flexible linker sequence such that it does not limit the effector molecule or polypeptide in a single undesired conformation.
  • a linker sequence can be used, for example, to separate the recognition site from the fusion protein.
  • the linker sequence is preferably composed primarily of amino acids having small side chains, such as glycine, alanine and serine, to provide said flexibility.
  • the amino acid in a ratio of about 80 or more of the linker sequence is a glycine, alanine or serine residue, in particular a glycine and a serine residue.
  • linker sequence is GGGGS (G 4 S), Gly Gly Gly Gly Ser, for example for ligation of the RGD polypeptide and Fc domain of the invention, and the Fc domain and IL-15Ra polypeptide; or SG 2 SG 4 SG 3 SG 4 SLQ, Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln, for example, for ligation of the IL-15 and IL-15Ra domains of the present invention.
  • Other different linker sequences can also be used, including a variety of flexible linker designs that have been successfully used to link different antibody variable regions.
  • the size and sequence composition of the linker sequence can be determined by conventional computer modeling and techniques.
  • polypeptide refers to a polymer of any length consisting essentially of any of the 20 natural amino acids.
  • protein or “protein” generally refers to a polymer with a relatively long amino acid length
  • peptide generally refers to a polymer with a small amino acid length, there is usually no clear boundary between the two terms, and often There is overlap on it.
  • Polypeptide variant generally refers to an amino acid sequence that has one or more amino acid substitutions, deletions or insertions as compared to a control polypeptide, yet is capable of maintaining the biological function of the polypeptide.
  • a "vector” is a nucleic acid molecule capable of autonomously replicating in a host cell and accepting foreign DNA.
  • the vector carries its own origin of replication and can be used to insert restriction endonuclease recognition sites for foreign DNA, as well as common selectable markers (such as genes encoding antibiotic resistance), often including expression
  • the recognition sequence of the inserted DNA such as a promoter and enhancer.
  • Common vectors include plasmid vectors and phage vectors.
  • Recombinant human IL-2 (AF-200-02) and granulocyte-macrophage colony stimulating factor (300-03) were purchased from Peprotech.
  • Recombinant human IL-15 (247-IL-105) was purchased from R&D Systems.
  • Anti-MsCD3e(145-2C11)-PerCP, anti-MsCD8a(53-6.7)-FITC, anti-MsNK1.1(PK136)-FITC, anti-MsCD44(IM7)-PE and anti-MsCD122(TM-Bta1)-PE were purchased from BD Pharmingen.
  • Anti-human CD51/61 ( ⁇ V ⁇ 3 integrin) purified monoclonal antibody was purchased from eBioscience.
  • Goat anti-human IgG (H+L)-AlexaFluor 488 and goat anti-mouse IgG (H+L)-AlexaFluor 488,647 were purchased from Invitrogen.
  • SKOV-3, CTLL-2 and Mo7e cells were obtained from the Shanghai Cell Bank. HUVEC cells were generously presented by Dr. Gao Huile from Sichuan University.
  • CTLL-2 cells were cultured in 20% fetal bovine serum (FBS) in RPMI 1640 medium with 30 ng/ml of IL-2 and 1% of non-essential amino acids.
  • Mo7e cells were cultured in 10% fetal bovine serum (FBS) in RPMI 1640 medium with 10 ng/ml GM-CSF and 1% non-essential amino acids.
  • FBS fetal bovine serum
  • RPMI 1640 medium fetal bovine serum
  • Both SKOV-3 and HUVEC cells were cultured in DMEM medium at 10% FBS.
  • PBMC peripheral blood mononuclear cells
  • the preparation of the fusion protein of the present invention can be prepared by the procedures and recombinant DNA techniques disclosed in the present invention, such as PCR, plasmid DNA extraction, restriction endonuclease digestion of DNA, DNA ligation, mRNA isolation, introduction of DNA. Suitable cells, transformation or transfection of host cells, culture of host cells, and the like. Further, the fusion protein can be isolated and purified using appropriate reagents and well-known methods such as electrophoresis, centrifugation, chromatography, and the like.
  • the gene sequence of the PFC-1 recombinant fusion protein was cloned into the pcDNA3.1 vector containing the signal peptide of the mouse kappa chain.
  • Expression vectors were transfected into 293 cells for expression using transient transfection techniques. After transiently transfected cells were cultured for 3 days in 100 ml of cell culture medium, the cells were harvested and purified by Protein-A agarose affinity purification to obtain a recombinant fusion protein.
  • the structure of the fusion protein PFC-1 is shown in Figure 1. It consists mainly of three modules: IL15/IL15Ra complex, Fc domain, and RGD polypeptide.
  • the modules are ligated by a GGGGS short peptide and a His-tag tag is added to the C-terminus of the protein ( Figure 1).
  • the DNA sequence was subcloned into the pcDNA3.1(+) vector, and the vector was introduced into HEK293 cells by transient transfection and expressed.
  • CTLL ⁇ 2 and Mo7e cells in logarithmic growth phase were harvested, washed twice with PBS, and cultured for 4 hours in assay medium (RPMI 1640 supplemented with 10% FBS, 1% NEAA) The medium) causes the experimental cells to reach a cytokine starvation state.
  • IL-15 and PFC-1 were diluted to a final concentration of 10 nM in assay medium and serially diluted.
  • CTLL-2 or Mo7e cells supplemented with IL-15 or PFC-1 were further cultured for 48 or 72 hours. Finally, the number of viable cells was determined using CCK-8 reagent and the cell proliferation ratio was calculated.
  • CTLL-2 is a mouse cytotoxic T lymphocyte line that is positive for IL-15R ⁇ chain and IL-15 ⁇ complex; while Mo7e is a human megakaryocyte leukemia cell line, which is a single positive expression of IL-15 ⁇ complex. Both of these cells rely on cytokines to stimulate cell proliferation and thus can be used to analyze the cytokine function of the IL-15 complex of the fusion protein PFC-1. Similar to the effect of IL-15, PFC-1 significantly stimulated the proliferation of Mo7e and CTLL-2 cell lines in vitro ( Figures 3 and 4), indicating that the purified PFC-1 fusion protein has cytokine activity.
  • rhIL-15 showed slightly higher cytokine activity than PFC-1, but as the concentration of cytokines increased, the difference in cytokine activity between the two proteins began to shrink. At 10 nM, the two proteins were There is almost no difference between them ( Figure 3). For CTLL-2 cells, the cytokine activity of PFC-1 was approximately 2 to 4 times that of rhIL-15 (Fig. 4).
  • PBMCs were adjusted to a single cell suspension of 2 x 10 6 cells/ml and stained with 5 ⁇ M CFSE (eBioscience).
  • the CFB-stained PBMCs were adjusted to 5 x 10 5 cells/ml and cultured for 6 days under stimulation with recombinant human IL-15 or PFC-1 at a final concentration of 1 nM or 10 nM.
  • Data collection was performed using a Cytomic FC500 (Beckman Coulter) flow cytometer, and data analysis was performed using Kaluza software (Beckman Coulter).
  • PBMC peripheral blood mononuclear cells
  • rhIL-15 cytokine activity
  • PFC-1 showed 10-fold higher cytokine activity than rhIL-15 in the PBMC proliferation assay.
  • the average proliferation rate of rhN-15 at 10 nM was 20.71%, and PFC-1 at 1 nm was 22.05% (Fig. 5). This may be due to the higher biological activity of the IL-15/IL-15R ⁇ complex in PFC-1 than rhIL-15.
  • HUVEC or the ovarian cancer cell line SKOV3 is an ⁇ V ⁇ 3 integrin high expression cell line.
  • flow cytometry showed that both HUVEC (Fig. 6) and ovarian cancer cell SKOV3 (Fig. 7) were positive for ⁇ V ⁇ 3 integrin expression.
  • the rectal cancer cell line LS174T was negative for V ⁇ 3 integrin expression (Fig. 8). Therefore, the above three cell lines were used to verify the colocalization of PFC-1 with integrins.
  • the HUVEC and SKOV3 cells in the logarithmic growth phase were trypsinized into a single cell suspension, adjusted to a density of 4 ⁇ 10 5 cells/ml, and cultured at 37 ° C for 2 hours to restore the expression of the cell surface marker protein. Subsequently, the cells were washed thoroughly with PBS washing, flow-through antibody or PFC-1 labeling was carried out in 2 x 10 5 cells per tube, and stained with the corresponding fluorescent secondary antibody, followed immediately by on-machine detection.
  • the HUVEC and SKOV3 cells in the logarithmic growth phase were inoculated one day in advance into a special dish for laser confocal microscopy, so that the cells were about 70% cell fusion when detected on the machine. On the next day, the cells were washed thoroughly with cold PBS, then fixed with 4% paraformaldehyde for 15 min at room temperature, and the fixed cells were incubated with 2 ⁇ g of anti-human CD51/61 ( ⁇ V ⁇ 3 integrin) antibody or PFC-1 for 1 hour at room temperature.
  • HUVEC and SKOV3 cells were used as models to incubate cells with PFC-1 and anti- ⁇ V ⁇ 3 integrin monoclonal antibodies. It was found that PFC-1 and anti- ⁇ V ⁇ 3 integrin monoclonal antibodies were found in two There was a significant colocalization on the cell surface (Fig. 9), indicating that PFC-1 binds to the same cell surface protein as the anti- ⁇ V ⁇ 3 integrin monoclonal antibody, but binds to different protein epitopes. In the context of this experiment, it is ⁇ V ⁇ 3 integrin.
  • PFC-1 has high anti-tumor effect in vivo
  • mice were transplanted subcutaneously with B16F10 melanoma and established a large tumor burden model. After the model was established, mice were intravenously injected with 10 ⁇ g of PFC-1 for two consecutive days (Fig. 11). PFC-1 showed good in vivo tumor growth inhibition. On the fifth day after the start of the experiment, the tumor was reduced to 75% of the original volume in the PFC-1 treated mice group compared to the tumor volume at the start of the experiment (Fig. 11), and on the sixth day, the PFC-1 treatment group was administered. The mice were injected intravenously with an additional PFC-1 and the tumor volume continued to shrink to 54% of the original volume.
  • peripheral venous blood was collected from the orbital vein and immediately subjected to anticoagulation and red blood cell lysis.
  • the spleen was taken out and the spleen cells were immediately collected, and subjected to filtration through a 70 uM nylon sieve (BD) and erythrocyte lysis treatment to obtain a spleen single cell suspension.
  • the tumor tissues were taken out, and the tumor tissue was gently disrupted with tweezers, and then digested with 0.2 mg/ml Collagenase IV and 0.1 mg/ml DNAse I at 37 ° C for 15 minutes. Single cell suspensions were collected and the remaining tumor tissue was continued to digest with the above enzyme solution for 25 minutes.
  • the combined single cell suspensions were filtered through a 70 micron nylon mesh.
  • Peripheral blood single cells, spleen cells and tumor cells were stained with flow cytometry and fixed with 4% paraformaldehyde solution and stored in the dark or immediately tested on the cell phenotype.
  • PFC-1 inhibits malignant metastasis of B16 tumor in mice
  • mice 5 ⁇ 10 5 B16F10 mouse melanoma cells were inoculated into 4-6 week old female C57BL/6 mice by tail vein injection. In model mice, tumor cells rapidly metastasize to the lungs and form visible tumor sinuses. On the second day, mice were administered 10 ⁇ g of PFC-1 or an equivalent volume of PBS (100 ul) by intraperitoneal injection. On day 21, the mice were humanely sacrificed, the lungs were removed, washed thoroughly with PBS and stored in 10% formaldehyde. The number of tumor metastases in the lungs of different groups of mice was then examined and counted using a binocular microscope (Leica M125).

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Abstract

本发明提供了一种肿瘤靶向融合蛋白，包括(i)IL-15多肽，其变体或功能性片段，(ii)IL-15Ra多肽，其变体或功能性片段，(iii)Fc结构域，其变体或功能性片段，和(iv)RGD多肽或其变体。所述融合蛋白的排列顺序为RGD多肽-Fc结构域-IL-15多肽-IL-15Ra多肽。该融合蛋白一方面能够提高IL-15的抗肿瘤功效，另一方面克服了IL-15的短半衰期问题，同时能够靶向肿瘤部位作用于肿瘤细胞。

Description

用于肿瘤靶向治疗的白细胞介素‐15融合蛋白 技术领域
本发明涉及细胞因子白介素‐15在肿瘤靶向治疗中的应用，具体涉及白介素‐15融合蛋白的抗肿瘤作用。
背景技术
细胞因子在调节人体免疫系统，包括抗肿瘤免疫反应中起重要作用。一些细胞因子已被证明具有抗肿瘤潜力。白细胞介素-15(IL-15)作为一个有前途的抗肿瘤候选药物已经被广泛研究。IL-15和IL-2共用受体(IL-2/15βγ)的β和γ链，但结合到不同的α受体链(IL-2Rα/IL-15Rα)。IL-15的作用主要是通过反式递呈模式，其中IL-15与表达于抗原呈递细胞表面的IL-15Rα结合，然后结合于临近效应细胞表面的IL-15βγ复合物，最终激活效应细胞。类似于IL-2，IL-15能够促进T细胞和自然杀伤(NK)细胞的增殖、促进细胞毒性T细胞扩增和NK细胞的激活。同时，与IL-2不同的是，IL-15不会导致激活诱导的细胞死亡，也不参与调节性T细胞的功能维持。因此，IL-15被美国国家癌症研究所列在最有潜力的癌症免疫治疗药物名单的第一位。
但是，IL‐15仍然有一定的不足。首先，研究表明，只有给予高剂量IL‐15才能在体内条件下达到抗肿瘤效果。其次，IL‐15的另一个不足是较短的半衰期。最后，IL‐15的作用是全身性，而不是肿瘤特异性的。细胞毒性T细胞或NK细胞在IL‐15刺激下将被扩增，但这一效果并不会仅局限于肿瘤部位，而是全身性的分布。由于免疫系统广泛性激活常常是致命性的，所以一种更理想的治疗药物应当靶向性作用于肿瘤部位同时不影响正常组织。
发明内容
本发明提供一种肿瘤靶向融合蛋白，其一方面能够提高IL‐15的抗肿瘤功效，另一方面克服了IL‐15的短半衰期问题，同时能够靶向肿瘤部位，针对性地作用于肿瘤细胞。
本发明提供的肿瘤靶向融合蛋白至少包括(i)IL‐15多肽、IL‐15多肽变体、或其功能性片段，(ii)IL‐15Ra多肽、IL‐15Ra多肽变体、或其功能性片段，(iii)Fc结构域、Fc变体、或其功能性片段，和(iv)RGD多肽或其变体。
在一个实施方式中，所述融合蛋白的各组分的排列顺序为RGD‐Fc‐IL‐15‐IL‐15Ra。
在本发明的一个实施方式中，Fc结构域由人IgG1的CH2和CH3构成，其具有SEQ ID NO.1所示的序列，或在SEQ ID NO.1所示序列经过取代、缺失或添加一个或多个氨基酸且具有所述Fc结构域相同功能的衍生序列。
在一个实施方式中，IL‐15Ra由IL‐15Rαsushi结构域及外显子3的随后12个氨基酸构 成，其具有SEQ ID NO.2所示的序列，或在SEQ ID NO.2所示序列经过取代、缺失或添加一个或多个氨基酸且具有IL‐15RA结构域相同功能的衍生序列。
在一个实施方式中，IL‐15具有SEQ ID NO.3所示的序列，或在SEQ ID NO.3所示序列经过取代、缺失或添加一个或多个氨基酸且具有IL‐15结构域相同功能的衍生序列。
在一个实施方式中，RGD多肽具有SEQ ID NO.4所示的序列，或在SEQ ID NO.4所示序列经过取代、缺失或添加一个或多个氨基酸且具有RGD多肽相同功能的衍生序列。
在优选的实施方式中，所述肿瘤靶向融合蛋白的氨基酸序列选自：(a)如SEQ ID NO.5所示的序列；(b)由SEQ ID NO.6所示的核酸序列编码的氨基酸序列；(c)由SEQ ID NO.6所示的序列的简并序列编码的氨基酸序列；和(d)在SEQ ID NO.5所示序列经过取代、缺失或添加一个或多个氨基酸且具有所述融合蛋白相同功能的衍生序列。
本发明另一方面提供一种药物组合物，其包含本发明所述的肿瘤靶向融合蛋白以及药学上可接受的附加剂，所述附加剂包括载体、稳定剂和/或赋形剂。
本发明的另一个方面提供一种药物组合物，其包含本发明所述的肿瘤靶向融合蛋白以及另一种抗癌剂。
在本发明的一个实施方式中，所述肿瘤为整合素阳性肿瘤，特别是αVβ3整合素阳性肿瘤，例如黑色素瘤、卵巢癌等。在一个实施方式中，所述肿瘤为进行性肿瘤、晚期肿瘤、具有高肿瘤负荷/负担的肿瘤、或转移性肿瘤。
本发明另一方面提供一种编码本发明所述肿瘤靶向融合蛋白的核酸序列、包含所述核酸序列的表达载体、或用所述表达载体转化或转染的宿主。
本发明另一方面还提供一种试剂盒，其包含本发明所述肿瘤靶向融合蛋白、编码本发明所述肿瘤靶向融合蛋白的核酸序列、包含所述核酸序列的表达载体、或用所述表达载体转化或转染的宿主。
本发明提供一种肿瘤靶向融合蛋白，其一方面能够提高IL‐15的抗肿瘤功效，另一方面克服了IL‐15的短半衰期问题，同时能够靶向肿瘤部位，针对性地作用于肿瘤细胞。此外，这种肿瘤靶向蛋白可以被高效的表达并纯化，同时具有高效的抗肿瘤活性，具有成为免疫抗肿瘤疗法药物的潜力。
附图说明
图1为本发明的肿瘤靶向融合蛋白的一个实施方式(PFC‐1)的蛋白结构示意图。SP，信号肽；RGD，精氨酸，甘氨酸‐天冬氨酸肽基序；Fc，人IgG1的CH 2和CH3；IL‐15Ra，IL‐15Rαsushi结构域+外显子3的随后12个氨基酸；L1：SS；L2：G4S；L4：SG2SG4SG3SG4SLQ。
图2为融合蛋白PFC‐1的在非还原(NR)或还原(R)的条件下用10％的SDS‐PAGE电泳展开并用考马斯蓝染色图。
图3显示PFC‐1和rhIL‐15对mo7e细胞的增值刺激实验。PFC‐1摩尔浓度按照单体分子量计算。实验结果为平均值加减标准差显示，实验结果代表至少三次独立实验。
图4显示PFC‐1和rhIL‐15对CTLL‐2细胞的增值刺激实验。PFC‐1摩尔浓度按照单体分子量计算。实验结果为平均值加减标准差显示，实验结果代表至少三次独立实验。
图5显示融合蛋白PFC‐1对PBMC的体外增殖刺激。PBMC细胞用CFSE标记后与各种浓度的rhIL‐15或PFC‐1共同培养6天，随后通过流式细胞术评估细胞增殖程度。A.代表性流式细胞术实验结果图。B.PBMC细胞增殖率，实验结果为平均值加减标准差显示，实验结果代表至少三次独立实验。
图6显示流式细胞术分析融合蛋白PFC‐1对HUVEC细胞系的结合作用。实验结果代表至少3次独立实验。
图7显示流式细胞术分析融合蛋白PFC‐1对SKOV‐3肿瘤细胞系的结合作用。实验结果代表至少3次独立实验。
图8显示流式细胞术分析融合蛋白PFC‐1对LS74T肿瘤细胞系的结合作用。实验结果代表至少3次独立实验。
图9显示激光共聚焦显微技术分析融合蛋白PFC‐1在HUVEC细胞和SKOV3肿瘤细胞模型上与抗人CD51/61(αVβ3整合素)抗体的共定位结果。
图10显示融合蛋白PFC‐1的小鼠体内抗肿瘤作用。背部皮下接种有B16F10小鼠黑色素瘤肿瘤的小鼠，在肿瘤长到100mm3体积后，每3天腹腔注射接受5或20μg的PFC‐1治疗，或者200ul的PBS。总共接受2次药物注射并相应测量肿瘤体积。实验结果为平均值加减标准差，每组有5～8只小鼠。通过T检验分析实验数据统计学显著性，**代表p<0.01。
图11显示融合蛋白PFC‐1的小鼠体内抗肿瘤作用。背部皮下接种有B16F10小鼠黑色素瘤肿瘤的小鼠，在肿瘤长到1000mm3体积后，在制定日期接受10μg PFC‐1的尾静脉注射治疗，或者200ul的PBS。总共接受3次药物注射并每天测量肿瘤体积。实验结果为平均值加减标准差，每组有5～8只小鼠。通过T检验分析实验数据统计学显著性，**代表p<0.01。
图12显示流式细胞术分析图10中实验小鼠接受PFC‐1治疗后体内CD8+T细胞表型变化。实验结果为平均值加减标准差，每组有5～8只小鼠。通过T检验分析实验数据统计学显著性，**代表p<0.01，***代表p<0.005。
图13显示流式细胞术分析图10中实验小鼠接受PFC‐1治疗后体内NK细胞表型变化。实验结果为平均值加减标准差，每组有5～8只小鼠。通过T检验分析实验数据统计学显著性，**代表p<0.01，***代表p<0.005。
图14显示流式细胞术分析图10中实验小鼠接受PFC‐1治疗后体内CD8+T细胞和NK细胞表面CD44抗原表型变化，以分析细胞激活比例。实验结果为平均值加减标准差，每组有5～8只小鼠。通过T检验分析实验数据统计学显著性，**代表p<0.01，***代表p<0.005。
图15显示融合蛋白PFC‐1在C57BL/6小鼠体内抗肿瘤恶心迁移实验结果。C57BL/6小鼠在day0通过尾静脉注射5*105B16F10黑色素瘤肿瘤细胞，并于同一天通过尾静脉注射接受10μg PFC‐1或者200ul等体积PBS。第21天，小鼠被安乐死并取出肺部在双目显微镜下检查肺部肿瘤窦的情况和数目。上图为代表性小鼠肺部照片。Mock，未接受肿瘤细胞注射小鼠肺部；vehicle，接受肿瘤细胞注射的对照组；PFC‐1，接受PFC‐1注射治疗组。实验结果为平均值加减标准差，每组有5只小鼠。通过T检验分析实验数据统计学显著性。
具体实施方式
融合蛋白
在本发明中，“融合蛋白”、“PFC-1”、“PFC-1重组融合蛋白”或“融合分子”可互换使用，它们是指生物活性多肽，通常为通过重组、化学或其他合适方法共价性连接(即融合)在一起的蛋白质或肽序列。融合蛋白可通过接头序列在一个或多个位点与其他肽或蛋白质序列融合。或者，接头序列可被用来协助构建融合分子。融合蛋白可以单体或多聚体(例如二聚体)的形式存在。
在本发明中，“融合蛋白”至少包括(i)IL-15多肽、IL-15多肽变体、或其功能性片段，(ii)IL-15Ra多肽、IL-15Ra多肽变体、或其功能性片段，(iii)Fc结构域、Fc变体、或其功能性片段，和(iv)RGD多肽或其变体。在该融合蛋白中，组分(i)(ii)共同构成了效应模块或效应分子，其能够引起效应细胞(细胞毒性T细胞、NK细胞)的激活。组分(iii)用于延长IL-15的循环半衰期。组分(iv)为靶向分子，其以高亲和性和特异性作用于肿瘤细胞表面表达的受体分子，从而将融合蛋白的其他部分富集于肿瘤灶，进而杀死肿瘤细胞。
在本发明中，融合蛋白中的各个组分以合理的顺序排列以使得融合蛋白整体上实现本发明的预期目的。在一个实施方式中，所述融合蛋白中各组分的排列顺序为RGD多肽-Fc结构域-IL-15多肽-IL-15Ra多肽。在另一个实施方式中，所述融合蛋白中各组分的排列顺序为RGD多肽-Fc结构域-IL-15Ra多肽-IL-15多肽。在另一个实施方式中，所述融合蛋白 中各组分的排列顺序为RGD多肽-IL-15Ra多肽-IL-15多肽-Fc结构域。在另一个实施方式中，所述融合蛋白中各组分的排列顺序为RGD多肽-IL-15多肽-IL-15Ra多肽-Fc结构域。本领域技术人员可通过基因工程技术和相关的技术得到不同顺序组成的所述融合蛋白并验证其生物学功能，而不需要付出创造性的劳动。
Fc结构域
“Fc结构域”或“Fc片段”是指免疫球蛋白重链的“可结晶片段”区。一般而言，Fc结构域能与另一Fc结构域相互作用而形成二聚体复合物。Fc结构域可结合细胞表面受体(Fc受体)和/或补体系统的蛋白质，或者可被修饰而削弱或增强这种结合活性。Fc结构域可来源于IgG、IgA、IgD、IgM或IgE抗体并产生免疫功能，例如调理作用、细胞裂解、肥大细胞脱颗粒等其他Fc受体依赖性进程。
IgG类型的免疫球蛋白是人类血液中含量最丰富的蛋白质之一，其循环半衰期可长达21天。已有报道过IgG的Fc区与另一蛋白质的结构域结合。原型融合蛋白为经由IgG的Fc铰链区中的半胱氨酸残基连接的同源二聚蛋白质，形成的分子类似于没有重链可变区、CH1结构域和轻链的IgG分子。包含Fc结构域的融合蛋白的二聚体性质可能有利于结合至其他分子(例如二价或双特异性结合)。由于结构上的同源性，Fc融合蛋白质显示与具有相似同种型的人类IgG相当的体内药代动力学曲线。为了延长IL-15或IL-15融合蛋白质的循环半衰期，本发明将IL-15/IL-15Rα复合物连接至人IgG蛋白重链的Fc部分。
天然Fc的原始免疫球蛋白来源优选为人源性的免疫球蛋白，优选为IgG1和IgG2。在本发明中，Fc结构域优选由人IgG1的CH2和CH3构成。
在某些实施方式中，“Fc变体”是指由天然Fc修饰而仍可结合Fc受体的分子或序列。“Fc变体”包括由非人类天然Fc经人源化的分子或序列。此外，由于某些结构特征或生物活性非本发明融合分子所需要的，所以天然Fc的某些位点可被移除。因此，在某些实施方式中，“Fc变体”包括缺乏一个或多个天然Fc位点或残基的分子或序列。“Fc结构域”包括如上所述的天然Fc及Fc变体的分子或序列，其包含单体形式或多聚体形式的分子，可由完整抗体分解得到，或通过基因重组表达得到或通过其他手段得到。
RGD多肽
精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽作为细胞粘附序列是Pierschbacher和Rouslahti于1984年在FN中发现的，他们发现RGD多肽能使整合素(intergrin)α5β1从亲和柱洗脱，固定在基质材料后能粘附细胞。其后，人们相继发现细胞外基质中很多糖 蛋白(如LM)、胶原蛋白、纤维蛋白原(Fb)等均含有高度保守的RGD多肽，并证实RGD多肽在介导细胞与细胞、细胞与胞外基质蛋白的相互作用中发挥重要作用。
RGD多肽与细胞的结合也是与细胞表面整合素的结合。整合素于20世纪90年代中期被发现，是一个依赖Ca2+的细胞表面受体家族，每一种整合素包括2个亚基：α亚基和β亚基，迄今发现18种α亚基和8种β亚基，构成24种整合素。能识别RGD多肽并与之结合的整合素受体有：α3β1、α5β1、αIIbβ3、α5β1、αvβ1、αvβ3、αvβ5、αvβ6、αvβ8等，尤其是对αvβ3整合素具有极强的选择性和亲和性。αvβ3整合素在多种肿瘤细胞和肿瘤相关的血管生成的内皮细胞中出现高度过表达。
本发明通过将药物与RGD多肽偶联或融合，利用RGD多肽引导融合的蛋白大分子药物在肿瘤组织部位选择性富集，增加局部浓度来增强肿瘤杀伤作用并限制全身性毒性。
单纯的RGD三肽生物活性很低，与RGD三肽相连的第4位氨基酸对其活性影响很大，与RGD三肽相连的第5位氨基酸对肽的结合专一性也起到重要的作用。已有研究证明，在RGD三肽的前端加残基不影响细胞的粘附，如RGD三肽与GRGD四肽对细胞粘附力无显著响应，但在其C末端增加氨基酸参加将影响它与细胞的粘附，如在RGD的天冬氨酸后有丝氨酸残基将增强其细胞粘附作用，若用右旋残基取代左旋残基将使得细胞粘附下降。
在本发明的一个实施方式中，RGD多肽序列为ACDCRGDCFCG，即Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly，其在第5至第7个氨基酸位置包含RGD基序。
在本发明中，RGD变体是指与本发明的RGD多肽序列具有至少一个氨基酸取代、缺失或插入的氨基酸序列，但仍能够维持RGD的整合素受体结合功能。本领域技术人员能够根据本发明给出的教导以及现有技术设计出其他满足本发明目的的一个或多个RGD多肽变体。示例性的RGD变体包括GRGD，GRGDSPC，GRGDDSY，EPRGDNYR等。
接头序列
本发明的融合蛋白还包括位于各组分之间的接头序列，通常为4-20个氨基酸构成的短肽。这些接头序列使得各组分之间被合理地定位而实现各组分的功能活性。
在一些实施方式中，在本发明的融合蛋白中，IL-15多肽通过接头序列共价连接至IL-15Ra多肽，从而使得IL-15和IL-15a结构域能够相互作用而形成复合物。在某些实施方式中，IL-15和IL-15Ra结构域的定位使得它们与免疫细胞之间相互作用，以启动或抑制免疫反应，或者抑制或刺激细胞发育。
在一些实施方式中，在本发明的融合蛋白中，IL-15或IL-15Ra结构域通过接头序列共价连接至免疫球蛋白Fc结构域。该接头序列应当使得Fc结构域、IL-15或IL-15Ra结构域能够合理地定位从而发挥每个结构域的功能活性。在某些实施方式中，Fc结构域被有效地定位从而能够形成适当的融合蛋白复合物以及使该融合蛋白复合物在体内有延长的半衰期。
在一些实施方式中，在本发明的融合蛋白中，RGD多肽通过接头序列共价地结合至Fc结构域。该接头序列应当使得RGD多肽和Fc结构域被合理地定位从而发挥各个结构域的功能活性。在某些实施方式中，RGD多肽被有效地定位从而能够以高亲和性和特异性结合肿瘤细胞表面的整合素分子。
优选地，接头序列包括2至20个氨基酸序列，更优选为5至20个氨基酸。接头序列优选为柔性接头序列，因而其不会限制效应分子或多肽在单个不期望的构象中。接头序列可被用来例如将识别位点与融合蛋白隔开。接头序列优选主要由具有小侧链的氨基酸构成，例如甘氨酸、丙氨酸和丝氨酸，从而提供所述柔性。优选地，接头序列的约80以上或更多比例的氨基酸是甘氨酸、丙氨酸或丝氨酸残基，特别是甘氨酸和丝氨酸残基。合适的接头序列的例子有GGGGS(G4S)，即Gly Gly Gly Gly Ser，例如用于连接本发明中的RGD多肽与Fc结构域，以及Fc结构域与IL-15Ra多肽；或SG2SG4SG3SG4SLQ，即Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln，例如用于连接本发明中的IL-15和IL-15Ra结构域。也可以使用其他不同的接头序列，包括已被成功地用于连接不同抗体可变区的多种柔性接头设计。接头序列的大小和序列组成可通过常规的电脑建模及技术来确定。
在本发明中，“多肽”是指基本上由20种天然氨基酸中的任何几个所组成的任何长度的聚合物。虽然“蛋白质”或“蛋白”通常是指氨基酸长度较大的聚合物，而“肽”通常是指氨基酸长度较小的聚合物，但是这两个术语之间通常没有明显的界限，而且经常范围上有重叠。“多肽变体”通常是指与对照多肽相比具有一个或多个氨基酸取代、缺失或插入，但仍能够维持多肽的生物学功能的氨基酸序列。
在本发明中，“载体”为能够在宿主细胞中自主复制且可接受外源DNA的核酸分子。载体带有其自身的复制起始位点，可用于插入外源DNA的限制性内切酶识别位点，以及通常的选择性标记(如编码抗生素抗性的基因)，常常还包括用于表达插入的DNA的识别序列(如启动子和增强子)。常见的载体包括质粒载体和噬菌体载体。
试剂与材料
抗体：重组人IL-2(AF-200-02)和粒细胞-巨噬细胞集落刺激因子(300-03)购买Peprotech公司。重组人IL-15(247-IL-105)得自R&D Systems公司购得。
抗MsCD3e(145-2C11)-PerCP，抗MsCD8a(53-6.7)-FITC，抗MsNK1.1(PK136)-FITC，抗MsCD44(IM7)-PE和抗MsCD122(TM-Bta1)-PE分别购自BD Pharmingen。抗人CD51/61(αVβ3整合素)纯化单克隆抗体购自eBioscience。山羊抗-人IgG(H+L)-AlexaFluor 488和山羊抗小鼠IgG(H+L)-AlexaFluor 488，647购自Invitrogen。
细胞系和实验动物：SKOV-3，CTLL-2和Mo7e细胞来自上海细胞库。HUVEC细胞由四川大学高会乐博士慷慨赠送。
CTLL-2细胞培养在20％胎牛血清(FBS)的RPMI 1640培养基中，同时添加30ng/ml的IL-2和1％非必需氨基酸。Mo7e细胞培养在10％胎牛血清(FBS)的RPMI 1640培养基中，同时添加10ng/ml GM-CSF，和1％非必需氨基酸。SKOV-3和HUVEC细胞均培养在10％FBS的DMEM培养基中。
PBMC从健康志愿者血液中利用Ficoll离心法提取，细胞培养在10％FBS的RPMI-1640培养基中。C57BL/6小鼠从中山大学动物实验中心购买。人体血液采集和动物实验，均经过学校主管部门的伦理审查和同意。
融合蛋白的表达与纯化
一般而言，本发明的融合蛋白的制备可通过本发明公开的程序和重组DNA技术来制备，例如PCR、质粒DNA提取、DNA的限制性内切酶消化、DNA连接、mRNA分离、将DNA导入合适的细胞、宿主细胞的转化或转染、宿主细胞的培养等。此外，融合蛋白可利用适当试剂和熟知的方法被分离和纯化，例如电泳、离心、色谱方法等。
PFC‐1重组融合蛋白的基因序列被克隆到含有小鼠κ链的信号肽的pcDNA3.1载体中。利用瞬时转染技术将表达载体转染到293细胞中进行表达。瞬时转染后的细胞在100ml细胞培养基中培养3天后，收集细胞并利用Protein‐A琼脂糖亲和纯化法纯化得到重组融合蛋白。
融合蛋白PFC-1的结构如图1所示，主要由IL15/IL15Ra复合物，Fc结构域，和RGD多肽三个模块构成。模块之间由GGGGS短肽连接，同时在蛋白C末端加入His-tag标签(图1)。DNA序列被亚克隆到pcDNA3.1(+)载体中，通过瞬时转染使载体进入HEK293细胞并被表达。
在还原性SDS‐PAGE电泳图(图2)中的60kDa处可观察到单一条带；在非还原性SDS‐PAGE电泳图中，约120kDa处可观察到一条主要条带，同时在约60kDa处有一个次要 条带。实验结果表明，通过哺乳动物细胞表达和亲和纯化，我们得到了单一均质的PFC‐1融合蛋白，并且绝大多数PFC‐1蛋白以二聚体形式存在。
细胞因子依赖性细胞增殖实验
在细胞因子依赖性细胞增殖实验中，收获处于对数生长期的CTLL‐2和Mo7e细胞，PBS洗涤两次，在检测培养基中培养4小时(补充有10％FBS，1％NEAA的RPMI 1640培养基)，使实验细胞达到细胞因子饥饿状态。在测定培养基中将IL‐15和PFC‐1稀释到终浓度为10nM并进行梯度稀释。将添加有IL‐15或PFC‐1的CTLL‐2或Mo7e细胞继续培养48或72小时。最后使用CCK‐8试剂测定活细胞的数量并计算细胞增殖比例。
CTLL-2是小鼠细胞毒性T淋巴细胞系，为IL-15Rα链和IL-15βγ复合物阳性表达；而Mo7e是人巨核细胞白血病细胞系，为IL-15βγ复合物单一阳性表达。这两种细胞都依赖细胞因子刺激细胞增殖，因此可用于分析融合蛋白PFC-1的IL-15复合物的细胞因子功能。与IL-15的效果类似，PFC-1在体外实验中能明显刺激Mo7e和CTLL-2细胞系的增殖(图3和4)，表明纯化得到的PFC-1融合蛋白具有细胞因子活性。
对于Mo7e细胞，rhIL-15表现出比PFC-1略高的细胞因子活性，但随着细胞因子浓度的增加，两种蛋白之间的细胞因子活性差异开始缩小，在10nM浓度下两种蛋白之间几乎没有差别(图3)。对于CTLL-2细胞，PFC-1的细胞因子活性大约是rhIL-15的2至4倍(图4)。
PBMC增殖实验
PBMC调整为2×106细胞/ml的单细胞悬浮液，用5μM的CFSE(eBioscience公司)进行染色。CFSE染色后的PBMC调整到5×105细胞/ml，并在1nM或10nM终浓度的重组人IL-15或PFC-1刺激下分别培养6天。用Cytomic FC500(Beckman Coulter公司)流式细胞仪进行数据收集，用Kaluza软件(Beckman Coulter公司)进行数据分析分析。
为了检测PFC-1对人源免疫细胞的细胞因子活性，PBMC用CFSE染色后与rhIL-15或PFC-1在体外共同培养6天。在1nm或10nM浓度下，rhIL-15和PFC-1均能显著刺激的PBMC的增殖(图5)。然而，与Mo7e和CTLL-2细胞因子依赖性增殖实验结果不同的是，PFC-1在PBMC增殖实验中表现出比rhIL-15高10倍的细胞因子活性。10nM的rhIL-15平均增殖率为20.71％的，1nm的PFC-1为22.05％(图5)。这可能是由于在PFC-1中IL-15/IL-15Rα复合物比rhIL-15有更高的生物活性。
PFC-1与整合素的共定位
根据文献可发现，HUVEC或卵巢癌细胞系SKOV3是αVβ3整合素高表达细胞系。同时，流式细胞分析术显示，无论HUVEC(图6)还是卵巢癌细胞SKOV3(图7)都是αVβ3整合素表达阳性。同时，直肠癌细胞系LS174T为Vβ3整合素表达阴性(图8)。因此，以上三种细胞系被用于验证PFC-1与整合素的共定位。
将对数生长期的HUVEC和SKOV3细胞用胰蛋白酶消化成单细胞悬液，调整为4×105细胞/ml密度，并37℃下培养2小时使细胞表面标志物蛋白表达得到恢复。随后用PBS洗涤充分洗涤细胞，以2×105细胞每管进行流式抗体或PFC-1标记，并用相应荧光二抗染色，随后立即上机检测。
PFC-1与HUVEC或SKOV3共孵育后，在流式细胞分析术中显示有明显阳性信号，提示PFC-1可以结合到HUVEC(图6)和SKOV3细胞(图7)上。PFC-1的阳性信号强度比抗αVβ3整合素单克隆抗体的信号弱。但是，无论是PFC-1还是抗αVβ3整合素单克隆抗体与LS174T细胞共孵育后均无阳性信号(图8)。流式细胞术分析结果可初步说明PFC-1可特异性结合于HUVEC或SKOV3细胞表面。
激光共聚焦显微实验
将对数生长期的HUVEC和SKOV3细胞提前一天接种至激光共聚焦显微镜专用培养皿中，使细胞在上机检测时约为70％细胞融合度。第二天，用冷PBS彻底洗涤细胞，随后用4％多聚甲醛室温固定15min，固定的细胞用为2μg抗人CD51/61(αVβ3整合素)抗体或PFC-1在室温下孵育1小时，随后用相应的荧光二抗分别在室温下染色1小时，用DAPI对细胞核进行染色后立即上机检测检测PFC-1和抗人CD51/61的共定位效应。本实验使用蔡司LSM710共聚焦显微镜。
在激光共聚焦显微实验中，以HUVEC和SKOV3细胞为模型，用PFC-1和抗αVβ3整合素单克隆抗体共同孵育细胞，可明显发现PFC-1和抗αVβ3整合素单克隆抗体在两种细胞表面均有明显的共定位现象(图9)，说明PFC-1与抗αVβ3整合素单克隆抗体结合同一种细胞表面蛋白，但是作用于不同的蛋白表位而结合。在本实验的情景下，为αVβ3整合素。
流式细胞术和激光共聚焦显微实验的结果共同表明，PFC-1可通过RGD模块特异性结合于αVβ3整合素，从而在体外实验条件下实现针对αVβ3整合素高表达的肿瘤细胞特异性结合。
PFC-1具有高效的体内抗肿瘤效果
为了评估的PFC-1的体内抗肿瘤功效，我们在4-6周龄雌性C57BL/6小鼠于右侧背部被注射5×105B16F10小鼠黑色素瘤细胞。大约10-12天后，肿瘤直径(day0)达到5-8mm。当移植瘤长到50-100mm3体积开始给予不同剂量的PFC-1或阴性对照(PBS)。并在指定日期测量小鼠肿瘤大小。实验结果显示，5μg PFC-1剂量组中肿瘤生长率出现70％抑制，而20μg PFC-1剂量组中则有100％抑制(图10)。
在一个类似的小鼠实验中，4-6周龄的雌性C57BL/6小鼠于背部皮下被移植B16F10黑色素瘤，并建立大肿瘤负担模型。模型建立之后，小鼠被连续两天静脉内注射10μg的PFC-1(图11)。PFC-1表现出良好的体内肿瘤生长抑制效果。实验开始后第五天，相比于实验开始时的肿瘤体积，在PFC-1治疗小鼠组中肿瘤缩小到原始体积的75％(图11)，在第六天，给予PFC-1治疗组小鼠一次额外的PFC-1静脉内注射，肿瘤体积持续缩小到原始体积的54％。
流式细胞分析细胞表型
小鼠麻醉后从眼眶静脉采集外周静脉血，并立即进行抗凝血和红细胞裂解处理。小鼠人道处死后，取出脾脏并立即采集脾脏细胞，并通过70uM尼龙筛(BD)的过滤和红细胞裂解处理后得到脾脏单细胞悬液。同时取出肿瘤组织，用镊子轻轻破坏肿瘤组织结构后在37℃下用0.2mg/ml Collagenase IV和为0.1mg/ml的DNAse I消化15分钟。收集单细胞悬液并且将剩余的肿瘤组织继续用上述酶溶液消化25分钟。合并得到的单细胞悬液用70微米的尼龙网过滤。外周血单细胞，脾脏细胞和肿瘤细胞用流式抗体染色后用4％多聚甲醛溶液固定并避光保存或立即上机检测细胞表型。
经过抗体染色后用流式细胞术分析可发现，经过PFC-1(图12)治疗的小鼠的外周血、脾脏和肿瘤组织中的CD8+T细胞比例急剧提高。同时，小鼠的外周血和脾脏细胞组织中的CD8+T细胞的表面抗原CD44表达量同样出现明显提高，在肿瘤组织中的CD8+T细胞的表面抗原CD44表达量出现相对微弱的提高。CD44在CD8+T细胞表面的表达量可反映CD8+T细胞的激活程度。在该实验结果提示两种融合蛋白不仅能够增加CD8+T细胞群落的比例，同时还能促进CD8+T细胞的激活。另外，流式细胞术分析还显示，PFC-1能够有效动员NK细胞能多的渗透进入肿瘤组织(图13)，这进一步提示PFC-1能有效激活免疫系统从而杀灭肿瘤细胞。
PFC-1抑制小鼠体内B16肿瘤恶性转移
将5×105B16F10小鼠黑色素瘤细胞通过尾静脉注射方式接种至4-6周龄雌性C57BL/6小鼠体内。在模型小鼠体内，肿瘤细胞迅速转移到肺部并且形成明显可见的肿瘤窦。第 二天，通过腹膜内注射方式给予小鼠10μg PFC-1或同等体积的PBS(100ul)。第21天，将小鼠人道处死，取出肺部，用PBS彻底清洗后保存在10％的甲醛中。随后用双目显微镜(Leica M125)检查并计数不同组小鼠肺部的肿瘤转移灶数量。
实验证明，单次腹腔注射10μg PFC-1可有效抑制肿瘤恶性迁移。相比于对照组，给药组的肺部肿瘤窦平均值从61下降到11.4，降低幅度超过80％(图15)。这表明，PFC-1可有效激活免疫系统从而杀灭转移的肿瘤细胞。抑制肿瘤恶性转移对于防止肿瘤复发和促进肿瘤预后有着积极的意义。
本领域技术人员会容易意识到，本发明可容易改造而获得本文所述的那些目的和优点以及隐含在本文中的那些目的和优点。在本文中以当前优选实施方式的代表的形式描述的方法、变体和组合物是示例性的，并不意在限制本发明的范围。对于本领域技术人员来说，可对它们做出改变或将其用于其他用途，但这都包括在如所附权利要求定义的本发明的范围内。
虽然本发明已通过优选实施方式和任选的特征具体公开，但本领域技术人员可对本文公开的想法做出修改和变化，而这些修改和变化仍属于所附权利要求定义出的本发明的范围之内。

Claims (16)

  1. 一种肿瘤靶向融合蛋白至少包括(i)IL-15多肽、IL-15多肽变体、或其功能性片段，(ii)IL-15Ra多肽、IL-15Ra多肽变体、或其功能性片段，(iii)Fc结构域、Fc变体、或其功能性片段，和(iv)RGD多肽或其变体。
  2. 根据权利要求1所述的肿瘤靶向融合蛋白，所述融合蛋白的各组分的排列顺序为RGD多肽-Fc结构域-IL-15多肽-IL-15Ra多肽。
  3. 根据权利要求1所述的肿瘤靶向融合蛋白，其中Fc结构域的氨基酸序列如SEQ ID NO.1所示，或在SEQ ID NO.1所示序列经过取代、缺失或添加一个或多个氨基酸且具有所述Fc结构域相同功能的衍生序列。
  4. 根据权利要求1所述的肿瘤靶向融合蛋白，其中IL-15RA的氨基酸序列如SEQ ID NO.2所示，或在SEQ ID NO.2所示序列经过取代、缺失或添加一个或多个氨基酸且具有IL-15RA结构域相同功能的衍生序列。
  5. 根据权利要求1所述的肿瘤靶向融合蛋白，其中IL-15的序列如SEQ ID NO.3所示，或在SEQ ID NO.3所示序列经过取代、缺失或添加一个或多个氨基酸且具有IL-15结构域相同功能的衍生序列。
  6. 根据权利要求1所述的肿瘤靶向融合蛋白，其中RGD多肽的序列SEQ ID NO.4所示的序列，或在SEQ ID NO.4所示序列经过取代、缺失或添加一个或多个氨基酸且具有RGD多肽结构域相同功能的衍生序列。
  7. 根据权利要求1所述的肿瘤靶向融合蛋白，其中所述肿瘤靶向融合蛋白的氨基酸序列选自：(a)如SEQ ID NO.5所示的序列；(b)由SEQ ID NO.6所示的核酸序列编码的氨基酸序列；(c)由SEQ ID NO.6所示的序列的简并序列编码的氨基酸序列；和(d)在SEQ ID NO.5所示序列经过取代、缺失或添加一个或多个氨基酸且具有所述融合蛋白相同功能的衍生序列。
  8. 根据权利要求1-7任一项所述的肿瘤靶向融合蛋白，其中所述肿瘤为整合素阳性表达肿瘤。
  9. 根据权利要求8所述的肿瘤靶向融合蛋白，其中所述肿瘤为αvβ3整合素阳性表达肿瘤。
  10. 根据权利要求1-7任一项所述的肿瘤靶向融合蛋白，其中所述肿瘤为进行性肿瘤、晚期肿瘤、具有高肿瘤负荷/负担的肿瘤、或转移性肿瘤。
  11. 一种药物组合物，其包含权利要求1-7任一项所述的肿瘤靶向融合蛋白以及药学上可接受的辅料。
  12. 一种药物组合物，其包含权利要求1-7任一项所述的肿瘤靶向融合蛋白以及另一种抗癌剂。
  13. 一种编码权利要求1-7任一项所述的肿瘤靶向融合蛋白的核酸序列。
  14. 一种包含权利要求13所述的核酸序列的表达载体。
  15. 一种用权利要求14所述的表达载体转化或转染的宿主。
  16. 一种试剂盒，其包含权利要求1-7任一项所述的肿瘤靶向融合蛋白、权利要求13所述的核酸序列、权利要求14所述的表达载体、或权利要求15所述的宿主。
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