WO2024051817A1 - 肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体 - Google Patents

肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体 Download PDF

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WO2024051817A1
WO2024051817A1 PCT/CN2023/117716 CN2023117716W WO2024051817A1 WO 2024051817 A1 WO2024051817 A1 WO 2024051817A1 CN 2023117716 W CN2023117716 W CN 2023117716W WO 2024051817 A1 WO2024051817 A1 WO 2024051817A1
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seq
structural unit
fusion protein
tumor
ngr
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傅阳心
高宇
任振华
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北京昌平实验室
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the field of biomedicine technology. Specifically, it relates to a tumor-targeted fusion protein-type drug prodrug with interleukin 2 as the active ingredient.
  • Interleukin-2 was discovered in the 1970s. Because it can promote T lymphocyte proliferation in vitro, it is also named T-cell growth factor [1].
  • purified IL-2 was first used in clinical trials to treat solid tumors (melanoma, sarcoma, adenocarcinoma, etc.) [2].
  • IL-2 did not show obvious effects. Tumor treatment effect.
  • Subsequent clinical treatment experiments used high-dose IL-2, which showed relatively good anti-tumor properties in patients with some tumor types. Therefore, high-dose IL-2 was first approved for metastatic kidney cells in 1992. cancer [3], and was subsequently approved for the treatment of melanoma in 1998 [4].
  • IL-2 high-dose IL-2
  • side effects such as capillary leak syndrome [5] and pulmonary edema [6] etc.
  • these side effects seriously limit the development of clinical application of IL-2.
  • Most patients cannot accept the planned dosage and discontinue the drug. To this day, this is still one of the problems that needs to be overcome in IL-2 tumor treatment, so development is safer. It is urgent to develop new IL-2 drugs.
  • the short blood circulation time of IL-2 is also one of the obstacles to its clinical application.
  • the blood half-life of IL-2 is only a few minutes [7], so patients have to receive frequent injections of large doses of IL-2 in a short period of time.
  • Each course of treatment consists of two consecutive doses (600,000 IU/kg administered every 8 hours for 5 consecutive days) [8]. This frequent and continuous administration will undoubtedly increase the side effects of the drug and aggravate the patient's pain.
  • NKTR-214 (Bempegaldesleukin) is a PEG-modified IL-2 drug that is currently the furthest in clinical trials. Through PEG modification, the half-life of NKTR-214 is significantly increased from 1.4 hours to 15.5 hours, and it prefers to bind tumor-killing T cells and NK cells rather than Tregs. cells, and PEG blocks the activity of IL-2, so NKTR-214 has no major side effects [9,10]. However, recent clinical trial results of NKTR-214 show that it failed to effectively improve patient survival.
  • IL-2 fusion proteins including IL-2 fusion proteins that simply extend the half-life, such as IL-2-human serum albumin fusion protein [11] and IL-2-Fc [12] fusion protein, etc.;
  • IL-2 fusion proteins that simply extend the half-life but also endow the fusion protein with new functions: ALKS 4230 (fusion of IL-2 and IL-2R ⁇ ) [13] and IL-2/S4B6 [14] (IL-2 and IL-2R ⁇ Antibody fusion) enhances the ability to promote the proliferation of CD8+ T cells and NK cells;
  • L19-IL-2 IL-2 fused with anti-extracellular domain B antibody
  • Hu14.18-IL-2 IL- 2 fused with an anti-disialoganglioside antibody
  • IL-2-related drugs Although there are various IL-2-related drugs, except for high-dose IL-2, there are currently no FDA-approved IL-2-related drugs on the market.
  • the biggest problem with polyethylene glycol-modified IL-2 is that the quality control of chemical modification is difficult to control, which also led to the failure of its clinical trials.
  • the slow release of PEG in the blood to restore activity may cause IL-2 to function in normal tissues and organs other than tumors, causing safety risks.
  • IL-2 fusion protein does not have the quality control problems of polyethylene glycol-modified IL-2, but due to its increased half-life and activity, even if the tumor targeting ability is improved to reduce its off-target harm, IL-2 fusion protein will still Before reaching the target tissue, normal tissues and organs are damaged, and safety is still a major issue.
  • IL-2 prodrug that only releases activity within the tumor as the main body.
  • This prodrug binds IL-2 and the IL-2 receptor ⁇ subunit through the cleavage site of the proteolytic enzyme (such as the MMP cleavage site). or CD13 enzyme cleavage site), the fused IL-2 receptor ⁇ subunit can block the binding of IL-2 to the IL-2 receptor on immune cells and reduce IL-2 activity.
  • the IL-2 prodrug is It exists in a closed, low-activity form in the blood and normal tissues and organs. Only after reaching the tumor site, it will be cleaved by MMP to release the IL-2 receptor ⁇ subunit, restore activity, and stimulate T cells in the tumor.
  • Fc is part of the antibody IgG. It can form homodimers and can also effectively increase the molecular weight of the protein it is fused with and increase the half-life. Fusion proteins that also contain Fc can be efficiently purified by Protein A.
  • Tumor targeting ability is the key to increasing the efficacy and reducing toxicity of anti-tumor drugs.
  • anti-tumor drugs when fused with other substances, their ability to penetrate tumor cells and their expression yield in practical applications are affected to varying degrees.
  • Tumor-targeting peptides are a type of oligopeptides that bind to specific receptors at tumor sites. Compared with antibodies and their fragments, this type of targeting peptide has a small molecular weight, weak immunogenicity, and is easy to express.
  • IL-2 we connect IL-2 to the receptor through a metalloproteinase substrate, which can reduce the binding of IL-2 to peripheral receptors. On the one hand, it reduces the peripheral consumption of IL-2, and on the other hand, it reduces the IL-2
  • the risk of prodrug activation in normal tissues and organs also reduces the toxic and side effects of the drug.
  • the targeting peptide and IL-2 prodrug are doubled, which improves the targeting effect and therapeutic effect. On the other hand, it also increases the half-life of the drug.
  • the specific tumor targeting peptides involved in the present invention are as follows:
  • CDCRGDCFC (SEQ ID NO: 1), CRGDC (SEQ ID NO: 2), CRGDKGPDC (SEQ ID NO: 3), CNGRCVSGCAGRC (SEQ ID NO: 4), CNGRC (SEQ ID NO: 5), TAASGVRSMH (SEQ ID NO:6), LTLRWVGLMS (SEQ ID NO:7), KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:8), CGNKRTRGC (SEQ ID NO:9), CNRRTKAGC (SEQ ID NO:10), CLSDGKRKC (SEQ ID NO:11), CREAGRKAC (SEQ ID NO:12), CAGRRSAYC (SEQ ID NO:13), CRSRKG (SEQ ID NO:14), CKAAKNK (SEQ ID NO:15), CRGRRST (SEQ ID NO:16), CSRPRRSEC (SEQ ID NO: 17), CGKRK (SEQ ID NO: 18), CDTRL (SEQ
  • the specifically selected tumor-targeting peptides of the present invention can effectively deliver drugs with the above structure to In tumors, the metalloproteinase substrate linking fragments in the middle of cytokines can be effectively cleaved by tumor-specific metalloproteinases, thereby releasing the powerful anti-tumor ability of cytokines. This not only improves the cure rate of tumors, but also reduces the toxic side effects of traditional cytokines. Therefore, the tumor-targeting peptide-IL-2 prodrug can effectively solve the clinical problems of weak IL-2 effect, high toxicity, and short half-life, and is easier to express and purify. Compared with unfused IL2 prodrugs, Obtaining equivalent or better expression yield has certain potential for clinical development and application.
  • a tumor-targeting fusion protein includes:
  • Structural unit 1 is a targeted binding region that specifically binds to tumor cells or tumor vascular endothelial cells, and the targeted binding region is composed of a polypeptide unit that binds to tumor cells or tumor vascular endothelial cells;
  • Structural unit 2 is an antibody Fc fragment
  • Structural unit 3 is interleukin-2 (IL-2) prodrug;
  • the fusion protein also contains a linker.
  • structural unit 1, structural unit 2 and structural unit 3 in the fusion protein can be connected in any order.
  • the order of each structural unit in the fusion protein from the N-terminus to the C-terminus is as follows:
  • X is not present, or X represents connector 1 or 2.
  • each structural unit in the fusion protein starts from the N-terminus and ends at the C-terminus, and the order is as follows:
  • each structural unit in the fusion protein starts from the N-terminus and ends at the C-terminus, and is arranged in the following order: structural unit 1, linker 1, structural unit 2, linker 2, and structural unit 3.
  • the structure unit 1 and the structure unit 2 preferably contain a proteolytic enzyme cleavage site (such as an MMP enzyme cleavage site or a CD13 enzyme cleavage site, preferably the MMP enzyme cleavage site
  • the sequence of the point is SGARYRWLTA (SEQ ID NO:74), SGRSENIRTA (SEQ ID NO:75), SGFIANPATA (SEQ ID NO:76), RSYAIL (SEQ ID NO:77) or RSPAIF (SEQ ID NO:78), preferably
  • the sequences of the CD13 restriction site are RVYIHPF (SEQ ID NO:79), VYIHPF (SEQ ID NO:80).
  • the targeting binding region is composed of one or more (preferably 2 or 3) CD13 ligand peptide units (i.e., NGR tumor-targeting peptides).
  • the ligand peptide unit is a linear or cyclic structure with a short peptide of NGR sequence. Most preferably, the ligand peptide unit is a cyclic structure with an NGR sequence. of short peptides.
  • the amino acid sequence of the ligand peptide unit is CNGRC (as shown in SEQ ID NO:5) or CNGRCVSGCAGRC (as shown in SEQ ID NO:4), or is SEQ ID NO:5 and SEQ
  • the amino acid sequence shown in ID NO:4 has at least 60% homology with the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:4 derived from substitution, deletion, or addition of one or more amino acids (for example, At least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homogeneous) and has specific binding to tumors Variant sequences for the activity of cells or
  • the ligand peptide units are connected through a linker, and the sequence of the linker is such as SEQ ID NO:72 or SEQ ID NO:73 shown.
  • the antibody Fc fragment is a native immunoglobulin Fc single chain or an immunoglobulin Fc single chain that knocks out the ADCC effect through genetic mutation (such as 'LALA-PG' mutation, See Rejuvenation of tumor-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells, or Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions, such as the sequence shown in SEQ ID NO:81); preferred
  • the antibody Fc fragment is the Fc single chain of natural human IgG (such as IgG1, IgG2, IgG3 or IgG4).
  • the amino acid sequence of the immunoglobulin Fc single chain is shown in SEQ ID NO: 82, most preferably Preferably, the amino acid sequence of the immunoglobulin Fc single chain is shown in SEQ ID NO: 83 (APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK).
  • the IL-2 prodrug comprises:
  • the IL-2 protein can be murine IL-2 protein or human IL-2 protein, preferably human IL-2 protein, more preferably, the human IL-2
  • the amino acid sequence of the protein is shown in SEQ ID NO:84;
  • the cleavage site of a proteolytic enzyme specifically expressed in the tumor microenvironment (such as an MMP cleavage site or a CD13 cleavage site).
  • the sequence of the MMP cleavage site is SGARYRWLTA (SEQ ID NO:74) , SGRSENIRTA (SEQ ID NO:75), SGFIANPATA (SEQ ID NO:76), RSYAIL (SEQ ID NO:77) or RSPAIF (SEQ ID NO:78), preferably the CD13 restriction site is RVYIHPF (SEQ ID NO:79), VYIHPF(SEQ ID NO:80);
  • the IL-2 receptor subunit extracellular domain can be an IL-2 receptor subunit extracellular domain that can effectively block IL-2 activity, and can be IL-2 receptor ⁇ (IL-2R ⁇ ) extracellular domain and/or IL-2 receptor ⁇ (IL-2R ⁇ ) extracellular domain;
  • the IL-2 receptor subunit extracellular domain can be from human or murine sources; preferably it is human IL-2 receptor body ⁇ and/or IL-2 receptor ⁇ (IL-2R ⁇ ) extracellular domain; more preferably, it is human IL-2 receptor ⁇ extracellular domain, and most preferably, the amino acid sequence of the IL-2 receptor ⁇ extracellular domain is as follows SEQ ID NO:85 is shown, and
  • a linker connecting IL-2 and the cleavage site of a proteolytic enzyme or connecting the enzyme cleavage site of a proteolytic enzyme and IL-2 receptor ⁇ (IL-2R ⁇ ).
  • the linker sequence As shown in SEQ ID NO:72 or 73.
  • the arrangement of each structural unit in the IL-2 prodrug is:
  • the C-terminus of the extracellular domain of the IL-2 receptor subunit is connected to the N-terminus of the proteolytic enzyme cleavage site (optionally, through a linker), and the C-terminus of the proteolytic enzyme cleavage site is connected to the IL-2 protein.
  • the N-terminus is connected (optionally, via a linker).
  • the arrangement of each structural unit in the IL-2 prodrug can also be:
  • the C-terminus of the IL-2 protein is connected to the N-terminus of the proteolytic enzyme cleavage site (optionally, connected through a linker), and the C-terminus of the proteolytic enzyme cleavage site is connected to the extracellular domain of the IL-2 receptor subunit.
  • the N-terminus is connected (optionally, via a linker).
  • the arrangement of each structural unit in the IL-2 prodrug is:
  • the C-terminus of the extracellular domain of the IL-2 receptor subunit is connected to the N-terminus of the proteolytic enzyme cleavage site (optionally, connected through a linker), and the C-terminus of the proteolytic enzyme cleavage site is connected to the IL-2 protein.
  • the N-terminus is connected (optionally, via a linker).
  • the fusion protein is a homodimer or a heterodimer; preferably, the tumor-targeting fusion protein is a homodimer.
  • the fusion protein is selected from the group consisting of:
  • Fusion protein 1 13P.NGR-Pro IL-2
  • the targeting binding region SEQ ID No:4
  • linker 1 SEQ ID No:72
  • antibody Fc fragment SEQ ID No:82
  • linker 2 SEQ ID No: 73
  • IL-2 prodrug SEQ ID No: 86
  • amino acid sequence structure of the fusion protein 1 is shown in SEQ ID No: 87;
  • Targeting binding region (SEQ ID No:88), linker 1 (SEQ ID No:72), antibody Fc fragment (SEQ ID No:82), linker 2 (SEQ ID No:73), interleukin-2 (IL-2 ) prodrug (SEQ ID No: 86), preferably, the amino acid sequence structure of the fusion protein 2 is shown in SEQ ID No: 89;
  • Targeting binding region (SEQ ID No:5), linker 1 (SEQ ID No:72), antibody Fc fragment (SEQ ID No:82), linker 2 (SEQ ID No:73), interleukin-2 (IL-2 ) prodrug (SEQ ID No: 86), preferably, the amino acid sequence structure of the fusion protein 3 is shown in SEQ ID No: 90;
  • Targeting binding region (SEQ ID No:91), linker 1 (SEQ ID No:72), antibody Fc fragment (SEQ ID No:82), linker 2 (SEQ ID No:73), interleukin-2 (IL-2) prodrug (SEQ ID No:86), preferably, the amino acid sequence structure of the fusion protein 4 is as SEQ Shown as ID No:92.
  • the present invention also relates to the use of the fusion protein for treating tumors, or the use of the fusion protein in preparing drugs for treating tumors.
  • the tumor is selected from the group consisting of prostate cancer, colon cancer, lung cancer, thyroid cancer, breast cancer, and pancreatic cancer.
  • the present invention also relates to a method for preparing the fusion protein, which includes the following steps:
  • (1) Construct an expression vector containing the nucleic acid sequence encoding the fusion protein.
  • the pEE12.4 expression vector is used for construction;
  • step 2 Construct a host cell containing the expression vector described in step 1 by transiently transfecting host cells.
  • the host cell is a 293F cell;
  • Figure 1 The binding effect of tumor-targeting peptide NGR on tumor cells.
  • Figures 1A and 1B show the binding effect of tumor-targeting peptide NGR (SEQ ID NO: 4) on multiple tumors of mouse origin ( Figure 1A) and human origin ( Figure 1B).
  • the control peptide is TYNL-RAW (TNYLFSPNGPIARAW, SEQ ID NO:65);
  • Figure 1C shows the ELISA identification of the tumor-targeting peptide NGR (SEQ ID NO:4) on A549 cells. Binding force.
  • Figures 1A-1C show that the control peptide TYNL-RAW has no target binding ability to the selected tumor cell lines.
  • FIG. 1 Tumor inhibitory effect of NGR-Pro IL-2 fusion protein (ie fusion protein 1).
  • Figure 2A shows the molecular structure of NGR-Pro IL-2 fusion protein. The dimer in the middle is the Fc region dimer of human IgG. Each Fc monomer is coupled to the N-terminus of a molecule of NGR polypeptide.
  • interleukin-2 prodrug the structure of interleukin-2 prodrug consists of the IL-2 receptor ⁇ extracellular domain shown in SEQ ID NO:85 and the MMP14 enzyme cleavage site shown in SEQ ID NO:74 dots and the IL-2 shown in SEQ ID NO:84 are sequentially connected;
  • Figure 2B shows the inhibitory effect of NGR-Pro IL-2 fusion protein on tumor growth.
  • Figure 3 Affinity of the fusion protein to tumor cells;
  • Figure 3A shows the molecular structure of the NGR 2 -Pro IL-2 fusion protein. Based on the NGR-Pro IL-2 structure shown on the left, the NGR 2 -Pro shown on the right IL-2 adds an NGR polypeptide to the N-terminus;
  • Figure 3B shows the optimization of the NGR polypeptide sequence. ization to remove excess cysteine;
  • Figure 3C shows the molecular sieve results of fusion proteins 1-4.
  • Figure 3D shows the cell ELISA to detect the affinity of NGR-Pro IL-2 (ie, fusion protein 1) and NGR 2 -Pro IL-2 (ie, fusion protein 4) to tumor cells A549.
  • Figure 4 Shows the tumor suppressive effects of NGR-Pro IL-2 (ie, fusion protein 1) and NGR 2 -Pro IL-2 fusion protein (ie, fusion protein 4).
  • Figure 5 Shows that NGR 2 -Pro IL-2 (i.e. fusion protein 4) fusion protein can effectively inhibit tumor growth;
  • Figure 5A shows that administration of NGR 2 -Pro IL
  • FIG. 5B shows the survival time of CT26 tumor-bearing mice
  • Figure 5C shows the tumor growth curve of mice in each group.
  • Figure 6 Shows that tumor-targeting peptides improve tumor uptake of fusion proteins.
  • Figure 6A shows small animal in vivo fluorescence imaging to detect the uptake of Pro IL-2, NGR-Pro IL-2 (ie fusion protein 1) and NGR 2 -Pro IL-2 (ie fusion protein 4) in tumors 48h after injection;
  • Figure 6B Analysis of tumor uptake results for Pro IL-2, NGR-Pro IL-2, and NGR 2 -Pro IL-2 is shown.
  • Figure 7 Shows that tumor-targeting peptides reduce fusion protein uptake in the liver.
  • Figure 7A shows the biodistribution of Pro IL-2, NGR-Pro IL-2 (i.e. fusion protein 1) and NGR 2 -Pro IL-2 (i.e. fusion protein 4) 72h after injection;
  • Figure 7B shows Pro IL-2, NGR -Pro IL-2 and NGR 2 -Mouse liver uptake 72h after injection of Pro IL-2.
  • Figure 8 Shows that NGR 2 -Pro IL-2 has no significant systemic toxicity.
  • Figure 8A shows the body weight monitoring results after treatment with Pro IL-2 and NGR 2 -Pro IL-2 (i.e. fusion protein 4);
  • Figure 8B shows the first and third times of Pro IL-2 and NGR 2 -Pro IL-2 Test results of mouse blood cytokines 24 hours after treatment.
  • Bacterial strain DH5 ⁇ E.coli competent cells (Beijing Quanshijin Biotechnology Co., Ltd.)
  • Pro IL-2 (the structure of Pro IL-2 consists of the Fc fragment shown in SEQ ID NO:82, the linker shown in SEQ ID NO:73, and the linker shown in SEQ ID NO:85
  • the IL-2 receptor ⁇ extracellular domain, the MMP14 restriction site shown in SEQ ID NO:74 and IL-2 shown in SEQ ID NO:84 are sequentially connected, and the sequence is shown in SEQ ID NO:93), 5P .NGR-Pro IL-2, 13P.NGR-Pro IL-2, 5P.NGR 2 -Pro IL-2 and 13P.NGR 2 -Pro IL-2.
  • Wild-type BALB/C mice were purchased from Vitong Lever Laboratory Animal Center, Beijing, China. Unless otherwise stated, all experiments used female mice aged 8-10 weeks. Mice were raised in a specific pathogen-free (SPF) barrier environment. Animal feeding and experimental operations comply with the relevant regulations of the Laboratory Animal Use and Management Committee of Tsinghua University.
  • SPF pathogen-free
  • MC38 was purchased from ATCC and is a mouse colorectal cancer cell line with C57 background
  • DMEM complete medium containing 10% inactivated fetal bovine serum, 2 mmol/L L-glutamine, 0.1 mmol/L non-essential amino acids, 100 U penicillin and 100 ⁇ g/mL streptomycin.
  • CT26 was purchased from ATCC and is a BALB/c background mouse colorectal cancer cell line
  • B16F10 was purchased from ATCC and is a C57 background mouse melanoma cell line
  • A549 was purchased from ATCC and is a human non-small cell lung cancer cell.
  • the above cell lines were cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2mmol/L L-glutamine, 0.1mmol/L non-essential amino acids, 100U penicillin and 100 ⁇ g/mL streptomycin).
  • FreeStyle TM 293F cell line (Invitrogen) is a suspension cell derived from the HEK293 cell line. It is cultured in SMM293-TII medium (Yiqiao Shenzhou) and is mainly used for transient transfection and expression of fusion proteins.
  • fusion proteins were transiently transfected and expressed in HEK293 cells and purified through two steps of Protein A affinity chromatography and molecular sieve superdex 200increase.
  • the tumor size was monitored twice a week, and the long diameter (a) and short diameter (b) of the tumor were measured using vernier calipers.
  • the mouse tumor volume a ⁇ b ⁇ b/2.
  • the fusion protein is injected intraperitoneally or intravenously, and the specific dosage will be described in the specific experiment.
  • Tumor-targeting peptides have high tumor uptake properties
  • polypeptides Compared with antibodies or antibody fragments, polypeptides have the advantages of small molecular weight, low immunogenicity, and easy fusion expression. Therefore, we adopt peptide fusion expression instead of antibody fusion expression as a method to enhance the tumor targeting of drugs.
  • We selected and synthesized tumor-targeting peptides (SEQ ID NO:4 and SEQ ID NO:65, of which SEQ ID NO:65 does not bind tumor cells LLC, CT26, MC38, B16F10 and A549 and can be used as a control). These The carboxyl termini of the targeting peptides were all coupled to biotin.
  • the tumor-targeting peptide with carboxyl-terminal conjugated biotin was synthesized at Gill Biochemical Company.
  • the tumor cell line was prepared into a 100 ⁇ L single cell suspension in phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS), and the number of cells was (1-10) ⁇ 10 5 .
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • A549 tumor cells were plated and cultured in a 96-well plate, and the experiment was started when the cell confluence reached 70%-80%. Wash twice with 200 ⁇ L Tris-Buffered Saline (TBS), fix the cells with 100 ⁇ L 4% paraformaldehyde at room temperature for 20 minutes, and wash three times with 200 ⁇ L phosphate buffer. After adding 100 ⁇ L of quenching buffer (1% hydrogen peroxide solution) to remove endogenous peroxidase activity, wash three times with phosphate buffer. Add 200 ⁇ L of PBS containing 5% FBS to each well and leave it at room temperature for 1 hour.
  • TBS Tris-Buffered Saline
  • NGR-Pro IL-2 fusion protein The tumor inhibitory effect of NGR-Pro IL-2 fusion protein is better than that of Pro IL-2
  • NGR Pro IL-2 in order to improve the tumor targeting effect of Pro IL-2, a prodrug for tumor treatment.
  • NGR-Pro IL-2 a homodimeric fusion protein NGR-Pro IL-2, namely fusion protein 1 ( Figure 2A)
  • Figure 2A fusion protein 1
  • the function of NGR-Pro IL-2 fusion protein was verified.
  • the average tumor size of mice in the NGR-Pro IL-2 treatment group was 670.9 mm 3
  • the average tumor size of mice in the Pro IL-2 treatment group was 1475.3 mm 3 .
  • treatment was started in random groups and intraperitoneal injection of 72 ⁇ g NGR-Pro IL-2 fusion protein or The same molar amount of Pro IL-2 fusion protein; mice in the control group were injected with 200 ⁇ L PBS. Dosing is given every 3 days.
  • Increasing the number of tumor-targeting peptide NGR in the fusion protein can improve the affinity of the fusion protein to tumor cells.
  • NGR-Pro IL-2 ie, fusion protein 1
  • NGR 2 -Pro IL-2 ie, fusion protein 4
  • Fig. 3A 13P.NGR was optimized.
  • the sequences containing disulfide bonds in the polypeptide were deleted, leaving only the smallest binding unit CNGRC ( Figure 3B).
  • the NGR 2 -Pro IL-2 described below is all 5P.NGR 2 -Pro IL-2 (i.e., fusion protein 4), that is, the truncated peptide CNGRC is used. Furthermore, we used cell ELISA experiments to measure that the affinity of NGR 2 -Pro IL-2 (ie, fusion protein 4) to human non-small cell lung cancer cells A549 was approximately 8.1 nM, which was higher than that of NGR-Pro IL-2 (ie, fusion protein 4). Protein 1, 41 nM) increased approximately 5-fold (Fig. 3D), and compared to the NGR polypeptide (i.e., SEQ ID NO: 4, 321 nM), it increased approximately 50-fold (Fig. 1C).
  • the fusion protein expression plasmid was transfected into 293F cells in the logarithmic growth phase, they were cultured for one week in an incubator at 37°C and 8% carbon dioxide at 135 rpm. Seven days later, the supernatant was collected by centrifugation at 10,000 rpm for 1 hour. After preliminary purification of the protein using Protein A (Cytiva) affinity chromatography, the highly aggregated protein was removed by superdex 200increase (Cytiva) gel filtration chromatography.
  • Fusion protein 13P.NGR-Pro IL-2 (fusion protein 1), 5P.NGR-Pro IL-2 (fusion protein 3), 5P.NGR 2 -Pro IL-2 (fusion protein 4) yield is about 30-40mg /L, there is no significant difference compared with the production of Pro IL-2 without targeting peptide fusion (30-40mg/L). .
  • the experimental method is the same as "Cell ELISA to detect polypeptide binding" in Part 1.
  • the tumor targeting effect of the drug therefore Only 13 ⁇ g of NGR 2 -Pro IL-2 (ie, fusion protein 4) or an equimolar amount of NGR-Pro IL-2 (ie, fusion protein 1) or Pro IL-2 was injected intravenously. Dosing is given every 3 days.
  • the average tumor size of mice in the NGR-Pro IL-2 (ie fusion protein 1) treatment group was 548.6mm 3
  • the average tumor size of mice in the Pro IL-2 treatment group was 625.7mm 3 .
  • mice in the NGR 2 -Pro IL-2 (ie, fusion protein 4) treatment group was 358.5mm 3 , and its tumor inhibitory effect was better than that of NGR-Pro IL-2 (ie, fusion protein 1) and Pro IL-2 ( Figure 4).
  • NGR 2 -Pro IL-2 ie, fusion protein 4
  • the therapeutic dose of Pro IL-2 was increased to 72 ⁇ g, and the control group was intravenously injected with an equal molar amount of Pro IL-2 fusion protein or an equal volume of PBS.
  • the treatment results showed that NGR 2 -Pro IL-2 (ie, fusion protein 4) can effectively inhibit the growth of CT26 tumors. 4 out of 7 mice were finally completely cured, while the tumors in the Pro IL-2 treatment group and the control group were completely cured. None of them were well inhibited, growing more than 1000mm 3 within one month ( Figures 5A, 5B and 5C).
  • Tumor-targeting peptide NGR improves the uptake of fusion proteins in tumors
  • Tumor-targeting peptide NGR reduces the uptake of fusion proteins in the liver
  • NGR 2 -Pro IL-2 had the lowest uptake in the liver (1.16E+10 ⁇ 1.28E+09), which was significantly lower than NGR-Pro IL-2 (1.58E+10 ⁇ 1.70 E+09), while the hepatic uptake of Pro IL-2 was the highest among all groups (2.55E+10 ⁇ 3.43E+09) (Fig. 7B). From this we concluded that NGR reduced the hepatic uptake of the fusion protein, and as the number of NGR in the fusion protein molecule increased, the hepatic uptake of the fusion protein was further reduced.
  • Pro IL-2-Cy5.5, NGR-Pro IL-2-Cy5.5 and NGR 2 -Pro IL-2-Cy5.5 with the same fluorescence intensity were injected into mice through the tail vein. 48 hours after injection, a small animal fluorescence imager was used to detect the fluorescence enrichment of tumor sites in mice in each group.
  • NGR-Pro IL-2 ie, fusion protein 1
  • NGR 2 -Pro IL-2 ie, fusion protein 4
  • NGR-Pro IL- In treatment group 2 the MCP-1 concentration in the blood 24 hours after the first administration was 36.968 ⁇ 8.332ng/mL, and the MCP-1 concentration in the blood 24 hours after the second administration was 36.685 ⁇ 13.115ng/mL; NGR-Pro The concentration of MCP-1 in the blood of the IL-2 (ie fusion protein 1) treatment group 24 hours after the first administration was 22.652 ⁇ 4.928ng/mL, and the concentration of MCP-1 in the blood 24 hours after the second administration was 23.51 ⁇ 8.44ng/mL; the concentration of MCP-1 in the blood of the P

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Abstract

涉及一种肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体。

Description

肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体 技术领域
本发明属于生物医药技术领域，具体的，涉及一种肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体。
背景技术
白介素-2(IL-2)于上世纪70年代被人们发现，因能够在体外促进T淋巴细胞增殖，也被命名为T细胞增殖因子(T-cell growth factor)[1]。1983年，纯化的IL-2首次应用于临床实验中用于治疗实体瘤(黑色素瘤，肉瘤，腺癌等)[2]，但因为药用剂量较小，IL-2并未展现出明显的肿瘤治疗效果。随后的临床治疗实验采用了大剂量的IL-2，在一些肿瘤类型的病人中表现出了比较好的抗肿瘤特性，因此在1992年高剂量的IL-2首先被批准用于转移性肾细胞癌的治疗[3]，随后在1998年被批准用于黑色素瘤的治疗[4]。
但高剂量的IL-2的临床应用受到了很大的限制，随着剂量的加大，病人出现了严重的副作用[2]，如毛细血管渗漏综合征[5]和肺水肿[6]等，这些副作用严重限制IL-2临床应用的发展，多数病人不能够接受计划的用药剂量而停药，时至今日，这仍然是IL-2肿瘤治疗需要攻克的难题之一，因此开发更安全的IL-2新药刻不容缓。
IL-2的血液循环时间短也是其临床应用的障碍之一，IL-2的血液半衰期只有几分钟[7]，因此病人不得不短时间内频繁接受大剂量的IL-2药物注射。每个疗程包含两次连续给药(600,000IU/kg每8小时给一次药，连续给药5天)[8]。这种频繁的连续给药无疑会对增加药物副作用，加重病人的痛苦。
针对IL-2临床使用的局限，目前IL-2药物研究的主要方向是降低副作用、提高药效和延长血液半衰期。策略包括:
聚乙二醇(PEG)修饰IL-2
NKTR-214(Bempegaldesleukin)是一种目前在临床实验中走的最远的PEG修饰的IL-2药物。通过PEG修饰，NKTR-214的半衰期从1.4小时显著提高到15.5小时，且更偏向于结合肿瘤杀伤T细胞和NK细胞而不是Treg 细胞，并且PEG封闭了IL-2的活性，因此NKTR-214没有较大的副作用[9,10]。但是近期NKTR-214临床实验结果显示，它未能有效提高病人的生存期。
IL-2融合蛋白
目前关于IL-2的融合蛋白多种多样，有单纯延长半衰期的IL-2融合蛋白，如IL-2-人血清白蛋白融合蛋白[11]和IL-2-Fc[12]融合蛋白等；亦有一些融合方法不仅延长了半衰期也赋予了融合蛋白新的功能：ALKS 4230(IL-2与IL-2Rα融合)[13]和IL-2/S4B6[14](IL-2与IL-2Rα抗体融合)增强了促进CD8+T细胞和NK细胞的增殖能力；L19-IL-2(IL-2与抗胞外结构域B抗体融合)[15]和Hu14.18-IL-2(IL-2与抗双唾液酸神经节苷脂抗体融合)[16]增加了IL-2分子的肿瘤靶向能力。
虽然IL-2相关的药物多种多样，但除了高剂量的IL-2，目前仍未有FDA批准的IL-2相关药物上市。聚乙二醇修饰IL-2的最大的问题是化学修饰的质控难以控制，这也导致了其临床试验的失败。其次其血液中缓慢释放PEG恢复活性的方式，可能使IL-2在除肿瘤外的正常组织和器官发挥作用产生安全隐患。IL-2融合蛋白没有聚乙二醇修饰IL-2的质控问题，但因其半衰期以及活性的提高，即使提高了肿瘤靶向能力以降低其脱靶的危害，IL-2融合蛋白仍然会在到达靶组织之前，损伤正常的组织和器官，安全性也仍是其主要问题。
发明内容
我们选择了只在肿瘤内释放活性的IL-2前药为主体，该前药将IL-2与IL-2受体α亚基通过蛋白水解酶的酶切位点(如MMP酶切位点或CD13酶切位点)连接表达，融合的IL-2受体α亚基可以遮蔽IL-2与免疫细胞上IL-2受体的结合，降低IL-2活性，该IL-2前药在血液及正常的组织器官中以封闭的低活性形式存在，只有到达肿瘤部位后，才会被MMP酶切释放IL-2受体α亚基，恢复活性，刺激肿瘤内T细胞。
Fc是抗体IgG的一部分，它可以形成同源二聚体，也可以有效的提高与其融合的蛋白的分子量，增加半衰期。同时含有Fc的融合蛋白可以通过Protein A高效的纯化。
肿瘤靶向能力是抗肿瘤药物增效减毒的关键。但抗肿瘤药物融合其他物质后，其穿透肿瘤细胞的能力，以及实际应用时的表达产量都不同程度受到影响。
肿瘤靶向肽是一类与肿瘤部位特异受体结合的寡肽。与抗体及其片段相比，这类靶向肽分子量小，免疫原性弱，易于表达。我们设计了一种新型的肿瘤靶向肽用来提高细胞因子前体药物的肿瘤靶向性，从而提高肿瘤治疗效果，降低毒副作用，基本保持或提高细胞因子前体药物的表达产量。
例如，我们将IL-2与受体通过金属蛋白酶底物相连，这样就可以降低IL-2与外周受体的结合，一方面降低了IL-2的外周消耗，另一方面降低了IL-2前药在正常组织和器官活化的风险，也降低了药物的毒副作用。通过Fc及其自身的二聚化，一方面使靶向肽和IL-2前体药物翻倍，提高了靶向效果和治疗效果，另一方面也提高了药物的半衰期。
本发明涉及的肿瘤靶向肽具体如下：
CDCRGDCFC(SEQ ID NO:1)，CRGDC(SEQ ID NO:2)，CRGDKGPDC(SEQ ID NO:3)，CNGRCVSGCAGRC(SEQ ID NO:4)，CNGRC(SEQ ID NO:5)，TAASGVRSMH(SEQ ID NO:6)，LTLRWVGLMS(SEQ ID NO:7)，KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK(SEQ ID NO:8)，CGNKRTRGC(SEQ ID NO:9)，CNRRTKAGC(SEQ ID NO:10)，CLSDGKRKC(SEQ ID NO:11)，CREAGRKAC(SEQ ID NO:12)，CAGRRSAYC(SEQ ID NO:13)，CRSRKG(SEQ ID NO:14)，CKAAKNK(SEQ ID NO:15)，CRGRRST(SEQ ID NO:16)，CSRPRRSEC(SEQ ID NO:17)，CGKRK(SEQ ID NO:18)，CDTRL(SEQ ID NO:19)，CGTKRKC(SEQ ID NO:20)；CVLNGRMEC(SEQ ID NO:21)，CVLNGRXEC(SEQ ID NO:22，其中X＝正亮氨酸)，NGRAHA(SEQ ID NO:23)，LNGRE(SEQ ID NO:24)，YNGRT(SEQ ID NO:25)，LNGRAHA(SEQ ID NO:26)，CNGRGEQC(SEQ ID NO:27)，CALNGRXEC(SEQ ID NO:28，其中X＝正亮氨酸)，CVANGRXEC(SEQ ID NO:29，其中X＝正亮氨酸)，CVLNGRAEC(SEQ ID NO:30)，CVLNGRXAC(SEQ ID NO:31，其中X＝正亮氨酸)，CVLNGRXEA(SEQ ID NO:32，其中X＝正亮氨酸)，CVLNGRXC(SEQ ID NO:33，其中X＝正亮氨酸)，CVLNGREC (SEQ ID NO:34)，CVLNGRC(SEQ ID NO:35)，CLNGRXEC(SEQ ID NO:36，其中X＝正亮氨酸)，CVNGRXEC(SEQ ID NO:37，其中X＝正亮氨酸)，CNGRXEC(SEQ ID NO:38，其中X＝正亮氨酸)，CLNGRXC(SEQ ID NO:39，其中X＝正亮氨酸)，CVNGREC(SEQ ID NO:40)，KCNGRC(SEQ ID NO:41)，KVLNGRXE(SEQ ID NO:42，其中X＝正亮氨酸)，GVLNGRMEG(SEQ ID NO:43)，GVLNGRXEG(SEQ ID NO:44，其中X＝正亮氨酸)，GNGRG(SEQ ID NO:45)，CILNGRXEC(SEQ ID NO:46，其中X＝正亮氨酸)，CTLNGRXEC(SEQ ID NO:47，其中X＝正亮氨酸)，CZLNGRXEC(SEQ ID NO:48，其中X＝正亮氨酸)，CVVNGRXEC(SEQ ID NO:49，其中X＝正亮氨酸)，CVZNGRXEC(SEQ ID NO:50，，其中X＝正亮氨酸，Z＝β-alanine)，CVXNGRXEC(SEQ ID NO:51，其中X＝正亮氨酸)，CVSNGRXEC(SEQ ID NO:52，其中X＝正亮氨酸)，CVLNGRSEC(SEQ ID NO:53)，CVLNGRXDC(SEQ ID NO:54，其中X＝正亮氨酸)，CVLNGRXKC(SEQ ID NO:55其中X＝正亮氨酸)，YHWYGYTPQNVI(SEQ ID NO:56，靶向肺癌、乳腺癌、结直肠癌和头颈癌细胞上表达的表皮生长因子受体EGFR)，QRHKPRE(SEQ ID NO:57)，MQLPLAT(SEQ ID NO:58)，AESGDDYCVLVFTDSAWTKICDWSHFRN(SEQ ID NO:59)，MARSGL(SEQ ID NO:60)，MARAKE(SEQ ID NO:61)，MSRTMS(SEQ ID NO:62)，ANTPCGPYTHDCPVKR(SEQ ID NO:63)，YSAYPDSVPMMS(SEQ ID NO:64)，TNYLFSPNGPIARAW(SEQ ID NO:65)，GGVSCMQTSPVCENNL(SEQ ID NO:66)，DPRHCQKRVLPCPAWL(SEQ ID NO:67)，FRERCDKHPQKCTKFL(SEQ ID NO:68)，HVGGSSV(SEQ ID NO:69)，WHPWSYLWTQQA(SEQ ID NO:70)，RRRPKGRGKRRREKQRPTDCHL(SEQ ID NO:71)，或者上述NGR类靶向肽的重复序列(优选2个重复)，优选NGR类靶向肽之间以接头连接，更优选所述接头为(GGGGS)n，n为1-4的整数(最优选n为2)，进一步优选地，所述NGR类靶向肽为CNGRCVSGCAGRCGGGGSGGGGSCNGRCVSGCAGRCSEQ ID NO:88)，或CNGRCGGGGSGGGGSCNGRC(SEQ ID NO:91)。
本发明具体选择的肿瘤靶向肽可以将上述结构的药物很有效的递送到 肿瘤内，在肿瘤内，细胞因子中间的金属蛋白酶底物连接片段可以被肿瘤内特有的金属蛋白酶有效的酶切，从而释放出细胞因子强大的抗肿瘤能力。这样既提高了肿瘤的治愈率，同时又降低了传统细胞因子的毒副作用。因此，肿瘤靶向肽-IL-2前药，很好的解决了临床IL-2效果弱、毒性大以及半衰期短的问题，并且更容易表达纯化，相比于未融合IL2前药而言，获得相当或更好的表达产量，具有一定的临床开发和应用的潜力。
一种靶向肿瘤的融合蛋白，所述靶向肿瘤的融合蛋白包括：
结构单元1：为特异性结合肿瘤细胞或肿瘤血管内皮细胞的靶向结合区，所述靶向结合区由结合肿瘤细胞或肿瘤血管内皮细胞的多肽单元组成；
结构单元2：为抗体Fc片段；和
结构单元3：为白介素-2(IL-2)前药；
任选地，所述融合蛋白还包含接头。
在一些实施方案中，所述融合蛋白中结构单元1，结构单元2和结构单元3可以任意顺序连接，优选地，所述融合蛋白中各结构单元从N端至C端的顺序如下：
(1)结构单元1-X-结构单元2-X-结构单元3；
(2)结构单元1-X-结构单元3-X-结构单元2；
(3)结构单元2-X-结构单元1-X-结构单元3；
(4)结构单元2-X-结构单元3-X-结构单元1；
(5)结构单元3-X-结构单元1-X-结构单元2；或
(6)结构单元3-X-结构单元2-X-结构单元1。
其中X不存在，或X表示接头1或2。
在一些实施方案中，X的序列选自GGGGSGGGGS(SEQ ID NO:72)或GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:73)所示的序列，并且，接头1和接头2的序列可以相同或不同。
在一些实施方案中，所述融合蛋白中各个结构单元从N端开始到C端结束，依次排列方式为：
(1)结构单元1，接头1，结构单元2，接头2，结构单元3；
(2)结构单元3，接头2，结构单元2，接头1，结构单元1；
(3)结构单元1，接头1，结构单元3，接头2，结构单元2；
(4)结构单元3，接头2，结构单元1，接头1，结构单元2；
(5)结构单元2，接头2，结构单元3，接头1，结构单元1；或
(6)结构单元2，接头1，结构单元1，接头2，结构单元3，
优选的，所述的融合蛋白中各个结构单元从N端开始到C端结束，依次排列方式为：结构单元1，接头1，结构单元2，接头2，结构单元3。在一些实施方案中，所述结构单元1和所述结构单元2之间优选含有蛋白水解酶的酶切位点(如MMP酶切位点或CD13酶切位点，优选所述MMP酶切位点的序列为SGARYRWLTA(SEQ ID NO:74)，SGRSENIRTA(SEQ ID NO:75)，SGFIANPATA(SEQ ID NO:76)，RSYAIL(SEQ ID NO:77)或RSPAIF(SEQ ID NO:78)，优选所述CD13酶切位点的序列为RVYIHPF(SEQ ID NO:79)，VYIHPF(SEQ ID NO:80)。
在一些实施方案中，所述的靶向结合区由1个或多个(优选2个或3个)CD13的配体肽段单元(即NGR类肿瘤靶向肽)组成。
在一些实施方案中，所述的配体肽段单元为线性或环状结构，带有NGR序列的短肽，最优选的，所述的配体肽段单元为环状结构，带有NGR序列的短肽。
在一些实施方案中，所述配体肽段单元的氨基酸序列为CNGRC(如SEQ ID NO:5所示)或CNGRCVSGCAGRC(如SEQ ID NO:4所示)，或为SEQ ID NO:5和SEQ ID NO:4所示的氨基酸序列经过取代、缺少、或添加一个或几个氨基酸衍生的与SEQ ID NO:5或SEQ ID NO:4所示的氨酸序列至少有60％同源性(例如至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性)且具有特异结合肿瘤细胞或者肿瘤血管内皮细胞的活性的变体序列。
当所述的靶向结合区含有两个或两个以上的配体肽段单元时，配体肽段单元的之间通过接头相连，接头的序列如SEQ ID NO:72或SEQ ID NO:73所示。
在一些实施方案中，所述的抗体Fc片段为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链(如‘LALA-PG’突变， 参见Rejuvenation of tumor-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells,或Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions，如SEQ ID NO:81所示的序列)；优选的，抗体Fc片段为天然的人IgG(如IgG1，IgG2，IgG3或IgG4)的Fc单链，更优选的，所述的免疫球蛋白Fc单链的氨基酸序列如SEQ ID NO:82所示，最优选地，所述的免疫球蛋白Fc单链的氨基酸序列如SEQ ID NO:83所示(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)。
在一些实施方案中，所述的IL-2前药包含：
野生型IL-2蛋白，所述的IL-2蛋白可以是鼠源IL-2蛋白或人源IL-2蛋白，优选为人源IL-2蛋白，更优选的，所述的人源IL-2蛋白的氨基酸序列如SEQ ID NO:84所示；
肿瘤微环境中特异性表达的蛋白水解酶的酶切位点(如MMP酶切位点或CD13酶切位点)，优选所述MMP酶切位点的序列为SGARYRWLTA(SEQ ID NO:74)，SGRSENIRTA(SEQ ID NO:75)，SGFIANPATA(SEQ ID NO:76)，RSYAIL(SEQ ID NO:77)或RSPAIF(SEQ ID NO:78)，优选所述CD13酶切位点为RVYIHPF(SEQ ID NO:79)，VYIHPF(SEQ ID NO:80)；
IL-2受体亚基胞外域，所述的IL-2受体亚基胞外域可以为能够有效封闭IL-2活性的IL-2受体亚基胞外域，可以是IL-2受体α(IL-2Rα)胞外域和/或IL-2受体β(IL-2Rβ)胞外域；所述IL-2受体亚基胞外域可来自人源或鼠源；优选为人源IL-2受体α和/或IL-2受体β(IL-2Rβ)胞外域；更优选为人源IL-2受体α胞外域，最优选的，所述IL-2受体α胞外域的氨基酸序列如SEQ ID NO:85所示，和
任选地，连接IL-2与蛋白水解酶的酶切位点或连接蛋白水解酶的酶切位点与IL-2受体α(IL-2Rα)的接头，优选的，所述的接头序列如SEQ ID NO:72或73所示。
在一些实施方案中，所述的IL-2前药中各个结构单元的排列方式为：
IL-2受体亚基胞外域C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2蛋白的N端连接(任选地，通过接头连接)。在一些实施方案中，所述的IL-2前药中各个结构单元的排列方式也可以为：
IL-2蛋白C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2受体亚基胞外域的N端连接(任选地，通过接头连接)。
在一些实施方案中，所述的IL-2前药中各个结构单元的排列方式为：
IL-2受体亚基胞外域C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2蛋白的N端连接(任选的，通过接头连接)。
在一些实施方案中，所述融合蛋白为同源或异源二聚体；优选的，所述的肿瘤靶向的融合蛋白为同源二聚体。
在一些实施方案中，所述融合蛋白选自以下各项组成的组：
(1)融合蛋白1(13P.NGR-Pro IL-2)，
从N端至C端方向，依次为靶向结合区(SEQ ID No:4)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白1的氨基酸序列结构如SEQ ID No:87所示；
(2)融合蛋白2(13P.NGR2-Pro IL-2)，
靶向结合区(SEQ ID No:88)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白2的氨基酸序列结构如SEQ ID No:89所示；
(3)融合蛋白3(5P.NGR-Pro IL-2)，
靶向结合区(SEQ ID No:5)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白3的氨基酸序列结构如SEQ ID No:90所示；
(4)融合蛋白4(5P.NGR2-Pro IL-2)，
靶向结合区(SEQ ID No:91)，接头1(SEQ ID No:72)，抗体Fc片段 (SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白4的氨基酸序列结构如SEQ ID No:92所示。
本发明还涉及所述融合蛋白用于治疗肿瘤的用途，或所述融合蛋白在制备治疗肿瘤的药物中的应用。
在本发明的一些实施方案中，所述肿瘤选自前列腺癌、结肠癌、肺癌、甲状腺癌、乳腺癌和胰腺癌。
本发明还涉及所述融合蛋白的制备方法，包括如下步骤：
(1)构建包含编码所述融合蛋白的核酸序列的表达载体，优选的，利用pEE12.4表达载体进行构建；
(2)通过瞬时转染宿主细胞的方法构建包含步骤1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ProteinA/G的亲和层析和分子筛层析纯化所述融合蛋白。
附图说明
图1.肿瘤靶向多肽NGR对肿瘤细胞的结合作用，其中图1A和1B显示肿瘤靶向肽NGR(SEQ ID NO:4)与鼠源(图1A)和人源(图1B)多个肿瘤细胞系结合的流式图与MFI分析统计结果，对照肽为TYNL-RAW(TNYLFSPNGPIARAW，SEQ ID NO:65)；图1C显示ELISA鉴定肿瘤靶向肽NGR(SEQ ID NO:4)对A549细胞的结合力。图1A-1C显示对照肽TYNL-RAW对选择的肿瘤细胞系无靶向结合能力。
图2.NGR-Pro IL-2融合蛋白(即融合蛋白1)的肿瘤抑制效果。其中图2A显示NGR-Pro IL-2融合蛋白的分子结构，中部的二聚体为人IgG的Fc区二聚体，每个Fc单体N端偶联了一分子的NGR多肽，Fc单体C端偶联了一分子的白介素-2前药(白介素-2前药的结构由SEQ ID NO:85所示的IL-2受体α胞外域、SEQ ID NO:74所示的MMP14酶切位点以及SEQ ID NO:84所示的IL-2依次连接组成)；图2B显示NGR-Pro IL-2融合蛋白对肿瘤生长的抑制效果。
图3.融合蛋白对肿瘤细胞的亲和力；其中图3A显示NGR2-Pro IL-2融合蛋白的分子结构，在左边所示NGR-Pro IL-2结构基础上，右边所示的NGR2-Pro IL-2在N端增加了一个NGR多肽；图3B显示NGR多肽序列的优 化，去除了多余的半胱氨酸；图3C显示融合蛋白1-4的分子筛结果。图3D显示细胞ELISA检测NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2(即融合蛋白4)对肿瘤细胞A549的亲和力。
图4.显示NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2融合蛋白(即融合蛋白4)的肿瘤抑制效果。
图5.显示NGR2-Pro IL-2(即融合蛋白4)融合蛋白可以有效抑制肿瘤生长；其中图5A显示给药NGR2-Pro IL
-2后的肿瘤体积；图5B显示CT26荷瘤小鼠生存期；图5C显示各组小鼠肿瘤生长曲线。
图6.显示肿瘤靶向肽提高了融合蛋白在肿瘤的摄取。图6A显示小动物活体荧光成像检测Pro IL-2、NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2(即融合蛋白4)注射后48h在肿瘤的摄取；图6B显示Pro IL-2、NGR-Pro IL-2和NGR2-Pro IL-2肿瘤摄取结果分析。
图7.显示肿瘤靶向肽降低了融合蛋白在肝脏的摄取。图7A显示Pro IL-2、NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2(即融合蛋白4)注射后72h的生物分布；图7B显示Pro IL-2、NGR-Pro IL-2和NGR2-Pro IL-2注射后72h小鼠肝脏摄取。
图8.显示NGR2-Pro IL-2没有显著的系统毒性。图8A显示Pro IL-2和NGR2-Pro IL-2(即融合蛋白4)治疗后的体重监测结果；图8B显示Pro IL-2和NGR2-Pro IL-2第一次和第三次治疗后24h小鼠血液细胞因子检测结果。
具体实施方式
为了进一步对本发明进行说明，下面将通过具体实施例进行说明，但是以下的实施例对本发明的保护范围不构成任何限制。同样，本申请不限于本文描述的任何具体优选的实施方案。本领域技术人员应该理解，对本申请技术特征所作的等同替换、或相应的改进仍属于本申请的保护范围之内。除特别说明的以外，以下实施例采用的试剂均为市售产品，溶液的配制可以采用本领域常规技术。
实验材料
1、菌种和质粒
菌种：DH5αE.coli感受态细胞(北京全式金生物技术有限公司)
质粒：
pEE12.4(Lonza)用于表达Pro IL-2(Pro IL-2的结构由SEQ ID NO:82所示的Fc片段，SEQ ID NO:73所示的接头，SEQ ID NO:85所示的IL-2受体α胞外域、SEQ ID NO:74所示的MMP14酶切位点以及SEQ ID NO:84所示的IL-2依次连接组成,序列如SEQ ID NO:93所示)、5P.NGR-Pro IL-2、13P.NGR-Pro IL-2、5P.NGR2-Pro IL-2和13P.NGR2-Pro IL-2。
2、实验动物
野生型BALB/C小鼠购于中国北京维通利华实验动物心。除特殊说明外，所有实验使用的均为8-10周龄的雌性小鼠。小鼠均在无特定病原微生物(specific pathogen-free，SPF)的屏障环境中饲养。动物的饲养和实验操作遵从清华大学实验动物使用和管理委员会的相关规定。
3、细胞系
LLC购买自ATCC，为C57背景小鼠肺癌细胞系；MC38购买自ATCC，为C57背景小鼠结直肠癌细胞系；
上述这些细胞系在DMEM完全培养基(含10％灭活胎牛血清，2mmol/L L-谷氨酰胺，0.1mmol/L非必需氨基酸，100U青霉素和100μg/mL链霉素)中培养。
CT26购买自ATCC，为BALB/c背景小鼠结直肠癌细胞系；
B16F10购买自ATCC，为C57背景小鼠黑色素瘤细胞系；
A549购买自ATCC，为人非小细胞肺癌细胞。
上述细胞系在RPMI1640完全培养基(含10％灭活胎牛血清，2mmol/L L-谷氨酰胺，0.1mmol/L非必需氨基酸，100U青霉素和100μg/mL链霉素)中培养。
FreeStyleTM 293F细胞系(Invitrogen)为悬浮细胞，源自于HEK293细胞株，培养在SMM293-TII培养基(义翘神州)中，主要用于瞬时转染表达融合蛋白。
基因及引物的设计与合成
实验中所用基因由安升达合成并构建。
融合蛋白制备
所有融合蛋白均由HEK293细胞瞬时转染表达，通过Protein A亲和层析和分子筛superdex 200increase两步纯化。
小鼠肿瘤接种及治疗
(1)肿瘤接种及测量：
肿瘤模型的建立，
2×105个CT26单细胞悬于100μL PBS中，皮下接种于BALB/C小鼠背部；
每周监测两次肿瘤大小，使用游标卡尺测量肿瘤长径(a)、短径(b)，小鼠肿瘤体积＝a×b×b/2。
(2)治疗：
融合蛋白采用腹腔注射方式或静脉的方式，具体给药剂量将在具体实验中叙述。
实施例1.肿瘤靶向多肽的融合显著改善Pro IL-2的肿瘤治疗效果
1.肿瘤靶向多肽具有肿瘤高摄取特性
相比于抗体或抗体片段，多肽具有分子量小、免疫原性低以及易于融合表达等优点。因此我们采取多肽融合表达，而不是抗体融合表达作为增强药物的肿瘤靶向的方法。我们挑选并合成了肿瘤靶向多肽(SEQ ID NO:4和SEQ ID NO:65，其中SEQ ID NO:65不结合肿瘤细胞LLC,CT26,MC38,B16F10和A549，可以用作对照使用)，这些靶向肽的羧基端都偶联了生物素，我们将生物素偶联的多肽与肿瘤细胞系孵育后，洗去未结合的多肽，加入avidin-APC染料，用流式检测了这些多肽对小鼠和人源肿瘤细胞的结合情况，结果显示NGR和多种小鼠肿瘤细胞(LLC,CT26,MC38,B16F10)和人源肿瘤细胞(A549)都有较高的结合(图1A和1B)。进一步地，我们用细胞ELISA实验测得NGR多肽对人非小细胞肺癌细胞A549的亲和力约为321nM(图1C)。这些结果显示了，肿瘤靶向多肽NGR作为抗肿瘤药物靶向目标的潜力。
具体方法如下：
流式检测多肽结合
羧基端偶联生物素的肿瘤靶向肽合成于吉尔生化公司。肿瘤细胞系用含2％胎牛血清(FBS)的磷酸缓冲盐溶液(PBS)制成100μL单细胞悬液，细胞个数为(1-10)×105个。我们将肿瘤靶向肽(终浓度为10μg/mL)和肿瘤细胞系冰上孵育半小时。200g离心除去未结合细胞的多肽，加入1mL含2％FBS的PBS重悬后离心。重复上述步骤一至两次，除去痕量的未结合多肽。100μL含2％FBS的PBS重悬细胞后加入streptavidin-APC(索莱宝)，冰上孵育半小时。洗去多余streptavidin-APC。200μL含2％FBS的PBS重悬，用流式细胞仪检测细胞荧光强度。检测结果使用flowjo分析得出平均荧光强度。
细胞ELISA检测多肽结合
将A549肿瘤细胞铺于96孔板中培养，待细胞汇合度为70％-80％时开始实验。用200μL三乙醇胺缓冲盐水溶液(Tris-Buffered Saline(TBS))清洗两次，用100μL 4％多聚甲醛室温固定细胞20分钟后，用200μL磷酸盐缓冲液清洗三次。加入100μL淬灭缓冲液(1％双氧水溶液)除去内源性的过氧化物酶的活性后，用磷酸盐缓冲液清洗三次。每孔加入200μL含有5％的FBS的PBS，室温放置1小时。后加入浓度梯度稀释的生物素偶联的肿瘤靶向肽，4℃过夜孵育。次日用磷酸盐缓冲液清洗三次。加入avidin-HRP(索莱宝)，室温孵育一个半小时。用PBS清洗三次后，加入50μL HRP底物(biolegend)，室温反应5-30分钟后，用酶标仪检测450nm的吸光。结果用graphpad prism 8拟合结合曲线，得出EC50值。
2.NGR-Pro IL-2融合蛋白的肿瘤抑制效果优于Pro IL-2
鉴于NGR具有较好的肿瘤靶向潜力，我们尝试了NGR融合表达Pro IL-2，以期提高肿瘤治疗前药Pro IL-2的肿瘤靶向效果。我们将1分子的NGR多肽和1分子的Pro IL-2亚基融合表达，得到同源二聚体融合蛋白NGR-Pro IL-2，即融合蛋白1(图2A)，并在CT26肿瘤模型中验证了NGR-Pro IL-2融合蛋白的功能。实验截止时，NGR-Pro IL-2治疗组小鼠肿瘤平均大小为670.9 mm3，而Pro IL-2治疗组小鼠肿瘤平均大小为1475.3mm3，该结果表明NGR-Pro IL-2的肿瘤治疗效果优于Pro IL-2，但并不能完全抑制肿瘤生长(图2B)。该结果提示我们，二聚体单体中单个NGR靶向肽的融合(因为NGR-Pro IL-2为同源二聚体，所以一个前药分子中含有两个NGR肿瘤靶向肽)，即可有效提高抗肿瘤前药Pro IL-2的抗肿瘤效果，那么增加前药分子单体中NGR肿瘤靶向肽的数量，其肿瘤效果可能会进一步增加。
具体方法如下：
BALB/C小鼠(n＝6/组)背部皮下接种2×105个CT26细胞，肿瘤生长至大小约为80mm3时，开始随机分组治疗，腹腔注射72μg NGR-Pro IL-2融合蛋白或者相同摩尔量的Pro IL-2融合蛋白；对照组小鼠注射200μL PBS。每3天给药一次。
3.融合蛋白中肿瘤靶向肽NGR数量的增加可以提高融合蛋白对肿瘤细胞的亲和力
为了进一步增加抗肿瘤前药NGR-Pro IL-2(即融合蛋白1)的治疗效果，我们设计了NGR2-Pro IL-2(即融合蛋白4)，将同源二聚体融合蛋白的单个亚基中NGR肽的数量增加到了2个(图3A)。对13P.NGR进行了优化，将多肽中除NGR motif外，含有二硫键的序列删除，只留下最小的结合单元CNGRC(图3B)。分子筛结果显示，连接截短NGR多肽(CNGRC)的融合蛋白(5P.NGR2-Pro IL-2，即融合蛋白4)相对于连接全长NGR多肽的融合蛋白(13P.NGR2-Pro IL-2，即融合蛋白2)的高聚现象显著减少(图3C)。高聚使得蛋白的产量减少，考虑到蛋白的产量问题，我们采取了截短肽CNGRC而不是全长NGR肽，作为NGR2-Pro IL-2的融合单元。以下所述的NGR2-Pro IL-2，除非有特别说明，都是5P.NGR2-Pro IL-2(即融合蛋白4)，即采用的都是截短肽CNGRC。进一步地，我们用细胞ELISA实验测得NGR2-Pro IL-2(即融合蛋白4)对人非小细胞肺癌细胞A549的亲和力约为8.1nM，相比于NGR-Pro IL-2(即融合蛋白1，41nM)提高了约5倍(图3D)，相比NGR多肽(即SEQ ID NO:4，321nM)提高了约50倍(图1C)。
具体方法如下：
融合蛋白的表达
融合蛋白表达质粒转染对数生长期的293F细胞后，于37℃和8％二氧化碳培养箱中135rpm培养一周。七天后，10000rpm离心1h取上清，使用Protein A(Cytiva)亲和层析初步纯化蛋白后，经superdex 200increase(Cytiva)凝胶过滤层析除去高聚蛋白。融合蛋白13P.NGR-Pro IL-2(融合蛋白1)，5P.NGR-Pro IL-2(融合蛋白3)，5P.NGR2-Pro IL-2(融合蛋白4)产量约为30-40mg/L，同未融合靶向肽的Pro IL-2的产量(30-40mg/L)相比未有明显差异。。
由此合理设计和融合靶向肽，不会降低融合蛋白产量。这主要得于靶向肽的分子量小(几个氨基酸到几十个氨基酸组成)以及结构简单、稳定，而目前融合整个蛋白或者蛋白结构域来增加靶向的方案，受限于所融合的蛋白或者结构域的分子过大以及结构复杂等，通常会使融合蛋白产量大大降低。这一点极不利于工业化生产。
分子筛鉴定蛋白高聚情况
我们使用superdex 200 increase(Cytiva)鉴定了蛋白高聚情况。蛋白经过Protein A(Cytiva)纯化并浓缩后，注入0.5ml的上样环，流动相为20mM Tris-HCl缓冲液，pH 8，含有150mM NaCl，检测280nm的紫外光吸收。多聚或高聚蛋白峰不能进入凝胶孔中，在分子筛中驻留时间短，因此比同源二聚体更早出峰。
细胞ELISA
实验方法同部分1中的“细胞ELISA检测多肽结合”。
4.NGR2-Pro IL-2的抗肿瘤效果优于NGR-Pro IL-2
体外实验证明，随着融合蛋白中NGR多肽数量的增加，其对肿瘤细胞的亲和力也逐步提高，也预示着NGR2-Pro IL-2(即融合蛋白4)融合蛋白可能有更好的肿瘤靶向和治疗效果。体内实验，我们用CT26小鼠模型验证了NGR2-Pro IL-2(即融合蛋白4)融合蛋白的肿瘤治疗效果，当小鼠肿瘤长至60mm3时开始随机分组，低剂量治疗更能体现药物的肿瘤靶向效果，因此 仅静脉注射13μg NGR2-Pro IL-2(即融合蛋白4)或等摩尔量的NGR-Pro IL-2(即融合蛋白1)或Pro IL-2治疗。每3天给药一次。实验截止时，NGR-Pro IL-2(即融合蛋白1)治疗组的小鼠肿瘤平均大小为548.6mm3，Pro IL-2治疗组的小鼠肿瘤平均大小为625.7mm3，NGR-Pro IL-2(即融合蛋白1)治疗组的小鼠肿瘤大小和Pro IL-2治疗组的小鼠肿瘤大小没有统计学差异。而NGR2-Pro IL-2(即融合蛋白4)治疗组的小鼠肿瘤平均大小为358.5mm3，其肿瘤抑制效果优于NGR-Pro IL-2(即融合蛋白1)和Pro IL-2(图4)。
5.NGR2-Pro IL-2能够有效抑制肿瘤生长
接下来我们用CT26小鼠模型，进一步全面地评估了NGR2-Pro IL-2(即融合蛋白4)的肿瘤抑制效果，当小鼠肿瘤长至80mm3左右时开始随机分组，我们提高了NGR2-Pro IL-2(即融合蛋白4)治疗剂量至72μg，对照组静脉注射等摩尔量Pro IL-2融合蛋白或等体积PBS。治疗结果显示，NGR2-Pro IL-2(即融合蛋白4)能够有效抑制CT26肿瘤生长，7只小鼠中最终有4只老鼠完全治愈，而Pro IL-2治疗组和对照组小鼠肿瘤都未能都到很好的抑制，在一个月内生长超过1000mm3(图5A、5B和5C)。
实施例2.肿瘤靶向多肽NGR的融合提高了Pro IL-2的肿瘤靶向，降低了肝脏摄取
1.肿瘤靶向肽NGR提高了融合蛋白在肿瘤的摄取
肿瘤靶向多肽NGR的融合显著改善了Pro IL-2的肿瘤治疗效果，我们推测这一效果的提升是由于NGR多肽的融合提高了Pro IL-2的肿瘤摄取。为了证明这一观点，我们用NHS-Cy5.5通过酯交联反应将药物非定点标记上荧光Cy5.5，并用小动物荧光成像仪观测其静脉注射入小鼠体内后的生物分布情况。在药物注射后的48h，检测肿瘤部位荧光信号强度(图6A)，结果显示，NGR多肽的融合的确提高了药物在肿瘤部位的摄取，且随着分子中NGR多肽数目的增加，小鼠肿瘤部位的平均荧光信号强度也在递增(Pro IL-2-Cy5.5、NGR-Pro IL-2-Cy5.5(即融合蛋白1)和NGR2-Pro IL-2-Cy5.5(即融合蛋白4)注射组小鼠肿瘤总发光效率分别为(7.86E+08)±(4.04E+08)，(2.04E+09)±(6.25E+08)和(2.39E+09)±(1.97E+09)) (图6B)，该结果与治疗结果一致。然而由于荧光成像的灵敏度不够，并未能观察到NGR-Pro IL-2和NGR2-Pro IL-2注射组存在明显差异。
2.肿瘤靶向肽NGR降低了融合蛋白在肝脏的摄取
以上结果我们可以看出肿瘤靶向肽NGR可以提高药物在肿瘤的摄取，因此药物在体内的代谢分布也必将改变。我们在注射后72小时，取小鼠肿瘤及各器官(心脏、肝脏、脾脏、肺和肾)做了荧光检测，结果显示Pro IL-2、NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2(即融合蛋白4)在心脏、脾脏和肺均无摄取，而在肝脏摄取最高，提示它们主要通过肝脏代谢(图7A)。对肝脏荧光信号强度分析的结果显示，NGR2-Pro IL-2在肝脏的摄取最低(1.16E+10±1.28E+09)，显著低于NGR-Pro IL-2(1.58E+10±1.70E+09)，而Pro IL-2的肝脏摄取在所有组中最高(2.55E+10±3.43E+09)(图7B)。由此我们得出，NGR降低了融合蛋白在肝脏摄取，且随着融合蛋白分子中NGR数量增加，融合蛋白在肝脏的摄取进一步降低。
综上，我们认为NGR多肽的融合，提高了药物在肿瘤的摄取，且降低了非靶的正常组织肝脏的摄取。且随着融合蛋白分子中NGR多肽数目的增加，这一趋势越加明显。最终表现为，融合蛋白抗肿瘤效果的提升。
具体方法如下：
蛋白荧光标记
NHS-Cy5.5(西安瑞禧)溶于DMSO后与0.1M碳酸氢钠溶液透析的蛋白混匀，投料比为10:1，4℃反应过夜，次日透析除去未反应的荧光。
小动物成像：
将相同荧光强度的pro IL-2-Cy5.5、NGR-Pro IL-2-Cy5.5和NGR2-Pro IL-2-Cy5.5尾静脉注射入小鼠体内。注射后48小时，使用小动物荧光成像仪检测各组小鼠肿瘤部位荧光富集情况。
生物分布：
将约100微克相同荧光强度的pro IL-2-Cy5.5、NGR-Pro IL-2-Cy5.5(即融合蛋白1)和NGR2-Pro IL-2-Cy5.5(即融合蛋白4)尾静脉注射入小鼠体 内。注射后72小时，取小鼠肿瘤及各器官(心脏、肝脏、脾脏、肺和肾)，使用小动物荧光成像仪检测荧光富集情况。
实施例3.治疗剂量的NGR2-Pro IL-2没有显著的系统毒性
临床上高剂量的IL-2常常引起强烈的副作用。因此我们采用了前药的设计，并融合了肿瘤靶向肽，增强其肿瘤靶向。肿瘤靶向的提高可以增强药效，同时也会降低正常组织器官的摄取，进一步增强药物的安全性。我们监测了NGR2-Pro IL-2(即融合蛋白4)治疗后小鼠的体重变化，并未发现有毒性(图8A)。为了进一步探究NGR2-Pro IL-2(即融合蛋白4)系统性的毒副作用，我们用小鼠炎症因子试剂盒(CBA)对小鼠第一次和第三个给药后24小时血液中的炎症因子：MCP-1、TNF、IL-6、IL-10、IL-12以及导致IL-2主要副作用的细胞因子IFNγ，进行了监测。仅在MCP-1一项中检测到了NGR-Pro IL-2(即融合蛋白1)和NGR2-Pro IL-2(即融合蛋白4)治疗后血液浓度有轻微上升(NGR2-Pro IL-2治疗组第一次给药24小时后血液中MCP-1浓度为36.968±8.332ng/mL，第二次给药24小时后血液中MCP-1浓度为36.685±13.115ng/mL；NGR-Pro IL-2(即融合蛋白1)治疗组第一次给药24小时后血液中MCP-1浓度为22.652±4.928ng/mL，第二次给药24小时后血液中MCP-1浓度为23.51±8.44ng/mL；PBS对照组第一次给药24小时后血液中MCP-1浓度为7.444±0.234ng/mL，第二次给药24小时后血液中MCP-1浓度为12.566±4.016ng/mL，而其他细胞因子特别是导致IL-2毒性的关键细胞因子IFNγ的血液浓度均低于检测阈值(20pg/mL)(图8B)。
具体方法如下：
CBA检测小鼠血液细胞因子浓度
使用CT26小鼠模型，当小鼠肿瘤长至80mm3左右时开始随机分组，治疗组注射72μg NGR2-Pro IL-2(即融合蛋白4)，对照组静脉注射等摩尔量Pro IL-2融合蛋白或等体积PBS。每三天给一次药，第一次给药和第三次给药后24小时，从小鼠眼底静脉丛取血100μL。将小鼠血液样本高速离心后取上清后，按照CBA检测试剂盒(BD)步骤检测。
以上，通过体重监测和给药后血液细胞炎症因子的检测并未发现严重的副反应。
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，不用于限制本发明的保护范围。
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Claims (13)

  1. 融合蛋白，其靶向肿瘤，其中所述融合蛋白包括：
    结构单元1：为肿瘤靶向肽；
    结构单元2：为抗体Fc片段；和
    结构单元3：为IL-2前药；
    任选地，所述融合蛋白还包含接头，
    其中所述的肿瘤靶向肽选自以下各项组成的组：CDCRGDCFC(SEQ ID NO:1)，CRGDC(SEQ ID NO:2)，CRGDKGPDC(SEQ ID NO:3)，TAASGVRSMH(SEQ ID NO:6)，LTLRWVGLMS(SEQ ID NO:7)，KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK(SEQ ID NO:8)，CGNKRTRGC(SEQ ID NO:9)，CNRRTKAGC(SEQ ID NO:10)，CLSDGKRKC(SEQ ID NO:11)，CREAGRKAC(SEQ ID NO:12)，CAGRRSAYC(SEQ ID NO:13)，CRSRKG(SEQ ID NO:14)，CKAAKNK(SEQ ID NO:15)，CRGRRST(SEQ ID NO:16)，CSRPRRSEC(SEQ ID NO:17)，CGKRK(SEQ ID NO:18)，CDTRL(SEQ ID NO:19)，CGTKRKC(SEQ ID NO:20)，YHWYGYTPQNVI(SEQ ID NO:56)，QRHKPRE(SEQ ID NO:57)，MQLPLAT(SEQ ID NO:58)，AESGDDYCVLVFTDSAWTKICDWSHFRN(SEQ ID NO:59)，MARSGL(SEQ ID NO:60)，MARAKE(SEQ ID NO:61)，MSRTMS(SEQ ID NO:62)，ANTPCGPYTHDCPVKR(SEQ ID NO:63)，YSAYPDSVPMMS(SEQ ID NO:64)，TNYLFSPNGPIARAW(SEQ ID NO:65)，GGVSCMQTSPVCENNL(SEQ ID NO:66)，DPRHCQKRVLPCPAWL(SEQ ID NO:67)，FRERCDKHPQKCTKFL(SEQ ID NO:68)，HVGGSSV(SEQ ID NO:69)，WHPWSYLWTQQA(SEQ ID NO:70)，RRRPKGRGKRRREKQRPTDCHL(SEQ ID NO:71)，或NGR类肿瘤靶向肽，所述NGR类肿瘤靶向肽由1个或多个(优选2个或3个)CD13配体肽段单元组成，优选地，所述的CD13配体肽段单元为线性或环状结构，且带有NGR序列的短肽，更优选地，当所述的靶向结合区含有两个或两个以上的CD13配体肽段单元时，CD13配体肽段单元的之间任选通过接头相连，优 选地，所述接头为(GGGGS)n，n为1-4的整数(最优选n为2)。
  2. 权利要求1所述的融合蛋白，其中所述融合蛋白中结构单元1，结构单元2和结构单元3可以任意顺序连接，优选地，所述融合蛋白中各结构单元从N端至C端的顺序如下：
    (1)结构单元1-X-结构单元2-X-结构单元3；
    (2)结构单元1-X-结构单元3-X-结构单元2；
    (3)结构单元2-X-结构单元1-X-结构单元3；
    (4)结构单元2-X-结构单元3-X-结构单元1；
    (5)结构单元3-X-结构单元1-X-结构单元2；或
    (6)结构单元3-X-结构单元2-X-结构单元1，
    其中X不存在，或X表示接头1或2。
  3. 权利要求2所述的融合蛋白，其中X的序列选自GGGGSGGGGS(SEQ ID NO:72)或GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:73)所示的序列，并且，接头1和接头2的序列可以相同或不同。
  4. 权利要求1-3任一项所述的融合蛋白，其中所述融合蛋白中各个结构单元从N端开始到C端结束，依次排列方式为：
    (1)结构单元1，接头1，结构单元2，接头2，结构单元3；
    (2)结构单元3，接头2，结构单元2，接头1，结构单元1；
    (3)结构单元1，接头1，结构单元3，接头2，结构单元2；
    (4)结构单元3，接头2，结构单元1，接头1，结构单元2；
    (5)结构单元2，接头2，结构单元3，接头1，结构单元1；或
    (6)结构单元2，接头1，结构单元1，接头2，结构单元3，
    优选的，所述的融合蛋白中各个结构单元从N端开始到C端结束，依次排列方式为：结构单元1，接头1，结构单元2，接头2，结构单元3。
  5. 权利要求1-3任一项所述的融合蛋白，其中所述结构单元1和所述结构单元2之间优选含有蛋白水解酶的酶切位点(如MMP酶切位点或CD13酶切位点，优选所述MMP酶切位点的序列为SGARYRWLTA(SEQ ID NO:74)，SGRSENIRTA(SEQ ID NO:75)，SGFIANPATA(SEQ ID NO:76)，RSYAIL(SEQ ID NO:77)或RSPAIF(SEQ ID NO:78)，优选所述CD13酶切位点的序列为RVYIHPF(SEQ ID NO:79)，VYIHPF(SEQ ID  NO:80)。
  6. 权利要求1-5任一项所述的融合蛋白，其中所述NGR类肿瘤靶向肽选自以下各项组成的组：CNGRCVSGCAGRC(SEQ ID NO:4)，CNGRC(SEQ ID NO:5)，CVLNGRMEC(SEQ ID NO:21)，CVLNGRXEC(SEQ ID NO:22，其中X＝正亮氨酸)，NGRAHA(SEQ ID NO:23)，LNGRE(SEQ ID NO:24)，YNGRT(SEQ ID NO:25)，LNGRAHA(SEQ ID NO:26)，CNGRGEQC(SEQ ID NO:27)，CALNGRXEC(SEQ ID NO:28，其中X＝正亮氨酸)，CVANGRXEC(SEQ ID NO:29，其中X＝正亮氨酸)，CVLNGRAEC(SEQ ID NO:30)，CVLNGRXAC(SEQ ID NO:31，其中X＝正亮氨酸)，CVLNGRXEA(SEQ ID NO:32，其中X＝正亮氨酸)，CVLNGRXC(SEQ ID NO:33，其中X＝正亮氨酸)，CVLNGREC(SEQ ID NO:34)，CVLNGRC(SEQ ID NO:35)，CLNGRXEC(SEQ ID NO:36，其中X＝正亮氨酸)，CVNGRXEC(SEQ ID NO:37，其中X＝正亮氨酸)，CNGRXEC(SEQ ID NO:38，其中X＝正亮氨酸)，CLNGRXC(SEQ ID NO:39，其中X＝正亮氨酸)，CVNGREC(SEQ ID NO:40)，KCNGRC(SEQ ID NO:41)，KVLNGRXE(SEQ ID NO:42，其中X＝正亮氨酸)，GVLNGRMEG(SEQ ID NO:43)，GVLNGRXEG(SEQ ID NO:44，其中X＝正亮氨酸)，GNGRG(SEQ ID NO:45)，CILNGRXEC(SEQ ID NO:46，其中X＝正亮氨酸)，CTLNGRXEC(SEQ ID NO:47，其中X＝正亮氨酸)，CZLNGRXEC(SEQ ID NO:48，其中X＝正亮氨酸)，CVVNGRXEC(SEQ ID NO:49，其中X＝正亮氨酸)，CVZNGRXEC(SEQ ID NO:50，，其中X＝正亮氨酸，Z＝β-alanine)，CVXNGRXEC(SEQ ID NO:51，其中X＝正亮氨酸)，CVSNGRXEC(SEQ ID NO:52，其中X＝正亮氨酸)，CVLNGRSEC(SEQ ID NO:53)，CVLNGRXDC(SEQ ID NO:54，其中X＝正亮氨酸)，CVLNGRXKC(SEQ ID NO:55其中X＝正亮氨酸)，
    进一步优选地，所述NGR类肿瘤靶向肽为CNGRCVSGCAGRCGGGGSGGGGSCNGRCVSGCAGRC(SEQ ID NO:88)，或CNGRCGGGGSGGGGSCNGRC(SEQ ID NO:91)。
  7. 权利要求1-6任一项所述的融合蛋白，其中所述靶向结合肽的氨基酸序列为CNGRC(如SEQ ID NO:5所示)或CNGRCVSGCAGRC(如SEQ  ID NO:4所示)，或为SEQ ID NO:5和SEQ ID NO:4所示的氨基酸序列经过取代、缺少、或添加一个或几个氨基酸衍生的与SEQ ID NO:5或SEQ ID NO:4所示的氨酸序列至少有60％同源性(例如至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性)，且具有特异结合肿瘤细胞或者肿瘤血管内皮细胞的活性的变体序列。
  8. 权利要求1-6任一项所述的融合蛋白，其中所述抗体Fc片段为天然免疫球蛋白Fc单链或通过基因突变敲除ADCC效应的免疫球蛋白Fc单链(如SEQ ID NO:81所示的序列)；优选的，抗体Fc片段为天然的人IgG(如IgG1，IgG2，IgG3或IgG4)的Fc单链，更优选的，所述的免疫球蛋白Fc单链的氨基酸序列如SEQ ID NO:82所示，最优选地，所述的免疫球蛋白Fc单链的氨基酸序列如SEQ ID NO:83所示(APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)。
  9. 权利要求1-6任一项所述的融合蛋白，其中所述的IL-2前药包含：
    野生型IL-2蛋白，所述的野生型IL-2蛋白可以是鼠源IL-2蛋白或人源IL-2蛋白，优选为人源IL-2蛋白，更优选的，所述的人源IL-2蛋白的氨基酸序列如SEQ ID NO:84所示；
    蛋白水解酶的酶切位点(如MMP酶切位点或CD13酶切位点)，优选所述MMP酶切位点的序列为SGARYRWLTA(SEQ ID NO:74)，SGRSENIRTA(SEQ ID NO:75)，SGFIANPATA(SEQ ID NO:76)，RSYAIL(SEQ ID NO:77)或RSPAIF(SEQ ID NO:78)，优选所述CD13酶切位点为RVYIHPF(SEQ ID NO:79)，VYIHPF(SEQ ID NO:80)；
    IL-2受体亚基胞外域，所述的IL-2受体亚基胞外域为能够有效封闭IL-2活性的IL-2受体亚基胞外域，优选为IL-2受体α(IL-2Rα)胞外域和/或IL-2 受体β(IL-2Rβ)胞外域；优选所述IL-2受体亚基胞外域来自人源或鼠源；优选为人源IL-2受体α和/或IL-2受体β(IL-2Rβ)胞外域；更优选为人源IL-2受体α胞外域，最优选地，所述IL-2受体α胞外域的氨基酸序列如SEQ ID NO:85所示，和
    任选地，连接IL-2蛋白与蛋白水解酶的酶切位点或连接蛋白水解酶的酶切位点与IL-2受体亚基胞外域的接头，优选地，所述的接头序列如SEQ ID NO:72或73所示；
    优选地，所述的IL-2前药中各个结构单元的排列方式为：
    IL-2受体亚基胞外域C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2蛋白的N端连接(任选地，通过接头连接)，
    更优选地，所述的IL-2前药中各个结构单元的排列方式也可以为：
    IL-2蛋白C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2受体亚基胞外域的N端连接(任选地，通过接头连接)，
    最优选地，所述的IL-2前药中各个结构单元的排列方式为：
    IL-2受体亚基胞外域C端与蛋白水解酶的酶切位点的N端连接(任选地，通过接头连接)，蛋白水解酶的酶切位点的C端与IL-2蛋白的N端连接(任选的，通过接头连接)。
  10. 权利要求1-9任一项所述的融合蛋白，其中所述融合蛋白为同源或异源二聚体；优选地，所述的融合蛋白型为同源二聚体。
  11. 权利要求1-10任一项所述的融合蛋白，其中所述融合蛋白选自以下各项组成的组：
    (1)融合蛋白1(13P.NGR-Pro IL-2)，
    从N端至C端方向，依次为靶向结合区(SEQ ID No:4)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白1的氨基酸序列结构如SEQ ID No:87所示；
    (2)融合蛋白2(13P.NGR2-Pro IL-2)，
    靶向结合区(SEQ ID No:88)，接头1(SEQ ID No:72)，抗体Fc片段 (SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白2的氨基酸序列结构如SEQ ID No:89所示；
    (3)融合蛋白3(5P.NGR-Pro IL-2)，
    靶向结合区(SEQ ID No:5)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白3的氨基酸序列结构如SEQ ID No:90所示；
    (4)融合蛋白4(5P.NGR2-Pro IL-2)，
    靶向结合区(SEQ ID No:91)，接头1(SEQ ID No:72)，抗体Fc片段(SEQ ID No:82)，接头2(SEQ ID No:73)，白介素-2(IL-2)前药(SEQ ID No:86)，优选的，所述融合蛋白4的氨基酸序列结构如SEQ ID No:92所示。
  12. 权利要求1-11任一项所述的融合蛋白用于治疗肿瘤的用途，或所述融合蛋白在制备治疗肿瘤的药物中的用途，优选地，所述肿瘤为前列腺癌、结肠癌、肺癌、黑色素瘤、甲状腺癌、乳腺癌或胰腺癌。
  13. 权利要求1-11任一项所述融合蛋白的制备方法，包括如下步骤：
    (1)构建包含编码所述融合蛋白的核酸序列的表达载体，优选的，利用pEE12.4表达载体进行构建；
    (2)通过瞬时转染宿主细胞的方法构建包含步骤1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ProteinA/G的亲和层析和分子筛层析纯化所述融合蛋白。
PCT/CN2023/117716 2022-09-09 2023-09-08 肿瘤靶向的以白介素2为活性成分的融合蛋白型药物前体 WO2024051817A1 (zh)

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