WO2018219301A1 - 一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途 - Google Patents

一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途 Download PDF

Info

Publication number
WO2018219301A1
WO2018219301A1 PCT/CN2018/089069 CN2018089069W WO2018219301A1 WO 2018219301 A1 WO2018219301 A1 WO 2018219301A1 CN 2018089069 W CN2018089069 W CN 2018089069W WO 2018219301 A1 WO2018219301 A1 WO 2018219301A1
Authority
WO
WIPO (PCT)
Prior art keywords
necrosis factor
tumor necrosis
related apoptosis
inducing ligand
htrail
Prior art date
Application number
PCT/CN2018/089069
Other languages
English (en)
French (fr)
Inventor
陶泽
杨浩
卢晓风
Original Assignee
四川大学华西医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川大学华西医院 filed Critical 四川大学华西医院
Publication of WO2018219301A1 publication Critical patent/WO2018219301A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • 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
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the invention relates to the field of biotechnology medicine, in particular to a PDGFR ⁇ targeting tumor pro-apoptosis-inducing ligand variant, a preparation method thereof and use thereof.
  • TNF-related apoptosis-inducing ligand belongs to the tumor necrosis factor (TNF) family, and its C-terminal 114-281 amino acids can be hydrolyzed by proteases into soluble extracellular segments, forming homotrimers. Body binding ability.
  • Membrane receptors for TRAIL include death receptors (DR4 and DR5) and decoy receptors (DcR1 and DcR2).
  • the death receptors DR4 and DR5 contain a death domain, which, when combined with TRAIL, transmits a death signal to the cell and induces apoptosis.
  • the decoy receptor DcR1 molecule does not contain a death domain, and the death domain of DcR2 is incomplete.
  • TRAIL binds to TRAIL but do not transmit a death signal and thus do not induce apoptosis. Both tumor cells and some aberrantly activated cells highly express death receptors, and TRAIL can promote apoptosis of these cells. Normal cells tend to be highly contagious to deny receptors from TRAIL damage. Therefore, TRAIL may be developed as an ideal therapeutic drug.
  • TRAIL has high expression of death receptors on the surface, so TRAIL showed superior tumor cell killing activity under in vitro conditions and is considered as a potential anti-tumor drug.
  • the in vivo antitumor effect of TRAIL does not match its in vitro killing activity against tumor cells. Possible reasons include: 1) TRAIL has a small molecular weight and a short half-life in vivo. Solutions include extending the half-life using methods such as polyethylene glycol modification or serum albumin fusion/binding. 2) Since the decoy receptor is widely expressed in normal tissues, TRAIL is consumed by normal tissues after being in the body, and the amount of tumor sites is small, so the anti-tumor effect is not good.
  • the improved method mainly uses the targeting molecule to deliver TRAIL, which is enriched in the tumor site, thereby improving the anti-tumor effect. It has been studied to deliver TRAIL using tumor cells or tumor neovascular endothelial cells as target cells. Pericytes are important vascular wall cells, which are distributed in the posterior side of vascular endothelial cells and play an important role in regulating the formation and stability of blood vessels. However, there has been no study on the use of pericytes as target cells to deliver TRAIL to tumors.
  • Hepatic stellate cell activation is the starting point of liver fibrosis. Inhibition or clearance of activated hepatic stellate cells can delay the process of liver fibrosis. Studies have found that death receptors DR4 and DR5 are highly expressed during hepatic stellate cell activation. Thus, TRAIL is capable of inducing apoptosis of activated hepatic stellate cells and exhibits anti-fibrotic effects. Long-acting TRAIL modified with PEG has been studied to alleviate the process of liver fibrosis in rats. However, due to the lack of targeting of TRAIL to hepatic stellate cells, the treatment effect is not good.
  • the present invention provides a PDGFR ⁇ -targeted tumor pro-apoptosis-inducing ligand variant, a preparation method thereof and use thereof.
  • the tumor necrosis factor-related apoptosis-inducing ligand variant of the present invention is a fusion protein of tumor necrosis factor-related apoptosis-inducing ligand and Z PDGFR ⁇ , and Z PDGFR ⁇ is linked by a linker in a tumor necrosis factor-related apoptosis-inducing ligand N End or C-end.
  • amino acid sequence of the Z PDGFR ⁇ is represented by SEQ ID NO: 1.
  • the Z PDGFR ⁇ is encoded by the nucleotide sequence set forth in SEQ ID NO: 2.
  • the amino acid sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 3.
  • the tumor necrosis factor-related apoptosis inducing ligand is encoded by the nucleotide sequence set forth in SEQ ID NO:4.
  • linker consists of 2-20 amino acids.
  • linker is a (G4S) 3 linker, the amino acid sequence of which is set forth in SEQ ID NO: 5.
  • linker is encoded by the nucleotide sequence set forth in SEQ ID NO: 6.
  • amino acid sequence of the variant is as shown in SEQ ID NO: 7.
  • the variant is encoded by the nucleotide sequence set forth in SEQ ID NO:8.
  • the present invention also provides a nucleotide sequence comprising a coding sequence for a tumor necrosis factor-related apoptosis inducing ligand and a coding sequence for Z PDGFR ⁇ , which are ligated 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: 4.
  • linker is a (G4S) 3 linker, the nucleotide sequence of which is set forth in SEQ ID NO: 6.
  • 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 tumor necrosis factor-related apoptosis inducing ligand variant, which is characterized in that it is prepared by genetic engineering using the aforementioned nucleotide sequence as a target fragment.
  • the present invention also provides the use of the aforementioned 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 tumor is a colorectal adenocarcinoma.
  • the present invention also provides an antitumor drug which is prepared by using the aforementioned tumor necrosis factor-related apoptosis inducing ligand variant as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • the present invention also provides the use of the aforementioned tumor necrosis factor-related apoptosis inducing ligand variant in the preparation of a medicament for treating an organ fibrotic disease.
  • the drug is a drug for treating liver fibrosis.
  • the present invention also provides a medicament for treating liver fibrosis, which is prepared by using the aforementioned tumor necrosis factor-related apoptosis-inducing ligand variant as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • the present invention also provides a method for treating a cell proliferative disorder, which is characterized in that it is administered to a patient with the aforementioned tumor necrosis factor-related apoptosis-inducing ligand variant.
  • the drug for treating a cell proliferative disease is a drug for treating a tumor or an autoimmune disease.
  • the tumor is a colorectal adenocarcinoma.
  • the present invention also provides a method for treating an organ fibrotic disease which is to give a patient a tumor necrosis factor-related apoptosis inducing ligand variant as described above.
  • the method is a method for treating liver fibrosis.
  • the present invention utilizes a PDGFR ⁇ affinity affibody Z PDGFR ⁇ as a targeting molecule, and fuses it with human TRAIL (hTRAIL) to construct a fusion protein Z-hTRAIL, and finds its anti-tumor and anti-fibrosis effects in vitro and in vivo.
  • hTRAIL is significantly enhanced and has good clinical application prospects.
  • FIG. 5 Comparison of tumor targeting of Z-hTRAIL and hTRAIL; A: In vivo observation of intratumoral enrichment B: tissue distribution;
  • Figure 8 The in vivo therapeutic effect of Z-hTRAIL and hTRAIL on HCT116 tumors
  • Figure 11 Sirius red staining of mouse liver tissue after Z-hTRAIL and hTRAIL treatment
  • Figure 12 Liver tissue fibrosis score (A) and hydroxyproline content (B) after Z-hTRAIL and hTRAIL treatment;
  • Z PDGFR ⁇ consists of 58 amino acids (Table 1).
  • hTRAIL is a fragment of the amino acid composition of the extracellular segment 114-281 of human TRAIL (see Table 1).
  • Z PDGFR ⁇ was ligated to the N-terminus of hTRAIL by (G4S) 3 Linker to construct the fusion protein Z PDGFR ⁇ -(G4S) 3 -hTRAIL (Z-hTRAIL for short) (Fig. 1).
  • the initial coding gene was designed, and then optimized by nucleic acid analysis software, the gene sequence was synthesized by Nanjing Jinsirui Company. At the time of synthesis, an EcoRI/BamHI restriction endonuclease site (EcoRI: gaattc/BamHIggatcc) was added to both ends of the sequence.
  • the gene sequence of ZPDGFR ⁇ was loaded onto the expression plasmid of pQE30-hTRAIL (the nucleotide sequence of the loaded Z-hTRAIL fragment is shown in Table 1) by double digestion and ligation. After the ligation product was transformed into TOP10 E. coli competent state, a recombinant plasmid was obtained.
  • the expression plasmid pQE30-Z-hTRAIL was transfected into M15 E. coli competent state, and the expression strain M15-pQE30-Z-hTRAIL was successfully constructed.
  • the monoclonal strain of the expression strain M15-pQE30-Z-hTRAIL (prepared in Example 1) was picked and inoculated into LB liquid medium with double resistance (containing ampicillin 100 ⁇ g/ml, kanamycin 30 ⁇ g/ml), and shaken at 37 ° C.
  • LB liquid medium with double resistance containing ampicillin 100 ⁇ g/ml, kanamycin 30 ⁇ g/ml
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • the cells were collected by centrifugation (7000 g, 10 min), resuspended in Lysis buffer (50 mM phosphate buffer, pH 8.0; 300 mM NaCl; 20 mM imidazole; 10 mM ⁇ -mercaptoethanol), and Phenylmethanesulfonyl fluoride (PMSF) was added. ) to a final concentration of 1 mM, ultrasonic disruption under ice bath conditions (power 300W, working 10s, interval 30s, a total of 40min). After the completion of the disruption, centrifugation (4 ° C, 25000 g, 10 min) was repeated 4 times, and the supernatant was collected.
  • Lysis buffer 50 mM phosphate buffer, pH 8.0; 300 mM NaCl; 20 mM imidazole; 10 mM ⁇ -mercaptoethanol
  • PMSF Phenylmethanesulfonyl fluoride
  • the combined gelatinous filler is poured into the chromatography column, and after the protein sample is completed, the gel column is washed with Wash buffer (50 mM phosphate buffer, pH 8.0; 300 mM NaCl; 40 mM imidazole; 10 mM ⁇ -mercaptoethanol). 30 times more than the column volume.
  • Protein samples were then collected by Elution buffer (50 mM phosphate buffer, pH 7.6; 300 mM NaCl; 300 mM imidazole; 10 mM ⁇ -mercaptoethanol) and displayed as a single band by SDS-PAGE electrophoresis (Figure 2). Gel filtration chromatography showed a single peak ( Figure 3).
  • Elution buffer 50 mM phosphate buffer, pH 7.6; 300 mM NaCl; 300 mM imidazole; 10 mM ⁇ -mercaptoethanol
  • Example 3 Z-TRAIL variants bind peripheral cells to kill surrounding tumor cells
  • Fig. 4A the surface of the pericytes highly expressed PDGFR ⁇ .
  • Pericytes were co-incubated with FAM-labeled Z-hTRAIL (prepared in Example 2 for Z-hTRAIL, FAM labeling) or hTRAIL, and then analyzed by flow cytometry, and it was found that Z-hTRAIL binds to pericytes, and this binding It can be blocked by PDGFR ⁇ -specific antibodies, indicating that fusion of Z PDGFR ⁇ allows hTRAIL to bind to pericytes.
  • Z-hTRAIL binds to pericytes to have tumor cell killing function
  • the pericytes were pre-incubated with Z-hTRAIL (prepared in Example 2) (1 ⁇ M) for 1 h, washed with PBS and then treated with tumor cells (LS174T and HCT116, 1.5*10 4 ; COLO 205, 2*10 4 ) co-culture overnight, CCK-8 was used to measure cell viability.
  • the survival rate of tumor cells decreased as the number of pericytes incubated with Z-hTRAIL increased.
  • the survival rate of tumor cells was not significantly related to the number of pericytes. This indicates that the Z-hTRAIL of the present invention binds to pericytes and kills tumor cells.
  • hTRAIL cannot bind to pericytes and therefore cannot kill tumor cells.
  • the pH of the Z-hTRAIL of the present invention (prepared in Example 2) was adjusted to 8.0 and mixed with the fluorescent dye CF750 at a molar ratio of 1:8. After reacting for 1 h at room temperature, the free CF750 fluorescent dye was removed by dialysis against PBS buffer. The labeled protein was injected into the LS174T tumor-bearing nude mouse model through the tail vein, and the tumor targeting of Z-hTRAIL was explored using the small animal living imaging system SPECTRAL Lago and Lago X Imaging Systems.
  • Z-hTRAIL of the present invention is more potent at the tumor site than hTRAIL, showing better tumor targeting.
  • the in vivo antitumor effect of the Z-hTRAIL of the present invention was further evaluated using the LS174T, HCT116 and COLO205 tumor-bearing nude mouse models.
  • LS174T tumor colonal adenocarcinoma
  • 10 mg/kg of hTRAIL and the same number of variants of Z-hTRAIL were administered three times through the tail vein.
  • the average size of the PBS group was 850. ⁇ 150mm 3
  • hTRAIL group was 390 ⁇ 80mm 3
  • Z-hTRAIL group was the smallest, only 102 ⁇ 75mm 3 ; average tumor weight was 0.652 ⁇ 0.2g in PBS group, 0.302 ⁇ 0.608g in hTRAIL group, Z-hTRAIL group Then only 0.091 ⁇ 0.039g ( Figure 6).
  • the COLO205 tumor (colorectal adenocarcinoma) model 5 mg/kg of hTRAIL and the same number of variants of Z-hTRAIL were administered twice through the tail vein, and on the 20th day, the average size of the tumor in the PBS group was 670 ⁇ 140mm 3 , 400 ⁇ 80mm 3 in the hTRAIL group, and the smallest in the Z-hTRAIL group, only 70 ⁇ 60mm 3 ; the average weight of the tumor was 0.266 ⁇ 0.114g in the PBS group and 0.23 ⁇ 0.053g in the hTRAIL group, Z-hTRAIL The group was only 0.038 ⁇ 0.035 g ( Figure 7).
  • HCT116 tumor colonal adenocarcinoma
  • 10 mg/kg of hTRAIL and the same number of variants of Z-hTRAIL were administered four times through the tail vein.
  • the average size of the tumor in the PBS group was 782 ⁇ 106.53mm 3 , 368.4 ⁇ 104.6mm 3 in the hTRAIL group, and the smallest in the Z-hTRAIL group, only 205.1 ⁇ 74.6mm 3 ;
  • the average weight of the tumor was 0.491 ⁇ 0.032g in the PBS group and 0.319 ⁇ 0.06g in the hTRAIL group.
  • the Z-hTRAIL group was only 0.11 ⁇ 0.036 g (Fig. 8).
  • Activated hepatic stellate cells are key cells that promote liver fibrosis. Activated stellate cells overexpress PDGFR ⁇ and death receptors, therefore, Z-hTRAIL may be more advantageous in activating stellate cell binding and killing than hTRAIL.
  • the FAM-labeled protein was incubated with activated stellate cells, and then flow-through detection revealed that Z-hTRAIL (prepared in Example 2) bound to activated stellate cells was more than hTRAIL.
  • Activated stellate cells were treated with different concentrations of protein, and the number of residual cells was determined by CCK8 the next day. The cell survival rate of the protein was 100%, and the cell killing rate of the protein was calculated.
  • the cell viability after 10 and 20 nM Z-hTRAIL treatment was 24.6 ⁇ 4.5% and 14.2 ⁇ 1.2%, respectively, and the cell viability after the same dose of hTRAIL was 81.4 ⁇ 4.8% and 59.7 ⁇ 0.25%, respectively. 9). This indicates that Z-hTRAIL is more potent than hTRAIL in killing activated stellate cells in vitro.
  • mice maintained carbon tetrachloride injection.
  • Mouse sera were collected at the sixth week and serum aspartate aminotransferase, alanine aminotransferase and total bilirubin were measured.
  • Liver tissue sections were stained with Sirius red to show collagen fibers and the degree of fibrosis was scored according to the ISHAK standard. The hydroxyproline in the liver was detected by a kit to reflect the collagen fiber content.
  • Figure 10 shows that the liver function index (serum aspartate aminotransferase, alanine aminotransferase and total bilirubin) levels in the model group were significantly different from those in the normal group, indicating that the hepatocyte injury was severe and the modeling was successful.
  • Z FcRn the affinity Z FcRn, which is similar to the Z PDGFR ⁇ sequence but with different recognition specificities, as a control.
  • Z FcRn was ligated to hTRAIL to construct the fusion protein Zf-hTRAIL. Further, in comparison with hTRAIL, it was determined whether the fusion control conjugate could also enhance the anti-tumor effect of hTRAIL.
  • Z FcRn was designed to be ligated to the N-terminus of hTRAIL to construct the fusion protein Zf-hTRAIL.
  • the fusion protein Zf-hTRAIL was prepared according to Example 2 using the E. coli expression system under the same conditions as Z-hTRAIL.
  • LS174T cells (1 ⁇ 10 4 cells) were first inoculated into 96-well plates, and proteins of different concentrations were added after overnight adherence. After the effect was overnight, CCK-8 was added to determine the number of remaining cells. The same volume of PBS was added to the control wells. The cell viability of the PBS-treated group was 100%, and the killing efficiency of Zf-hTRAIL on tumor cells was calculated. Compared with hTRAIL, it was judged whether fusion Z FcRn affected the killing activity of hTRAIL on tumor cells. As a result, as shown in Fig. 13A, the killing efficiency of Zf-hTRAIL against LS174T cells was similar to that of hTRAIL, indicating that the fusion Z FcRn had no significant effect on the activity of hTRAIL.
  • Zf-hTRAIL and TRAIL were compared as described in Example 5. Seven days after subcutaneous inoculation of LS174 cells in nude mice, Zf-hTRAIL, hTRAIL or PBS was injected into the tail vein. The tumor volume was then measured daily and a growth curve was drawn. At the end of the observation, the mice were sacrificed and the tumors were stripped and weighed. As shown in Fig. 13B, the tumor growth rate of the Zf-hTRAIL and hTRAIL treatment groups was slower than that of the PBS treatment group, and the tumor weight was also lighter than that of the PBS group. However, there was no significant difference in growth rate or tumor weight between Zf-hTRAIL and hTRAIL-treated tumors, indicating that the fusion affinity Z FcRn did not significantly enhance the in vivo anti-tumor effect of hTRAIL.
  • the present invention utilizes a fusion protein Z-hTRAIL which can specifically recognize the PDGFR ⁇ affinity Z PDGFR ⁇ as a targeting molecule and is fused with human TRAIL (hTRAIL), and has antitumor and anti-fibrosis effects in vitro and in vivo.
  • hTRAIL is significantly enhanced, whereas fusion proteins constructed using other recognition factors, such as Z FcRn , are not effective.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Transplantation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种肿瘤坏死因子相关凋亡诱导配体变异体，它是肿瘤坏死因子相关凋亡诱导配体与Z PDGFRβ的融合蛋白，Z PDGFRβ通过连接子连接在肿瘤坏死因子相关凋亡诱导配体的N末端或者C末端。本发明还公开了一种如核苷酸序列以及包括它的重组载体、重组菌，还公开了前述变异体的制备方法和用途。本发明的TRAIL变异体蛋白Z-hTRAIL具有良好的肿瘤杀伤活性，并且对肝脏纤维化也有明确疗效，临床应用前景良好。

Description

一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途 技术领域
本发明涉及生物技术药物领域，具体涉及PDGFRβ靶向性肿瘤促凋亡诱导配体变异体及其制备方法和用途。
背景技术
肿瘤坏死因子凋亡相关诱导配体(TRAIL)属于肿瘤坏死因子(TNF)家族成员，其C末端114-281位氨基酸可以被蛋白酶水解为可溶性的胞外段，形成同源三聚体，具有受体结合能力。TRAIL的膜受体包括死亡受体(DR4和DR5)和诱骗受体(DcR1和DcR2)。死亡受体DR4和DR5分子中含有死亡结构域，与TRAIL结合后能向胞内传递死亡信号，诱导细胞凋亡。相反，诱骗受体DcR1分子不含死亡结构域，DcR2的死亡结构域不完整，二者能与TRAIL结合，但不能传递死亡信号，因而不诱导细胞凋亡。肿瘤细胞和一些异常活化细胞均高表达死亡受体，TRAIL能促进这些细胞凋亡。正常细胞往往高表达诱骗受体而免于TRAIL的伤害。因此，TRAIL可能被发展为理想的治疗性药物。
研究发现，肿瘤细胞表面高表达死亡受体，因而TRAIL体外条件下显示了超强的肿瘤细胞杀伤活性，被认为是潜在的抗肿瘤药物。但是，TRAIL的体内抗肿瘤效果与其体外对肿瘤细胞的杀伤活性不匹配。可能的原因包括：1)TRAIL分子量小，体内半衰期短。解决的办法包括利用聚乙二醇修饰或血清白蛋白融合/结合等方法延长半衰期。2)由于诱骗受体广泛表达于正常组织，TRAIL进入体内后被正常组织大量消耗，到达肿瘤部位量少，因而抗肿瘤效果不佳。改善的方法主要是利用导向分子递送TRAIL，使其富集于肿瘤部位，进而提高抗肿瘤效果。已有研究以肿瘤细胞或肿瘤新生血管内皮细胞为靶细胞进行TRAIL的递送。周细胞是重要的血管壁细胞，其分布于血管内皮细胞后侧，对血管的形成及稳定有重要的调控作用。但是，尚无利用周细胞作为靶细胞向瘤内提送TRAIL的研究。
肝星状细胞活化是肝纤维化的始动环节。抑制或清除活化的肝星状细胞能延缓肝纤维化过程。研究发现，肝星状细胞活化过程中会高表达死亡受体DR4和DR5。因此，TRAIL能够诱导活化的肝星状细胞凋亡而显示抗纤维化作用。已有研究用PEG修饰的长效化TRAIL缓解了大鼠肝纤维化进程。但是，由于TRAIL对肝星状细胞缺乏靶向性，治疗效果不佳。
发明内容
为了解决上述问题，本发明提供了一种PDGFRβ靶向性肿瘤促凋亡诱导配体变异体及其制备方法和用途。
本发明肿瘤坏死因子相关凋亡诱导配体变异体，它是肿瘤坏死因子相关凋亡诱导配体 与Z PDGFRβ的融合蛋白，Z PDGFRβ通过连接子连接在肿瘤坏死因子相关凋亡诱导配体的N末端或者C末端。
其中，所述Z PDGFRβ的氨基酸序列如SEQ ID NO：1所示。优选地，所述Z PDGFRβ由SEQ ID NO：2所示的核苷酸序列编码。
其中，所述肿瘤坏死因子相关凋亡诱导配体的氨基酸序列如SEQ ID NO：3所示。优选地，所述肿瘤坏死因子相关凋亡诱导配体由SEQ ID NO：4所示的核苷酸序列编码。
其中，所述连接子由2～20个氨基酸组成。
其中，所述连接子是(G4S) 3连接子，其氨基酸序列如SEQ ID NO：5所示。优选地，所述连接子由SEQ ID NO：6所示的核苷酸序列编码。
其中，所述变异体的氨基酸序列如SEQ ID NO：7所示。优选地，所述变异体由SEQ ID NO：8所示的核苷酸序列编码。
本发明还提供了一种核苷酸序列，它包括肿瘤坏死因子相关凋亡诱导配体的编码序列与Z PDGFRβ的编码序列，二者之间通过连接子的编码序列连接。
其中，所述Z PDGFRβ的编码序列如SEQ ID NO：2所示。
其中，所述肿瘤坏死因子相关凋亡诱导配体的编码序列如SEQ ID NO：4所示。
其中，所述连接子是(G4S) 3连接子，其核苷酸序列如SEQ ID NO：6所示。
其中，其如SEQ ID NO：8所示。
本发明还提供了前述核苷酸序列的重组载体或重组菌。
本发明还提供了一种制备前述肿瘤坏死因子相关凋亡诱导配体变异体的方法，其特征在于：它是以前述核苷酸序列为目标片段，采用基因工程的方法制备得到的。
本发明还提供了前述肿瘤坏死因子相关凋亡诱导配体变异体在制备治疗细胞增生性疾病的药物中的用途。
其中，所述治疗细胞增生性疾病的药物是治疗肿瘤或自身免疫性疾病的药物。所述肿瘤为结直肠腺癌。
本发明还提供了一种抗肿瘤药物，它是以前述肿瘤坏死因子相关凋亡诱导配体变异体为活性成分，加上药学上可接受的辅料制备而成的制剂。
本发明还提供了前述肿瘤坏死因子相关凋亡诱导配体变异体在制备治疗器官纤维化疾病的药物中的用途。
其中，所述药物是治疗肝脏纤维化的药物。
本发明还提供了一种治疗肝脏纤维化的药物，它是以前述肿瘤坏死因子相关凋亡诱导配体变异体为活性成分，加上药学上可接受的辅料制备而成的制剂。
本发明还提供了一种治疗细胞增生性疾病的方法，其特征在于：它是给予患者前述肿瘤坏死因子相关凋亡诱导配体变异体。
其中，所述治疗细胞增生性疾病的药物是治疗肿瘤或自身免疫性疾病的药物。其中，所述肿瘤为结直肠腺癌。
本发明还提供了一种治疗器官纤维化疾病的方法它是给予患者前述肿瘤坏死因子相关凋亡诱导配体变异体。其中，所述方法是治疗肝脏纤维化的方法。
本发明利用能特异性识别PDGFRβ亲和体Z PDGFRβ为导向分子，将其与人源TRAIL(hTRAIL)融合，构建了融合蛋白Z-hTRAIL，并发现其体内外抗肿瘤和抗肝纤维化作用比hTRAIL明显增强，临床应用前景良好。
根据本发明的上述内容，按照本发明相关领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
附图说明
图1 变异体Z-hTRAIL的分子设计；
图2 Z-hTRAIL和hTRAIL的SDS-PAGE电泳；
图3 Z-hTRAIL和hTRAIL凝胶过滤层析；
图4 Z-hTRAIL和hTRAIL与周细胞结合后对肿瘤细胞的杀伤；
图5 Z-hTRAIL和hTRAIL的肿瘤靶向性比较；A:瘤内富集的活体观察B:组织分布；
图6 Z-hTRAIL和hTRAIL对LS174T肿瘤的体内治疗效果；
图7 Z-hTRAIL和hTRAIL对COLO205肿瘤的体内治疗效果；
图8 Z-hTRAIL和hTRAIL对HCT116肿瘤的体内治疗效果；
图9 Z-hTRAIL和hTRAIL对肝星状细胞的结合(A)和杀伤(B)；
图10 Z-hTRAIL和hTRAIL治疗后小鼠肝功能指标；
图11 Z-hTRAIL和hTRAIL治疗后小鼠肝组织天狼星红染色；
图12 Z-hTRAIL和hTRAIL治疗后小鼠肝组织纤维化评分(A)和羟脯氨酸含量(B)；
图13 对照蛋白Zf-TRAIL和TRAIL的体外细胞杀伤(A)和体内抗肿瘤效果(B,C)比较。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1 Z-hTRAIL变异体的分子设计及克隆构建
1、Z-hTRAIL变异体的分子设计
Z PDGFRβ由58个氨基酸组成(表1)。hTRAIL为人TRAIL胞外段114-281的氨基酸组成的片段(见表1)。通过(G4S) 3Linker将Z PDGFRβ连接在hTRAIL的N末端，构建融合蛋白Z PDGFRβ-(G4S) 3-hTRAIL(简称Z-hTRAIL)(图1)。
2、Z-TRAIL变异体表达载体的构建
根据Z PDGFRβ的氨基酸序列，设计其初始编码基因，再经核酸分析软件优化后，由南京金斯瑞公司进行基因序列的合成。合成时在序列的两端分别添加EcoRI/BamHI限制性内切酶酶切位点(EcoRI:gaattc/BamHIggatcc)。通过双酶切和连接作用，将ZPDGFRβ的基因序列装载到pQE30-hTRAIL(装载 后的Z-hTRAIL片段的核苷酸序列如表1所示)的表达质粒上。连接产物转化TOP10大肠杆菌感受态后，获得重组质粒。通过对重组质粒进行测序后发现Z PDGFRβ片段成功装载到表达质粒载体上。将表达质粒pQE30-Z-hTRAIL转入M15大肠杆菌感受态中，成功构建表达菌株M15-pQE30-Z-hTRAIL。
表1本发明涉及的融合蛋白氨基酸和核苷酸序列
Figure PCTCN2018089069-appb-000001
实施例2 Z-TRAIL变异体的蛋白表达及分离纯化
挑取表达菌株M15-pQE30-Z-hTRAIL(实施例1制备)的单克隆接种到双抗性(含氨苄西林100μg/ml，卡那霉素30μg/ml)LB液体培养基中，37℃振荡培养，当菌液浓度A 600至0.8左右时，加入0.05mM异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-1-thiogalactopyranoside,IPTG)，26℃振荡诱导培养14-16小时。离心(7000g，10min)收集菌体，用Lysis buffer(50mM磷酸盐缓冲液，pH8.0；300mM NaCl；20mM咪唑；10mMβ-巯基乙醇)重悬，加入苯甲基磺酰氟(Phenylmethanesulfonyl fluoride，PMSF)至终浓度为1mM，冰浴条件下超声破菌(功率300W，工作10s，间隔30s，共40min)。破菌完成后，离心(4℃，25000g，10min)，重复4次，收集破菌上清。获得的上清与Ni-NTA树脂凝胶(购自Qiagen)按照适量体积比(如V/V＝50:1)混合，4℃振荡结合2h。将结合后的凝胶填料灌入层析柱，待蛋白样品过柱完成后，用Wash buffer(50mM磷酸盐缓冲液，pH8.0；300mM NaCl；40mM咪唑；10mMβ-巯基乙醇)洗涤凝胶柱30倍柱体积以上。然后用Elution buffer(50mM磷酸盐缓冲液，pH7.6；300mM NaCl；300mM咪唑；10mMβ-巯基乙醇)洗脱收集蛋白样品，SDS-PAGE电泳显示为单一条带(图2)。凝胶过滤层析显示为单峰(图3)。
结果表明我们获得了纯的变异体蛋白Z-hTRAIL，采用磷酸盐缓冲液PBS(10mM Na 2HPO 4,137mMNaCl,2.68mMKCl,2mM KH 2PO 4,pH 7.4)透析过夜备用。
实施例3 Z-TRAIL变异体结合周细胞后杀伤周围的肿瘤细胞
利用PDGFRβ特异性抗体与周细胞孵育后，再用流式细胞术分析。结果如图4A所示，周细胞表面高表达PDGFRβ。将周细胞与FAM标记的Z-hTRAIL(取实施例2制备Z-hTRAIL，FAM标记)或hTRAIL共孵育，然后用流式细胞术分析，发现Z-hTRAIL能与周细胞结合，且这种结合能被PDGFRβ特异性抗体封闭，说明融合Z PDGFRβ可让hTRAIL与周细胞结合。为检测与Z-hTRAIL与周细胞结合后是否还具有肿瘤细胞杀伤功能，先将周细胞与Z-hTRAIL(实施例2制备)(1μM)预孵育1h，PBS洗涤后再与肿瘤细胞(LS174T和HCT116，1.5*10 4；COLO205，2*10 4)共培养过夜，CCK-8检测细胞存活率。
结果如图4B所示，肿瘤细胞的存活率随与Z-hTRAIL孵育的周细胞数目的增多而降低。与hTRAIL孵育的周细胞共培养后，肿瘤细胞的存活率与周细胞数目多少无明显关系。这说明本发明Z-hTRAIL能与周细胞结合并杀伤肿瘤细胞。而hTRAIL不能与周细胞结合，因此无法杀伤肿瘤细胞。
实施例4 Z-TRAIL变异体的肿瘤靶向性分析
本发明Z-hTRAIL(实施例2制备)的pH调至8.0后与荧光染料CF750按照摩尔比1:8进行混合标记。室温反应1h后，用PBS缓冲液透析排除游离的CF750荧光染料。标记后的蛋白通过尾静脉注射入LS174T荷瘤裸鼠模型中，利用小动物活体成像系统SPECTRAL Lago and Lago X Imaging Systems探究Z-hTRAIL的肿瘤靶向性。
结果显示，给药后0.5h，Z-hTRAIL组的肿瘤部位有较强的荧光信号，其强度明显高于hTRAIL组，两组荷瘤裸鼠肾脏部位均能检测到很强的荧光信号。在随后的时间点(1,2,4,6h)，Z-hTRAIL组肿瘤部位依然检测到较强的荧光信号，而hTRAIL组荧光信号迅速减弱直至消失(图5A)。6h后处死荷瘤鼠，剥离其主要脏器及肿瘤组织后进行扫描，结果显示，Z-hTRAIL组肿瘤组织中可检测到较强的荧光信号，其信号强度约为hTRAIL组的3倍(图5B)。
这些结果表明，本发明Z-hTRAIL比hTRAIL能够在肿瘤部位富集，显示出更好的肿瘤靶向性。
实施例5 Z-TRAIL变异体的体内抗肿瘤效果
利用LS174T，HCT116以及COLO205荷瘤裸鼠模型进一步评价本发明Z-hTRAIL(实施例2制备)的体内抗肿瘤效果。
在LS174T肿瘤(结直肠腺癌)模型中，10mg/kg的hTRAIL以及相同摩尔数的变异体Z-hTRAIL通过尾静脉给药三次，第16天时处死裸鼠时，PBS组瘤体平均大小为850±150mm 3，hTRAIL组为390±80mm 3，而Z-hTRAIL组则最小，仅有102±75mm 3；瘤体平均重量为PBS组0.652±0.2g，hTRAIL组0.302±0.608g，Z-hTRAIL组则仅有0.091±0.039g(图6)。
在COLO205肿瘤(结直肠腺癌)模型中，5mg/kg的hTRAIL以及相同摩尔数的变异体Z-hTRAIL通过尾静脉给药两次，第20天时处死裸鼠时，PBS组瘤体平均大小为670±140mm 3，hTRAIL组为400±80mm 3，而Z-hTRAIL组则最小，仅有70±60mm 3；瘤体平均重量为PBS组0.266±0.114g，hTRAIL组0.23±0.053g，Z-hTRAIL组则仅有0.038±0.035g(图7)。
在HCT116肿瘤(结直肠腺癌)模型中，10mg/kg的hTRAIL以及相同摩尔数的变异体Z-hTRAIL通过尾静脉给药四次，第19天时处死裸鼠时，PBS组瘤体平均大小为782±106.53mm 3，hTRAIL组为368.4±104.6mm 3，而Z-hTRAIL组则最小，仅有205.1±74.6mm 3；瘤体平均重量为PBS组0.491±0.032g，hTRAIL组0.319±0.06g，Z-hTRAIL组则仅有0.11±0.036g(图8)。
这些结果表明，本发明Z-hTRAIL比hTRAIL具有更强的体内抗肿瘤能力，说明融合Z PDGFRβ能够显著增强hTRAIL的体内抗肿瘤效果，尤其是对结直肠癌的疗效明确。
实施例6 Z-TRAIL变异体的抗肝纤维化作用
活化的肝星状细胞是促进肝纤维化的关键细胞。活化星状细胞高表达PDGFRβ和死亡受体，因此，与hTRAIL相比，Z-hTRAIL可能在活化星状细胞的结合和杀伤方面更具优势。将FAM标记的蛋白与活化星状细胞孵育，然后通过流式检测发现，结合到活化星状细胞的Z-hTRAIL(实施例2制备)比hTRAIL多。用不同浓度蛋白处理活化星状细胞，第二天再用CCK8测定残余细胞数量。以PBS处理的细胞存活率为100％，计算蛋白的细胞杀伤率。显示，10和20nM的Z-hTRAIL作用后的细胞存活率分别为24.6±4.5％和14.2±1.2％，而相同剂量hTRAIL作用后的细胞存活率分别为81.4±4.8％和59.7±0.25％(图9)。这说明Z-hTRAIL比hTRAIL体外条件下杀伤活化星状细胞的能力更强。
为了比较Z-hTRAIL和hTRAIL的体内抗纤维化效果，我们建立了小鼠肝纤维化模型。将四氯化碳溶于橄榄油中，浓度为25％。对四周龄雌性C57小鼠腹腔注射溶解好的四氯化碳，注射频率为每周 两次，注射剂量为首次2.5ml/kg，之后均为5ml/kg。建模4周时，将实验鼠分为模型组和治疗组。模型组注射PBS，治疗组分别给予Z-hTRAIL或hTRAIL蛋白(10mg/kg)，每周两次。治疗期间，三组小鼠维持四氯化碳注射。在第六周收取小鼠血清，检测血清谷草转氨酶、谷丙转氨酶和总胆红素。肝脏组织切片，用天狼星红染色显示胶原纤维并对纤维化程度按ISHAK标准进行评分，同时用试剂盒检测肝脏中羟脯氨酸，反映胶原纤维含量。
图10显示，模型组肝功能指标(血清谷草转氨酶、谷丙转氨酶及总胆红素)水平与正常组有显著差异，说明肝细胞损伤严重，建模成功。
hTRAIL治疗组三种血清指标水平与模型组无明显差异。但Z-hTRAIL治疗组三种血清指标均低于hTRAIL治疗组。天狼星红染色显示Z-hTRAIL治疗组肝内胶原纤维沉积少于hTRAIL治疗组(图11)，纤维化评分和羟脯氨酸含量均低于hTRAIL治疗组(图12)。
以上结果说明，本发明Z-hTRAIL相对于hTRAIL对小鼠肝脏纤维化具有更好的治疗效果。
对比例1 融合其它亲合体对TRAIL抗肿瘤效果的影响
为了说明融合Z PDGFRβ对增强TRAIL活性的重要性，我们选择了与Z PDGFRβ序列相似，但识别特异性不同的亲合体Z FcRn为对照。将Z FcRn与hTRAIL连接，构建融合蛋白Zf-hTRAIL。进一步与hTRAIL比较，确定融合对照亲合体是否同样可以增强hTRAIL的抗肿瘤效果。
1、Zf-hTRAIL变异体的分子设计和制备
按照实施例1，设计将Z FcRn连接在hTRAIL N末端，构建融合蛋白Zf-hTRAIL。按照实施例2，用与Z-hTRAIL相同的条件，用大肠杆菌表达系统制备融合蛋白Zf-hTRAIL。
2、Zf-hTRAIL变异体的对肿瘤细胞的体外杀伤及体内抗肿瘤作用
为了测试Zf-hTRAIL对肿瘤细胞的杀伤活性，先将LS174T细胞(1×10 4个)接种于96孔板，贴壁过夜后加入不同浓度蛋白。作用过夜后，再加CCK-8测定残存细胞的数量。对照孔中加入相同体积的PBS。以PBS处理组的细胞存活率为100％，计算Zf-hTRAIL对肿瘤细胞的杀伤效率。与hTRAIL进行比较，判断融合Z FcRn是否影响了hTRAIL对肿瘤细胞的杀伤活性。结果如图13A所示，Zf-hTRAIL对LS174T细胞的杀伤效率与hTRAIL相似，说明融合Z FcRn对hTRAIL的活性没有显著影响。
按照实施例5描述的方法，比较Zf-hTRAIL与TRAIL对LS174T肿瘤的治疗效果。在裸鼠皮下接种LS174细胞后7天，尾静脉注射Zf-hTRAIL、hTRAIL或PBS。然后每天测量肿瘤体积，绘制生长曲线。观察结束，处死小鼠，剥取瘤体称重。如图13B所示，Zf-hTRAIL和hTRAIL处理组肿瘤生长速度慢于PBS处理组，瘤体重量也轻于PBS组。但是，Zf-hTRAIL和hTRAIL处理的肿瘤无论是生长速度还是瘤重都没有明显差异，说明融合亲合体Z FcRn并没有显著增强hTRAIL的体内抗肿瘤效果。
表2对照蛋白氨基酸和核苷酸序列
Figure PCTCN2018089069-appb-000002
综上，本发明利用能特异性识别PDGFRβ亲和体Z PDGFRβ为导向分子，将其与人源TRAIL(hTRAIL)融合构建的融合蛋白Z-hTRAIL，其体内外抗肿瘤和抗肝纤维化作用比hTRAIL明显增强，而采用其他识别因子，如，Z FcRn构建的融合蛋白则效果不佳。

Claims (22)

  1. 一种肿瘤坏死因子相关凋亡诱导配体变异体，其特征在于：它是肿瘤坏死因子相关凋亡诱导配体与Z PDGFRβ的融合蛋白，Z PDGFRβ通过连接子连接在肿瘤坏死因子相关凋亡诱导配体的N末端或者C末端。
  2. 根据权利要求1所述的肿瘤坏死因子相关凋亡诱导配体变异体，其特征在于：所述Z PDGFRβ的氨基酸序列如SEQ ID NO：1所示；
    和/或，所述肿瘤坏死因子相关凋亡诱导配体的氨基酸序列如SEQ ID NO：3所示；
    和/或，所述连接子由2～20个氨基酸组成，优选所述连接子是(G4S) 3连接子，其氨基酸序列如SEQ ID NO：5所示。
  3. 根据权利要求23所述的肿瘤坏死因子相关凋亡诱导配体变异体，其特征在于：所述Z PDGFRβ由SEQ ID NO：2所示的核苷酸序列编码；和/或，所述肿瘤坏死因子相关凋亡诱导配体由SEQ ID NO：4所示的核苷酸序列编码；和/或，所述连接子由SEQ ID NO：6所示的核苷酸序列编码。
  4. 根据权利要求1～3任意一项所述的肿瘤坏死因子相关凋亡诱导配体变异体，其特征在于：其氨基酸序列如SEQ ID NO：7所示。
  5. 根据权利要求4所述的肿瘤坏死因子相关凋亡诱导配体变异体，其特征在于：其由SEQ ID NO：8所示的核苷酸序列编码。
  6. 一种核苷酸序列，其特征在于：它包括肿瘤坏死因子相关凋亡诱导配体的编码序列与Z PDGFRβ的编码序列，二者之间通过连接子的编码序列连接。
  7. 根据权利要求6所述的编码序列，其特征在于：所述Z PDGFRβ的编码序列如SEQ ID NO：2所示；和/或，所述肿瘤坏死因子相关凋亡诱导配体的编码序列如SEQ ID NO：4所示；和/或，所述连接子是(G4S) 3连接子，其核苷酸序列如SEQ ID NO：6所示。
  8. 根据权利要求7所述的核苷酸序列，其特征在于：其如SEQ ID NO：8所示。
  9. 包含权利要求6～8任意一项所述核苷酸序列的重组载体或重组菌。
  10. 一种制备权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体的方法，其特征在于：它是以权利要求6～8任意一项所述核苷酸序列为目标片段，采用基因工程的方法制备得到的。
  11. 权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体在制备治疗细胞增生性疾病的药物中的用途。
  12. 根据权利要求11所述的用途，其特征在于：所述治疗细胞增生性疾病的药物是治疗肿瘤或自身免疫性疾病的药物。
  13. 根据权利要求12所述的用途，其特征在于：所述肿瘤为结直肠腺癌。
  14. 一种抗肿瘤药物，其特征在于：它是以权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体为活性成分，加上药学上可接受的辅料制备而成的制剂。
  15. 权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体在制备治疗器官纤维化疾病的药物中的用途。
  16. 根据权利要求15所述的用途，其特征在于：所述药物是治疗肝脏纤维化的药物。
  17. 一种治疗肝脏纤维化的药物，其特征在于：它是以权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体为活性成分，加上药学上可接受的辅料制备而成的制剂。
  18. 一种治疗细胞增生性疾病的方法，其特征在于：它是给予患者权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体。
  19. 根据权利要求18所述的方法，其特征在于：所述治疗细胞增生性疾病的药物是治疗肿瘤或自身免疫性疾病的药物。
  20. 根据权利要求19所述的方法，其特征在于：所述肿瘤为结直肠腺癌。
  21. 一种治疗器官纤维化疾病的方法，其特征在于：它是给予患者权利要求1～5任意一项所述肿瘤坏死因子相关凋亡诱导配体变异体。
  22. 根据权利要求21所述的方法，其特征在于：所述方法是治疗肝脏纤维化的方法。
PCT/CN2018/089069 2017-05-31 2018-05-30 一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途 WO2018219301A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710398226 2017-05-31
CN201710398226.5 2017-05-31

Publications (1)

Publication Number Publication Date
WO2018219301A1 true WO2018219301A1 (zh) 2018-12-06

Family

ID=64455744

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/089069 WO2018219301A1 (zh) 2017-05-31 2018-05-30 一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途

Country Status (2)

Country Link
CN (1) CN109400711B (zh)
WO (1) WO2018219301A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112870390B (zh) * 2021-01-29 2022-04-22 四川大学华西医院 一种镓68标记的亲合体蛋白pet显像剂及应用
CN113444182B (zh) * 2021-06-22 2022-07-19 四川大学华西医院 一种靶向递送IgG类抗体的融合蛋白载体及其用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009077175A1 (en) * 2007-12-19 2009-06-25 Affibody Ab Polypeptide derived from protein a and able to bind pdgf
WO2013098755A2 (en) * 2011-12-28 2013-07-04 Adamed Sp. Z O.O. Anticancer fusion protein

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009025846A2 (en) * 2007-08-22 2009-02-26 The Regents Of The University Of California Activatable binding polypeptides and methods of identification and use thereof
CN101157729B (zh) * 2007-10-23 2011-01-12 南京大学 一种肿瘤坏死因子相关凋亡配体变体及其应用
US8124725B2 (en) * 2007-12-19 2012-02-28 General Electric Company PDGF-Rβ binders
WO2011047180A1 (en) * 2009-10-14 2011-04-21 Merrimack Pharmaceuticals, Inc. Bispecific binding agents targeting igf-1r and erbb3 signalling and uses thereof
US20130150566A1 (en) * 2011-07-06 2013-06-13 Targetpharma Laboratories (Changzhou) Co., Ltd Tumor-targeted tnf-related apoptosis-inducing ligand's variant and the application thereof
CN102775497B (zh) * 2012-07-13 2014-07-23 浙江大学 肿瘤坏死因子相关的凋亡诱导配体融合蛋白及其制备方法
CN104342444B (zh) * 2013-07-23 2017-11-17 四川大学华西医院 一种重组trail蛋白及其制备方法和用途
US9901620B2 (en) * 2014-04-21 2018-02-27 Theraly Pharmaceuticals, Inc. Trail receptor agonists for treatment of fibrotic disease
CN105985445B (zh) * 2015-02-11 2020-02-04 四川大学华西医院 一种肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途
CN105985447B (zh) * 2015-03-02 2019-10-22 四川大学华西医院 一种白蛋白结合型肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途
CN106188311B (zh) * 2016-07-18 2020-08-04 山东大学齐鲁医院 一种重组肿瘤坏死因子相关细胞凋亡诱导配体蛋白的制备方法与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009077175A1 (en) * 2007-12-19 2009-06-25 Affibody Ab Polypeptide derived from protein a and able to bind pdgf
WO2013098755A2 (en) * 2011-12-28 2013-07-04 Adamed Sp. Z O.O. Anticancer fusion protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAO, Z. ET AL.: "Targeted Delivery to Tumor-associated Pericytes via an Affibody with High Affinity for PDGFR(3 Enhances the in vivo Antitumor Effects of Human TRAIL", THERANOSTICS, vol. 7, no. 8, 1 June 2017 (2017-06-01), pages 2261 - 2276, XP055551028 *

Also Published As

Publication number Publication date
CN109400711A (zh) 2019-03-01
CN109400711B (zh) 2022-02-08

Similar Documents

Publication Publication Date Title
JP6018648B2 (ja) キメラのコイルドコイル（二重コイル）分子を使用する方法
ES2279649T3 (es) Compuestos trombopoyeticos.
JP2017529059A5 (zh)
EP3453401A1 (en) Interleukin combination and use thereof
Heimbrook et al. Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts.
KR101841211B1 (ko) 세포 투과성 펩티드 및 이를 이용한 생물학적 활성 물질의 전달방법
WO1999051643A1 (en) Mutagenized il13-based chimeric molecules
JP6209616B2 (ja) ｒＴＲＡＩＬ突然変異体およびそのモノメチルアウリスタチンＥコンジュゲート
WO2003093478A1 (en) Immunoconjugates for the treatment of tumours
PT98048B (pt) Metodo de tratamento de celulas cancerosas da bexiga utilizando proteinas hibridas, nomeadamente pe40 modificado ligado a tgf-alfa
JP2003530360A (ja) 薬剤送達のためのペプチド複合体
WO2018219301A1 (zh) 一种PDGFRβ靶向性肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途
CN103805621B (zh) 靶向性抗肿瘤融合蛋白质lpo的新型制备工艺
JP2007527206A (ja) がん治療のためのペプタボディ
WO2021083077A1 (zh) 半衰期延长的药物及其文库、以及制备方法和应用
CN109157656B (zh) 一种双靶标肿瘤疫苗及其制备方法和应用
KR20080076622A (ko) 올리고머화된 단백질 전달체와 이를 이용한 세포내바이러스 벡터 전달 방법
US10428132B2 (en) Tumor necrosis factor-related apoptosis-inducing ligand variant, as well as a preparation method and use thereof
WO2022241167A1 (en) Delivery constructs derived from bacterial toxins and uses thereof
CN110386985B (zh) 一种肿瘤靶向性促凋亡融合蛋白及其用途
CN103865899B (zh) 具有vegfr2/kdr受体特异性的融合毒素及其编码基因与应用
CN102260352B (zh) 靶向性白细胞介素融合蛋白及其制备方法与应用
KR20130126549A (ko) 항종양괴사인자 수용체 2를 발현하도록 설계된 미니서클 벡터를 이용한 자가면역질환 치료
CN113817071B (zh) 一种egfr靶向的trail融合蛋白及其制备方法和用途
CN105985445B (zh) 一种肿瘤坏死因子相关凋亡诱导配体变异体及其制备方法和用途

Legal Events

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

Ref document number: 18810221

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18810221

Country of ref document: EP

Kind code of ref document: A1