WO2019227266A1 - Polypeptide pour prolonger le temps de circulation sanguine d'un phage - Google Patents

Polypeptide pour prolonger le temps de circulation sanguine d'un phage Download PDF

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WO2019227266A1
WO2019227266A1 PCT/CN2018/088626 CN2018088626W WO2019227266A1 WO 2019227266 A1 WO2019227266 A1 WO 2019227266A1 CN 2018088626 W CN2018088626 W CN 2018088626W WO 2019227266 A1 WO2019227266 A1 WO 2019227266A1
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phage
polypeptide
bcp1
phages
blood
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PCT/CN2018/088626
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Chinese (zh)
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温龙平
金佩佩
沙锐
张云娇
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华南理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to a polypeptide that prolongs the blood circulation time of phage.
  • Phage therapy is a treatment method that uses phage to fight bacterial infections and has been used in clinical practice since the early 20th century. For example, in 1919, d'Herelle tried to use phage to treat avian influenza (Salmonella), rabbit dysentery (Shigella), and bovine hemorrhagic septicemia (Pasteurium), which was the first attempt to use phage therapy. In 1921, Richard Bruynoghe and Joseph Maisin used bacteriophage to treat human skin diseases caused by S. aureus.
  • phage therapy currently has multiple limiting factors.
  • the main problem hindering its clinical application is that the phage is rapidly cleared in the blood circulation after administration, and the short blood retention time prevents it from effectively entering the target site for treatment. Therefore, the engineering treatment of phages that prolongs the blood circulation time is the focus of research. It will be a more effective phage therapy to treat existing bacterial infections or prevent bacterial reinfection.
  • the strategies reported in related studies to extend the blood circulation time of phages include: through naturally occurring mutations, chemical modification of phage surfaces with polyethylene glycol, and avoiding complement-mediated inactivation by changing phage capsid proteins Wait. Nonetheless, the above schemes still have their limitations. The application of more innovative strategies to obtain long-term circulating phages with better antibacterial effects remains to be broken.
  • Phage display as a powerful technique has been used to screen peptide sequences with certain characteristics.
  • phage libraries including phage displaying> 109 different sequence polypeptides
  • phage display technology has been successfully applied to the screening of tumor vascular targeting peptides, blood-brain barrier penetrating peptides, transdermal peptides, and the like.
  • the inventors of the present application used New England Biolabs' phage display peptide library Ph.D. TM -C7C to perform a series of screenings, and finally obtained a short peptide with the function of extending the blood circulation time of the phage.
  • a polypeptide that prolongs the blood circulation time of a phage is provided.
  • the polypeptide that prolongs the blood circulation time of the phage contains the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 or an analog thereof.
  • the polypeptide that prolongs the blood circulation time of the phage can be used to prepare a phage that has the function of extending the blood circulation time.
  • a phage having a function of extending blood circulation time is provided.
  • the phage having the function of extending the blood circulation time carries the polypeptide for extending the blood circulation time of the phage.
  • the application of the phage with the function of extending blood circulation time is provided.
  • the phage with the function of extending blood circulation time can be used for preparing phage antibacterial drugs.
  • a pharmaceutical composition is provided.
  • the pharmaceutical composition includes at least one of the polypeptides that prolong the blood circulation time of phage, and at least one pharmaceutically active protein, drug, or material that is effective in treating a disease.
  • a phage antibacterial drug is provided.
  • the phage antibacterial drug comprises the phage having the function of extending blood circulation time.
  • a time-delayed antibacterial phage is provided.
  • the delayed antibacterial phage carries the polypeptide that prolongs the blood circulation time of the phage and the nucleotide sequence corresponding to the antibacterial protein BglII.
  • a method for screening a polypeptide having the ability to extend the circulating time of a phage in vivo including the following steps:
  • the phage bank is injected into the animal through the tail vein;
  • a polypeptide capable of significantly prolonging the circulation time of the phage is screened by phage display technology, and it proves that the function has sequence specificity, and its principle of action is achieved by binding with platelets; the phage displaying the polypeptide is genetically engineered. It is engineered to express the antibacterial protein BglII at the same time, to obtain a phage with a delayed function and antibacterial activity, which has good antibacterial activity in vitro and in vivo in rats.
  • the polypeptide for prolonging the circulation time of phages in the present invention phages with the function of extending blood circulation time, and phages with both time-delay and antibacterial functions are of great significance for phage therapy.
  • Figure 2 shows the comparison of the in vivo time-delay ability of long-cycle phage monoclonals.
  • Figure 3 shows the long-cycle characteristics of delayed phages.
  • the equivalent (1 ⁇ 10 11 ) BCP1 and SC phages were injected into different rats through the tail vein.
  • Figure 4 shows the mean and SEM (n ⁇ 3, * p ⁇ 0.05, ** p ⁇ 0.01, *) of the number of two types of phage per ml of blood at different time points in rats injected with tail veins mixed with BCP1 and REW phage. ** p ⁇ 0.001).
  • Figure 5 shows the sequence specificity of delayed phages. Equal amounts (1 ⁇ 10 11 ) of different phage BCP1, SC and three BCP1 mutant phages were injected into the tail vein of different rats, and the number of phages per milliliter of blood at different time points was measured. Mean and SEM.
  • Figure 6 shows the BCP1 and three BCP1 mutant phages mixed with REW phages at a 1: 1 ratio.
  • Figure 7 shows the complement system-mediated inactivation of phages.
  • Figures 10A-10C show the distribution of BCP1, SC, and TB2 phages by finely separating the three components of blood;
  • Figure 10A shows the distribution of BCP1 phages in blood in vivo experiments;
  • Figure 10B shows the in vivo experiments of SC phages in blood
  • Figure 11 shows the effect of BCP and SC short peptides on the in vitro binding of phages to blood cells in vitro.
  • 1 ⁇ 10 9 BCP1 phages were incubated with 1 ml of rat whole blood for 1.5 h at 37 ° C in vitro. 500 ⁇ g / ml) and the absence of short peptides in the binding of phage to different blood cells.
  • Figure 18 shows HE staining of rat liver tissue sections showing different cell infiltration effects after treatment with different combinations.
  • phage display library refers to a collection of genetically engineered phage displayed on the surface by expressing a series of polypeptides.
  • a "display peptide” consists of a continuous sequence of amino acids that includes proteins displayed on the surface of a phage.
  • polypeptide refers to a chain consisting of at least three amino acids connected by peptide bonds. This chain can be linear, branched, circular, or a combination thereof.
  • the research object of phage library is to use genetic engineering to express a large number of display peptides of different amino acids. After using this object, collect and identify phage particles in the research object.
  • the "subject” refers to mammals, such as mice, rabbits, and humans.
  • Phage particles are usually collected from one or more organ tissues, cells, blood, urine, or other various body fluids.
  • the phage particles are injected into the animal through the tail vein, and after a period of time Residual phage particles are collected from the blood circulation. It is those phage particles containing peptides that can extend the circulation time in the blood.
  • the peptide sequences expressed on the surface of phage particles collected from experimental animals can be separated by solid medium plates, that is, in cili-positive bacteria, phage particles can be propagated in vitro on biological plates. Bacteria are not lysed by the phage, but instead secrete multiple copies of the phage to display the inserted peptide.
  • the amino acid sequence of the inserted display peptide is determined by the DNA sequence corresponding to the inserted peptide in the phage genome.
  • polypeptide analog refers to other short peptides having a time-delay function obtained by phage screening, or obtained by adding, deleting, changing the order or replacing the amino acid of the polypeptide sequence shown in SEQ ID NO: 1. Peptide.
  • the library used for screening specific time-lapse peptides was provided by New England Biolabs as a heptapeptide library (Ph.D. TM -C7C) containing disulfide bonds.
  • the random fragments in this library are flanked by cysteine residues, which can be oxidized during phage assembly to form disulfide bonds, thus forming a cyclic peptide to interact with the target.
  • This library contains more than two billion clones.
  • the random peptides in the library are at the amino terminus of the small coat protein pIII, so each phage particle expresses five copies. The position of the phage-expressing random sequence in the Ph.D.
  • TM- C7C library was preceded by alanine-cysteine.
  • a short linker (glycine-glycine-glycine-serine) is contained between the random peptide and the pIII protein. (Ph.D. TM -C7C phage peptide display kit, http://www.neb.com/nebecomm/products/productE8120.asp).
  • 1 ⁇ 10 11 phages were dissolved in 300 ⁇ l of physiological saline, and 150 g of SD rats were injected into the tail vein.
  • Whole blood was taken from the body 48 hours later, and spread on X-gal (5-bromo-4-chloro-3-indolyl) -bD-galactoside) and IPTG (isopropyl-bD-thiogalactoside) LB plate, 6 rats obtained 300 plaques. All of them were picked and amplified, and 1 ⁇ 10 11 phages were subjected to the second round of in vivo screening.
  • the 500 plaques obtained after 96 hours were mixed and amplified for the third round of screening, and the time was extended to 120 hours.
  • Figure 1 shows the number of phage in the whole blood of rats after 48 hours of three rounds of screening. It can be seen that with the progress of the screening, the number of phages in each round of the same time point has increased significantly. It reaches 3.5 ⁇ 10 4 pieces.
  • BCP1 CNARGDMHC (SEQ ID NO: 1);
  • BCP2 CIVRGDNVC (SEQ ID NO: 2);
  • BCP6 CVPRGDMHC (SEQ ID NO: 3).
  • Example 2 Take 1 ⁇ 10 11 phages carrying the display peptides BCP1, BCP2, and BCP6 obtained in Example 1, mix them with random phage SC (display peptide sequence: CNATLPHQC, SEQ ID NO: 4), and inject them into the tail vein of rats. Blood samples were taken at 0h (3min) and 72h to detect phage.
  • Figure 2 shows the number of phages at 0h and 72h. It can be seen from the figure that the number of the four kinds of phages was similar at 0h, but SC phages were gone at 72h, and BCP1 accounted for the highest proportion, followed by BCP6 and BCP2. It can be seen that the phages carrying the three display peptides obtained in Example 1 all have the ability to significantly extend the circulation time of phages in the blood of rats.
  • BCP1 and white spot phage REW (display peptide sequence: CTARSPWIC, SEQ ID NO: 5) were mixed 1: 1 and injected into the same rat. The ratio of the two in blood was measured at different time points. The results are shown in Figure 4 As shown.
  • Example 3 Verifying the phage's time-delay function is sequence-specific
  • TB1 CRNHDMGAC (SEQ ID NO: 6, disrupts the BCP1 sequence);
  • TB2 CNAAGAMHC (SEQ ID NO: 7, mutation of RGD to AGA);
  • TB3 CAARGDAAC (SEQ ID NO: 8, RGD is retained, and the remaining four amino acids are mutated to A).
  • Example 4 The phage delayed function is not achieved by the resistance complement system
  • Example 5 Bacteriophage achieves time-delay by interacting with blood cells
  • Figures 9A, 9B, and 9C respectively show the numbers of SC, BCP1, and TB2 phages in plasma and blood cells at different time points.
  • the number of BCP1 phages in plasma is 10 times higher than that of blood-bound phages, and close to 24h. The same, but the number of phages bound to blood cells was much higher than that in plasma at 36h, and continued to 72h.
  • Example 6 Bacteriophage achieves time delay by interacting with platelets in blood cells
  • the blood was taken from the heart at different time points, mixed with 100 ⁇ l CPD, left at room temperature for 15 minutes, and centrifuged at 200g for 20 minutes.
  • the sample is divided into three layers. From top to bottom, the first layer is a platelet-rich region, the second layer is a leukocyte-rich region, and the third layer is a red-cell-rich region.
  • the white blood cell layer and the red blood cell layer can directly detect the titer representing the number of phage binding. Platelets were obtained by centrifuging the first layer of the supernatant at 2000g for 10 minutes.
  • three components can be obtained more accurately by flow sorting.
  • 1 ml of blood was taken from the heart at different time points, mixed with 100 ⁇ l CPD, left at room temperature for 15 minutes, and centrifuged at 200g for 20 minutes.
  • the sample is divided into three layers. From top to bottom, the first layer is a platelet-rich region, the second layer is a leukocyte-rich region, and the third layer is a red-cell-rich region.
  • 0.2M adenosine triphosphate diphosphatase was added to the supernatant platelet layer to prevent platelet activation, and platelets were enriched by centrifugation at 2000g and 4 ° C for 10min.
  • the middle layer of white blood cells is further enriched and recovered by adding red blood cell lysate to remove red blood cells.
  • the three cell components were resuspended in PBS containing 1% FBS, CD16 / 32 antibody was added, and after standing on ice for 10 minutes, FITC-anti-rat CD45, PE-anti-rat Erythroid Cell, and PerCP / Cy5.5 were labeled, respectively.
  • -anti-mouse / rat CD42d antibody was sorted and recovered. Centrifuge at 2000g and 4 ° C for 5min to recover the enriched and sorted cells and check the number of phage. The results are shown in Figure 10, where PLT represents platelets, WBC is white blood cells, and RBC is red blood cells.
  • FIG. 10A show that BCP1 phage binds more to platelets and white blood cells than red blood cells at different time points, while the results of FIGS. 10B and 10C show that the amount of SC and TB2 bound to blood cells is greatly reduced.
  • Example 7 BCP1 phage interacts with platelets via display peptide
  • BCP1 phages were mixed with 1 ml of rat blood in vitro, and the other two groups were added with a synthetic BCP short peptide (sequence: ACNARGDMHCG, SEQ ID NO: 9) or SC short peptide (sequence: ACNATLPHQCG, SEQ ID NO: 10) After incubation at 37 ° C for 1.5 hours, the number of phage binding in the three cell components of blood was detected by flow sorting. The results are shown in FIG. 11.
  • Example 8 Delayed antibacterial phage BCP1-BGL
  • the restriction enzymes EcoR I and Hind III were used to digest the PCR product containing the BglIIR gene (bactericidal gene, which specifically recognizes and cleaves the host cell's BglII site 5'-AGATCT-3 '), and then recovered.
  • the fragment was ligated with M13KE vector (purchased from New England Biolabs) at 16 ° C overnight, and it was determined whether the BCP1 nucleic acid sequence was included between the digestion sites of Eag I and Kpn I on the M13KE vector.
  • the recombinant phage containing the BCP1 nucleic acid sequence was BCP1-BGL. , BGL is not included.
  • the PCR product containing the BglIIR gene is using the following primers:
  • the PMRB1 plasmid was used as a template and obtained by the following PCR conditions:
  • the recombinant phage BCP1-BGL is transformed into ER2738 competent cells containing the PBM1 plasmid (containing the BglII gene, which can methylate the BglII site of the host DNA so as not to be cleaved by the BglII protein).
  • PBM1 plasmid containing the BglII gene, which can methylate the BglII site of the host DNA so as not to be cleaved by the BglII protein.
  • PMRB1 plasmid and PBM1 plasmid are all by Professor Armin Resch of the University of Vienna.
  • Example 9 In vitro antibacterial capacity of antibacterial phage BGL and BCP1-BGL
  • E. coli MC4100F cultured with OD600 to 0.2 and BCP1, SC, BGL, and BCP1-BGL with different ratios of MOI (number of E. coli / number of phage) continued to be cultured in LB medium containing 0.003mol / L IPTG.
  • MOI number of E. coli / number of phage
  • Example 10 In vitro antibacterial release of endotoxin by antibacterial phage BGL and BCP1-BGL
  • Endotoxin detection kit (ToxinSensor TM Chromogenic LAL Endotoxin Assay Kit) was used to detect the content of endotoxin in the culture supernatant at 0, 1, 2 and 4 hours, respectively, and the results are shown in FIG. 13.
  • Example 11 Detecting the in vivo delay ability of antibacterial phage BGL and BCP1-BGL
  • Figures 14A and 14B are the numbers of blood and ascites phages at different time points of the four types of phages. It can be seen that compared to SC and BGL phages, BCP1-BGL phages containing BCP1 still have a longer delay in blood and ascites. Time, indicating that the addition of BGL did not destroy the delay function of BCP1.
  • FIGS. 15A and 15B show that the BCP1-BGL phage-treated group, which has both time-delay and antibacterial ability, has the least amount of E. coli in ascites and liver, which indicates that it has antibacterial ability.
  • Example 13 Detecting the effect of BCP1-BGL treatment on IFN- ⁇ in blood of rats after bacterial infection
  • Example 14 Effects of BCP1-BGL treatment on liver function in rats after bacterial infection
  • liver tissue was coated with paraffin, and sections of about 5 mm were subjected to H & E staining. The results are shown in FIG. 18.
  • FIGS. 17A and 17B show that the BCP1-BGL treatment group with both time-delay and antibacterial ability has less increase in ALT and AST values in the blood compared to the remaining three phages after infection with bacteria, indicating that the liver function of the rats in this group There is less damage.
  • liver tissue section results in FIG. 18 show the same results, indicating that BCP1-BGL has a certain antibacterial activity in vivo.

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Abstract

L'invention concerne un polypeptide pour prolonger le temps de circulation sanguine d'un phage, le polypeptide contenant les séquences d'acides aminés telles que représentées dans SEQ ID NO : 1, 2 ou 3. L'invention concerne également un phage qui porte le polypeptide et a pour fonction de prolonger un temps de circulation sanguine, un procédé de criblage du polypeptide, un médicament antimicrobien contenant le phage, et l'utilisation de tels polypeptides et phages.
PCT/CN2018/088626 2018-05-28 2018-05-28 Polypeptide pour prolonger le temps de circulation sanguine d'un phage WO2019227266A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596219A (zh) * 2009-09-03 2012-07-18 Cj第一制糖株式会社 新型噬菌体和包含所述噬菌体的抗菌组合物
CN106497899A (zh) * 2015-09-04 2017-03-15 佛教慈济医疗财团法人 衍生自鲍氏不动杆菌噬菌体的新颖抗菌肽及其用途
CN108707185A (zh) * 2018-05-24 2018-10-26 华南理工大学 一种延长噬菌体血液循环时间的多肽

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596219A (zh) * 2009-09-03 2012-07-18 Cj第一制糖株式会社 新型噬菌体和包含所述噬菌体的抗菌组合物
CN106497899A (zh) * 2015-09-04 2017-03-15 佛教慈济医疗财团法人 衍生自鲍氏不动杆菌噬菌体的新颖抗菌肽及其用途
CN108707185A (zh) * 2018-05-24 2018-10-26 华南理工大学 一种延长噬菌体血液循环时间的多肽

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MA, XIAOLI ET AL: "Basic Study on Innovation of Phage in Serum and its Mechanism", JOURNAL OF UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA, vol. 1, no. 39, 15 January 2009 (2009-01-15) *

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