WO2016138610A1 - Variant de ligand induisant l'apoptose apparenté au facteur de nécrose des tumeurs conjugué à l'albumine, procédé de préparation associé et utilisation associée - Google Patents

Variant de ligand induisant l'apoptose apparenté au facteur de nécrose des tumeurs conjugué à l'albumine, procédé de préparation associé et utilisation associée Download PDF

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WO2016138610A1
WO2016138610A1 PCT/CN2015/073483 CN2015073483W WO2016138610A1 WO 2016138610 A1 WO2016138610 A1 WO 2016138610A1 CN 2015073483 W CN2015073483 W CN 2015073483W WO 2016138610 A1 WO2016138610 A1 WO 2016138610A1
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albumin
necrosis factor
tumor necrosis
inducing ligand
related apoptosis
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PCT/CN2015/073483
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Chinese (zh)
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卢晓风
杨浩
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四川大学华西医院
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Publication of WO2016138610A1 publication Critical patent/WO2016138610A1/fr

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to the field of genetic engineering, in particular to an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant, a preparation method thereof and use thereof.
  • TNF related apoptosis-inducing ligand is a protein with high selectivity to kill tumor cells. It transmits death signals through the death receptors DR4 and DR5 on the surface of tumor cells, and induces tumor cell apoptosis.
  • the death receptors DR4 and DR5 are highly expressed on the surface of tumor cells and are lowly expressed on the surface of normal cells.
  • the normal cell surface also highly expresses TRAIL decoy receptors DcR1 and DcR2.
  • TRAIL binds to decoy receptors but does not transmit death signals. Therefore, TRAIL is highly toxic or low-toxic to most normal cells while killing tumor cells efficiently.
  • hTRAIL human TRAIL
  • hTRAIL human TRAIL
  • results showed that hTRAIL has good safety and many tumor patients respond to the treatment of hTRAIL.
  • hTRAIL has a small molecular weight (about 20 KD per monomer) and is easily cleared by the kidneys in the body, and its half-life in plasma is less than 30 minutes (Xiang H, et al. Drug metabolism and disposition, 2004, 32(11): 1230-1238.).
  • the "slow half-life" characteristic greatly impairs the in vivo anti-tumor effect of hTRAIL. Therefore, prolonging the half-life of hTRAIL and increasing its anti-tumor effect has become a new research hotspot.
  • Serum albumin as an antitumor drug carrier may increase the half-life of the tumor and may increase the drug absorption of the tumor.
  • Serum albumin is the most abundant protein in mammalian serum that binds to the FcRn receptor.
  • albumin can also be enriched in tumors and sites of inflammation. Therefore, after the drug forms a complex with albumin, it may not only prolong the half-life of the drug, but also enrich the tumor site, thereby enhancing the anti-tumor effect (Kratz F. Journal of Controlled Release, 2014).
  • Methods for preparing hTRAIL-albumin complexes include: 1) direct coupling of hTRAIL to serum albumin; 2) recombinant expression of hTRAIL and albumin encoding gene (Müller N, et al. Biochemical and Biophysical Research Communications, 2010) , 396(4): 793-799).
  • the direct coupling method consumes a large amount of albumin, and the recombinant expression of TRAIL and albumin gene is limited.
  • the introduction of exogenous serum albumin increases the risk of serious infection.
  • the present invention provides an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant, a preparation method thereof and use thereof.
  • the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant of the invention is a fusion protein of a tumor necrosis factor-related apoptosis-inducing ligand and an albumin binding domain, and the albumin binding domain is linked to the tumor necrosis factor via a linker The N-terminus of the related apoptosis-inducing ligand.
  • amino acid sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 2 or 4.
  • amino acid sequence of the albumin binding domain is as shown in SEQ ID NO: 6.
  • albumin binding domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 5.
  • the linker consists of 2-20 amino acids.
  • the linker is a (G4S) 3 linker, the amino acid sequence of which is set forth in SEQ ID NO: 8.
  • albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant is encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or 11. Its amino acid sequence is shown as SEQ ID NO: 10 or 12.
  • the present invention also provides a nucleotide sequence comprising a coding sequence of a tumor necrosis factor-related apoptosis inducing ligand and a coding sequence of an albumin binding domain, which are linked by a coding sequence of a linker.
  • the coding sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 1 or 3.
  • albumin binding domain coding sequence is as shown in SEQ ID NO: 5.
  • linker is a (G4S) 3 linker, and the nucleotide sequence thereof is shown in SEQ ID NO: 7.
  • nucleotide sequence is as shown in SEQ ID NO: 9 or 11.
  • the present invention also provides a recombinant vector or recombinant strain of the aforementioned nucleotide sequence.
  • the present invention also provides a method for preparing the aforementioned albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant, which is prepared by genetic engineering using the aforementioned nucleotide sequence as a target fragment.
  • the present invention also provides the use of the aforementioned albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant in the preparation of a medicament for treating a cell proliferative disorder.
  • the drug for treating a cell proliferative disease is a drug for treating a tumor or an autoimmune disease.
  • the present invention also provides an antitumor drug which is prepared by using the aforementioned albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • the method utilizes the method of adding albumin binding domain to make TRAIL enter the body and use endogenous albumin to prolong the half-life and enhance the anti-tumor effect, and the cost is low, and there is no risk of infection.
  • albumin binding domain itself may affect the activity of the target protein.
  • the modified target protein may also affect its activity after binding to albumin, and the structure of the protein is complex, and the interaction between protein and protein is also It is unclear, therefore, which albumin binding domain is selected, the fusion protein which is obtained by extending the half-life of the target protein and having excellent pharmacodynamic activity is uncertain, and it is highly probable that an inactive fusion protein is obtained.
  • the present inventors screened an albumin binding domain SA21 in the early stage, and did not significantly prolong the half-life of hTRAIL after ligation with hTRAIL; in addition, the albumin binding domain ABD screened by the present invention was ligated to the C-terminus of hTRAIL, not only Significantly prolonged half-life also reduced the in vivo anti-tumor activity of hTRAIL.
  • the present invention obtains a specific albumin binding domain and optimizes its nucleotide sequence, and at the same time, the fusion protein prepared by fusion with TRAIL under a specific linkage mode has a long half-life and excellent antitumor activity, and has achieved unexpected results. Less technical effects.
  • the invention prepares pure albumin-bound tumor necrosis factor by genetic engineering method
  • Sub-related apoptosis-inducing ligand variants ABD-hTRAIL and ABD-mmTRAIL, compared with hTRAIL and mmTRAIL, their half-life is significantly prolonged, and the anti-tumor activity in vivo is also significantly enhanced, and the pharmacodynamic and pharmacokinetic properties are excellent.
  • the clinical application prospects are good.
  • FIG. 1 ABD-hTRAIL purified SDS-PAGE gel electrophoresis.
  • M protein molecular weight standard
  • 1 hTRAIL
  • 2 ABD-hTRAIL
  • FIG. 1 ABD-mmTRAIL purified SDS-PAGE gel electrophoresis.
  • M protein molecular weight standard; 1: ABD-mmTRAIL; 2: mmTRAIL
  • FIG. 3 ABD-hTRAIL and hTRAIL are compared to albumin binding ability.
  • Figure 4 is a comparison of the binding ability of ABD-mmTRAIL and mmTRAIL to albumin.
  • Figure 9 ABD-hTRAIL and hTRAIL tumor uptake and tissue distribution comparison.
  • 1 heart, 2: liver, 3: spleen, 4: lung, 5: kidney, 6: small intestine, 7: colon, 8: muscle, 9: brain, 10: tumor
  • Figure 10 ABD-mmTRAIL and mmTRAIL tumor uptake and tissue distribution comparison.
  • 1 heart, 2: liver, 3: spleen, 4: lung, 5: kidney, 6: small intestine, 7: colon, 8: brain, 9: muscle, 10: tumor
  • FIG. 11 Comparison of anti-tumor effects of ABD-hTRAIL and hTRAIL in vivo (arrow indicates administration time).
  • FIG. 12 Comparison of in vivo antitumor effects of ABD-mmTRAIL and mmTRAIL (arrows indicate administration time).
  • albumin-binding domain was ligated to hTRAIL or mmTRAIL to prepare albumin-bound TRAIL variants, ABD-hTRAIL and ABD-mmTRAIL.
  • hTRAIL and mmTRAIL are fragments of human and monkey full-length TRAIL 114-281 amino acids.
  • the ABD was ligated to the N-terminus of hTRAIL or mmTRAIL via (G4S)3, and the resulting fusion proteins were named ABD-hTRAIL and ABD-mmTRAIL, respectively. Fusion protein for cloning BamHI and NotI restriction sites were added to both ends of the coding gene. The gene was commissioned by Nanjing Jinsirui Company for synthesis.
  • the DNA fragment was recovered by agarose gel, and then ligated with the pQE30 vector which was also digested and recovered by gel, and the ligated product was transformed into TOP10. Positive monoclonal strains were initially identified by ampicillin screening (100 ⁇ g/ml) and double restriction enzyme digestion, and positive clones were further verified by DNA sequence analysis.
  • the plasmid containing the ABD-hTRAIL or ABD-mmTRAIL coding gene was designated as pQE30-ABD-hTRAIL or pQE30-ABD-mmTRAIL, respectively.
  • Table 1 The present invention relates to amino acid and nucleic acid sequences
  • the expression plasmid was extracted, the expression strain M15 was transformed, and a positive clone strain was obtained by screening with LB plate medium containing ampicillin (100 ⁇ g/ml) and kanamycin (30 ⁇ g/ml).
  • the positive clone strain was inoculated into LB liquid medium containing the same antibiotic, and cultured at 37 ° C until the bacterial solution A 600 reached 0.5 to 1, and 0.05-1 mM isopropylthiogalactoside (IPTG) was added to induce expression. After 4 hours, the cells were collected by centrifugation. The obtained cells were resuspended in phosphate buffer (50 m M, pH 8.0).
  • Ni-NTA Super Flow gel was added to the supernatant and slowly shaken at 4 ° C for 3 h. The gel was packed, the heteroprotein was washed with 40 mM imidazole, and eluted with 300 mM imidazole to obtain the protein of interest.
  • the purified protein was endotoxin removed using a de-endotoxin kit (Nanjing Kingsray).
  • the molecular weights of hTRAIL and mmTRAIL were about 20 KD, and the albumin-bound TRAIL variants formed by fusion of ABD, that is, ABD-hTRAIL and ABD-mmTRAIL had molecular weights of about 25 KD.
  • the experimental results show that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants ABD-hTRAIL and ABD-mmTRAIL were prepared by the present invention.
  • ABD-hTRAIL or ABD-mmTRAIL and human serum albumin (HSA) were diluted to the same level with phosphate buffer (10 mM Na 2 HPO 4 , 2.68 mM KCl, 2 mM KH 2 PO 4 ) containing 500 mM NaCl. The molar concentration is then mixed in equal volumes. After 10 to 30 min of binding at room temperature, the mixture sample was analyzed on a gel filtration column Superdex 75 (GE Healthcare). hTRAIL and mmTRAIL were used as controls for the fusion proteins ABD-hTRAIL and ABD-mmTRAIL, respectively.
  • HSA was dissolved to 20 mg/ml with phosphate buffer PBS (137 mM NaCl, 10 mM Na 2 HPO 4 , 2.68 mM KCl, 2 mM KH 2 PO 4 ). HSA (100 ⁇ l/well) was added to the plate and coated overnight at 4 °C. The plate was washed twice with PBS (200 ⁇ l/well), and 100 ⁇ l of different concentrations of fusion protein were added. After incubating for 2 h at 37 ° C, the plate was washed 4 times with PBST (PBS + 0.075% Tween 20), and then incubated with mouse anti-TRAIL antibody for 1 h.
  • PBST PBS + 0.075% Tween 20
  • hTRAIL and mmTRAIL were incubated with HSA and detected with an anti-TRAIL antibody, and the color reaction did not change with an increase in protein concentration.
  • concentration of ABD-hTRAIL and ABD-mmTRAIL increased, the A 450 nm absorption value also increased accordingly. This result indicates that hTRAIL and mmTRAIL hardly bind to HSA.
  • ABD-hTRAIL and ABD-mmTRAIL can be combined with HSA.
  • the experimental results indicate that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention: ABD-hTRAIL and ABD-mmTRAIL all have albumin binding ability.
  • Colon cancer Colo205 cells were seeded in RPMI 1640 and cultured at 37 ° C, 5% CO 2 .
  • ABD-hTRAIL and ABD-mmTRAIL were mixed with has 1:1, respectively, and added to the cells (20000 cells/well) after 30-60 min at room temperature. After overnight action, 10 ul of CCK8 was added to the wells to determine cell viability.
  • ABD-hTRAIL and ABD-mmTRAIL bind to proteins and have a killing effect on tumor cells.
  • the experimental results show that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention: ABD-hTRAIL and ABD-mmTRAIL have tumor cell killing activity after binding to albumin, and can be used for anti-tumor in vivo. .
  • mice were divided into two groups. One group was injected with hTRAIL or mmTRAIL protein at a tail vein of 10 mg/kg. The other group injected the same amount of ABD-hTRAIL or ABD-mmTRAIL. Blood was taken at different time points after injection, heparin was anticoagulated, and mouse plasma was obtained by centrifugation. After the mouse plasma was diluted 25, 50, 100 and 250 times with RMPI1640 medium, the killing activity was measured using Colo205 cells. The rate of protein metabolism in vivo is monitored by changes in residual protein activity in plasma.
  • albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention have slow metabolism and long half-life in vivo compared with hTRAIL and mmTRAIL.
  • the retention time in the body is long.
  • ABD-hTRAIL and hTRAIL were labeled with CF750 fluorescent dye, respectively.
  • 5 ⁇ 10 5 Colo205 cells 100 ⁇ l were inoculated subcutaneously into the right hind limb of nude mice.
  • 50 ug of CF750-labeled protein was administered by tail vein injection, and then at 2, 4, 8 , 24h in vivo imaging scan to observe the protein intake by the tumor.
  • the mice were sacrificed and the organs and tissues were removed for scanning to examine the tissue distribution of the protein.
  • hTRAIL and ABD-hTRAIL were detected in the tumor 2 hours after intravenous injection.
  • the amount of hTRAIL uptake by the tumor increased compared with 2h.
  • the amount of hTRAIL in the tumor was significantly reduced.
  • ABD-hTRAIL uptake reached the peak during 4-8h, but the amount of protein did not decrease significantly after 24h.
  • the mouse tissue was removed and found to have more ABD-hTRAIL content than hTRAIL.
  • the two proteins are mainly distributed in the liver and kidney of the metabolic organs. Consistent with in vivo imaging observations, tissue-organ scans also demonstrated that ABD-hTRAIL is more readily enriched in tumor tissue than hTRAIL.
  • ABD-mmTRAIL and mmTRAIL show the same pattern by tumor absorption and tissue distribution. As a result, as shown in FIG. 10, both in vivo imaging and tissue distribution showed that ABD-mmTRAIL was more easily enriched in tumor tissues than mmTRAIL.
  • albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants ABD-hTRAIL and ABD-mmTRAIL prepared by the present invention were more targeted than hTRAIL and mmTRAIL.
  • 5 ⁇ 10 5 Colo205 cells (100 ⁇ l) were inoculated subcutaneously into the right hind limb of nude mice and randomly divided into groups. At a certain time after inoculation, 5 mg/kg protein was administered by tail vein injection, and the control group was given the same volume of PBS. Tumor size was recorded daily and mice were sacrificed at 24 days and tumors were taken.
  • the tumor growth trend of the ABD-hTRAIL group was significantly delayed compared with the hTRAIL group.
  • the sizes of the PBS control group, hTRAIL group and ABD-hTRAIL group were: 674.3 ⁇ 194.1 mm 3 , 546.1 ⁇ 265.1 mm 3 and 65.6 + 41.7 mm 3 (Fig. 11A). Tumor size observations were consistent with growth curve results ( Figure 11B).
  • the tumor weights of the hTRAIL group and the ABD-hTRAIL group were 0.367 ⁇ 0.06 g, 0.32 ⁇ 0.09 g, and 0.016 ⁇ 0.02 g, respectively.
  • Figure 12 shows that ABD-mmTRAIL is similar to ABD-hTRAIL treatment.
  • the tumor growth was significantly slower than that of the mmTRAIL treatment group in the ABD-mmTRAIL group.
  • the tumor sizes of the PBS control, mmTRAIL group and ABD-mmTRAIL group were 925.7 ⁇ 222.7 mm 3 , 642.4 ⁇ 194.5 mm 3 and 152.3 ⁇ 44.7 mm 3 , respectively (Fig. 12A).
  • Tumor size was consistent with tumor growth curve results (Figure 12B).
  • the average weight of the tumor in the PBS control group, mmTRAIL group and ABD-mmTRAIL group was 0.539 ⁇ 0.069 g, 0.341 ⁇ 0.089 g, and 0.088+0.028 g, respectively.
  • albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention ABD-hTRAIL and ABD-mmTRAIL have stronger in vivo antitumor activity than hTRAIL and mmTRAIL.
  • the invention provides a pure albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant: ABD-hTRAIL and ABD-mmTRAIL by genetic engineering, and their half-life is significantly prolonged compared with hTRAIL and mmTRAIL.
  • ABD-hTRAIL and ABD-mmTRAIL by genetic engineering, and their half-life is significantly prolonged compared with hTRAIL and mmTRAIL.
  • the anti-tumor activity in the body is also obviously enhanced, the pharmacodynamics and pharmacokinetic performance are excellent, and the clinical application prospect is good.

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Abstract

L'invention concerne un variant de ligand induisant l'apoptose apparenté au facteur de nécrose des tumeurs conjugué à l'albumine, et le variant est une protéine de fusion ligand induisant l'apoptose apparenté au facteur de nécrose des tumeurs et domaine de liaison de l'albumine. L'invention concerne également une séquence nucléotidique codant pour le variant, un vecteur recombinant et une bactérie recombinante comprenant la séquence nucléotidique, et un procédé de préparation dudit variant et une utilisation du variant.
PCT/CN2015/073483 2015-03-02 2015-03-02 Variant de ligand induisant l'apoptose apparenté au facteur de nécrose des tumeurs conjugué à l'albumine, procédé de préparation associé et utilisation associée WO2016138610A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012130471A1 (fr) * 2011-04-01 2012-10-04 Universität Stuttgart Polypeptides recombinés membres de la famille des ligands tnf ayant un domaine de liaison à l'anticorps et leurs utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012130471A1 (fr) * 2011-04-01 2012-10-04 Universität Stuttgart Polypeptides recombinés membres de la famille des ligands tnf ayant un domaine de liaison à l'anticorps et leurs utilisations

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