WO2025007748A1 - 纳米递送系统、其制备方法和用途 - Google Patents

纳米递送系统、其制备方法和用途 Download PDF

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WO2025007748A1
WO2025007748A1 PCT/CN2024/100135 CN2024100135W WO2025007748A1 WO 2025007748 A1 WO2025007748 A1 WO 2025007748A1 CN 2024100135 W CN2024100135 W CN 2024100135W WO 2025007748 A1 WO2025007748 A1 WO 2025007748A1
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ferritin
nanoparticles
seq
antibody
igg
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WO2025007748A9 (zh
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陈海燕
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陈海燕
广州纳普生物研究有限公司
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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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

Definitions

  • the present invention belongs to the field of drug delivery systems for disease diagnosis and treatment.
  • Small molecule drugs are widely used in disease treatment, such as cancer chemotherapy.
  • the clinical application of small molecule drugs is limited by their water solubility, tissue distribution, cell specificity and toxicity (Z.G.Chen, Small-molecule delivery by nanoparticles for anticancer therapy.
  • This antibody-mediated targeted therapy includes CAR T-cell therapy, antibody-drug conjugates (ADCs), bispecific antibodies, etc.
  • ADC antibody-drug conjugates
  • the currently developed CAR-T cell therapy is only targeted at hematological tumors, has large side effects, is expensive, takes a long time, and is only targeted at specific subjects; ADC exposes cytotoxic drugs to normal cells or tissues, so it is not stable enough and has large side effects; bispecific antibodies may cause some adverse reactions and have problems such as antibody stability. Therefore, there is still a need for safe, convenient, stable, less toxic, highly targeted and universal targeted delivery systems and targeted therapies.
  • the purpose of the present invention is to provide a targeted delivery system and targeted therapy that is safe, stable, less toxic, highly targeted, multi-specific, and convenient to prepare.
  • ferritin nanoparticles are composed of 24 monomers (or units), they can theoretically bind to 24 antibody molecules through sortase A, thereby improving the affinity of the antibody; in addition, multiple different antibodies can be connected to achieve targeting and multi-specificity for multiple targets.
  • TfR1 human transferrin receptor 1
  • Human TfR1 is expressed at high levels in tumor cells to achieve the purpose of targeted drug delivery.
  • human TfR1 is also commonly expressed in healthy human tissues, such as bone marrow, lungs, colon and liver, to import iron into cells. Therefore, the use of human ferritin poses a risk of drug accumulation in healthy tissues, as well as a risk of autoimmunity, which is undesirable.
  • the inventors unexpectedly discovered that by modifying the ferritin encoding gene from Helicobacter pylori and expressing it in mammalian cells (such as CHO cells), the above-mentioned problems can be avoided while making the ferritin conformation expressed by mammalian cells closer to its natural state, while achieving high yield (>80 mg/L cells).
  • a nanodelivery system which comprises ferritin nanoparticles, a targeting protein and an active molecule, wherein the targeting protein molecule is connected to the surface of the ferritin nanoparticle via SEQ ID NO.19 (LPXTGGGG, wherein X can be any amino acid), and the active molecule is encapsulated inside the ferritin nanoparticle.
  • amino acid sequence of ferritin in the nanodelivery system can be as shown in SEQ ID NO.11.
  • the targeting protein can be any protein with targeting properties (eg, targeting specific cells, such as targeting tumor cells), such as an antibody molecule (eg, IgG antibody) or an antigen-binding fragment thereof.
  • targeting properties eg, targeting specific cells, such as targeting tumor cells
  • an antibody molecule eg, IgG antibody
  • an antigen-binding fragment thereof e.g, IgG antibody
  • the active molecule can be any molecule desired for targeted delivery, which can be encapsulated inside the ferritin nanoparticles.
  • a suitable active molecule can be any suitable therapeutic agent, such as a small molecule drug, such as an anti-tumor drug, as long as it can be encapsulated inside the ferritin nanoparticles used herein.
  • the ferritin nanoparticles and the targeting protein are connected by transferase A, thereby forming a linker (N-terminus-targeting molecule-linker-ferritin-C-terminus) shown in SEQ ID NO.19 (LPXTGGGG, where X can be any amino acid) between the two.
  • a method for preparing a nanodelivery system comprising:
  • steps 1) and 2) may be performed in one reaction or separately in two reactions.
  • the method further includes the step of providing a ferritin unit having SEQ ID NO.20 (GGGG) connected to the N-terminus and a targeting protein having SEQ ID NO.21 (LPXTGG) connected to the C-terminus.
  • GGGG ferritin unit having SEQ ID NO.20
  • LXTGG targeting protein having SEQ ID NO.21
  • amino acid sequence of ferritin in the nanodelivery system can be as shown in SEQ ID NO.11.
  • the targeting protein in the nano-delivery system can be any protein with targeting (e.g., targeting specific cells, such as targeting tumor cells), such as an antibody molecule (e.g., IgG antibody) or an antigen-binding fragment thereof.
  • targeting e.g., targeting specific cells, such as targeting tumor cells
  • an antibody molecule e.g., IgG antibody
  • SEQ ID NO.21 LXTGG
  • LXTGG can be connected to the C-terminus of its heavy chain.
  • the active molecule can be any molecule desired for targeted delivery, which can be encapsulated inside the ferritin nanoparticles.
  • a suitable active molecule can be any suitable therapeutic agent, such as a small molecule drug, such as an anti-tumor drug, as long as it can be encapsulated by the ferritin nanoparticles used herein.
  • a modified ferritin is provided, whose sequence is shown in SEQ ID NO.11.
  • a polynucleotide encoding a modified ferritin protein as shown in SEQ ID NO.11 is provided, and its sequence can be, for example, SEQ ID NO.2.
  • modified ferritin or a polynucleotide encoding the modified ferritin described herein in preparing a nanodelivery system.
  • nanodelivery system described herein for use in treating a tumor.
  • a method of treating a tumor comprising administering to a subject a nanodelivery system as described herein.
  • tumor-targeting antibodies, anti-tumor small molecule drugs and nanoparticle technology are combined to produce an antibody-mediated antibody-ferritin nanoparticle with targeted delivery of small molecule drugs, which can be used as an anti-tumor nanoparticle drug with precise targeting, strong stability and low toxicity.
  • the targeted antibodies for B cell non-Hodgkin's lymphoma or lymphocytic leukemia are coupled to the surface of nanoparticles coated with anti-tumor small molecule drugs through transpeptidase A, and the antibodies are used to accurately target tumor cells and mediate the delivery of small molecule drugs, thereby reducing toxicity to normal cells and enhancing stability. It can be used as a targeted therapeutic drug for B cell non-Hodgkin's lymphoma or lymphocytic leukemia, with targeted precision. It has the characteristics of high stability and low toxicity.
  • Figure 1 Schematic diagram showing the passive permeation of ferritin at high temperature and the dissociation-reassembly under strong acid and strong base.
  • FIG. 2 As an example, a flow chart of the preparation of a nanoparticle delivery system encapsulating the anti-tumor drug doxorubicin is shown. Under specific temperature or pH conditions, small molecule drugs such as doxorubicin (DOX) are encapsulated into ferritin nanoparticles. Then, using Sortase A, IgG antibodies or antigen-binding fragments Fab are coupled to the drug-encapsulated ferritin nanoparticles to form nanoparticles with targeted drug delivery.
  • DOX doxorubicin
  • FIG. 3 shows the preparation of ferritin nanoparticles.
  • A Schematic diagram of ferritin nanoparticles. One nanoparticle is composed of 24 ferritin subunits, and the N-terminus of ferritin is connected to SEQ ID NO.20 (GGGG) for connecting to targeting protein molecules through transpeptidase A.
  • B High performance liquid chromatography separation of ferritin nanoparticles. The peaks indicated by arrows prove that the ferritin nanoparticles have high purity.
  • C The morphology of ferritin nanoparticles under negative staining electron microscopy.
  • FIG. 4 Ferritin nanoparticles can dissociate or become loose after being treated with strong acid, strong alkaline or high temperature conditions, and reassemble or return to normal under neutral pH or room temperature conditions. This suggests its potential for encapsulating small molecule drugs.
  • B HPLC analysis shows that ferritin nanoparticles are treated with an alkaline solution with pH > 10.0 and dissociate into ferritin monomers.
  • FIG. 5 Ferritin nanoparticles coated with DOX (Ferritin-Dox) are shown as an example.
  • A Schematic diagram of ferritin nanoparticles. One nanoparticle is composed of 24 subunits.
  • B High performance liquid chromatography separation of ferritin nanoparticles. The peaks indicated by arrows prove that ferritin nanoparticles have high purity.
  • C The morphology of ferritin nanoparticles encapsulated with DOX under negative staining electron microscopy.
  • CD19 IgG antibody-coupled ferritin (denoted as F) nanoparticles coated with DOX (denoted as D) (abbreviated as CD19 IgG-FD) are shown.
  • A Schematic diagram of ferritin nanoparticles coated with DOX and conjugated with IgG antibodies. IgG antibodies are linked to ferritin subunits through transpeptidase A enzyme, and up to 24 IgG antibodies can be linked to the surface of each ferritin nanoparticle.
  • B HPLC analysis and purification.
  • C SDS-PAGE analysis of the purified CD19 IgG-FD product.
  • D The morphology of the nanoparticles before coating with DOX under negative staining electron microscopy (left: CD19 IgG-F, before coating with DOX; right: CD19 IgG-FD, after coating with DOX).
  • FIG. 7 As an example, ferritin nanoparticles coated with DOX (abbreviated as CD19 Fab-FD) coupled to the antigen-binding fragment (Fab) of the CD19 antibody are shown.
  • A Schematic diagram of ferritin nanoparticles coated with DOX coupled to the Fab fragment of the antibody IgG. Fab is linked to the ferritin subunit via transpeptidase A, and up to 24 Fabs can be linked to the surface of each ferritin nanoparticle.
  • B Analysis and purification by HPLC.
  • C SDS-PAGE analysis of the purified CD19 Fab-FD product.
  • D The morphology of the nanoparticles before DOX coating (left: CD19 Fab-F, before DOX coating; right: CD19 Fab-FD, after DOX coating) under negative staining electron microscopy.
  • Figure 8 Test results of thawing of ferritin nanoparticles under different conditions after freezing after coupling with antibodies.
  • FIG. 9 SDS-PAGE Coomassie blue staining to test the stability of antibody-coupled ferritin nanoparticles-DOX.
  • the samples were stored in PBS and 4°C for 1, 2, 3, 5, and 7 weeks before being stained with SDS-PAGE Coomassie blue to detect the stability of the components.
  • Figure 10 Stability of ferritin nanoparticles-DOX without antibody coupling. Samples were stored in PBS and detected by FPLC after storage at 4°C for 1, 2, 3, 5, and 7 weeks.
  • Figure 11 Stability of ferritin nanoparticles-DOX after antibody coupling. Samples were stored in PBS or Tris buffer (10 mM Tris pH 8, 500 mM NaCl) at 4°C for 1, 2, 3, 5, and 7 weeks and then detected by FPLC.
  • FIG. 12 CD20 IgG antibody-coupled ferritin nanoparticles coated with DOX (abbreviated as CD19IgG-FD).
  • A HPLC analysis and purification.
  • B SDS-PAGE analysis of the purified CD20 IgG-FD product.
  • FIG. 13 CD22 antibody IgG coupled ferritin nanoparticles coated with DOX (abbreviated as CD22IgG-FD).
  • A HPLC analysis and purification.
  • B SDS-PAGE analysis after purification CD22 IgG-FD product.
  • FIG. 14 Antibody-conjugated ferritin nanoparticles-DOX specifically binds to tumor cell lines.
  • Flow cytometry was used to detect the binding of various antibodies and nanoparticle antibodies to Raji cell lines (B cells) or K562 cell lines (myeloid leukemia cells). The concentrations of various antibodies and nanoparticle antibodies were 10, 2, and 0.4 ⁇ g/mL, respectively.
  • A Flow cytometric detection of antibody binding.
  • B Bar graph of MFI of each group.
  • FIG. 15 The result that antibody-coupled ferritin nanoparticles-DOX can be internalized by tumor cells.
  • Figure 16 Antibody-coupled ferritin nanoparticles-DOX tumor drug cell-specific killing results.
  • CD19 IgG, CD19IgG-F, CD19IgG–FD, CD19 Fab, CD19Fab-F, CD19Fab–FD, CD20 IgG, CD20IgG-F, CD20IgG–FD, CD22 IgG, CD20IgG-F, CD22IgG-FD were tested for cell killing by CCK-8 at different concentrations.
  • Figure 17 Results of the B cell tumor treatment experiment in mice.
  • A The mortality rate of mice after drug treatment in each group;
  • B The weight change of mice after drug treatment in each group;
  • C The fluorescence intensity of tumor cells in vivo shown by in vivo imaging.
  • the nano-delivery system involved in this article comprises ferritin nanoparticles, targeting proteins and active molecules (e.g., therapeutic agents), wherein the targeting protein molecules are connected to the ferritin unit through SEQ ID NO.19, so as to be presented on the surface of the ferritin nanoparticles to bind to the target, which is, for example, a target cell surface molecule; and the active molecules (e.g., therapeutic agents) are encapsulated inside the ferritin nanoparticles.
  • the targeting protein specifically binds to the cell surface target
  • the nanoparticles are internalized into the target cells and release the active molecules (e.g., therapeutic agents) to exert the desired active effects.
  • Ferritin nanoparticles are spherical particles with a diameter of about 12-20 nm composed of 24 ferritin subunits. They are the main storage protein and carrier protein of iron ions in the body. Ferritin can efficiently encapsulate small molecule anti-tumor drugs, thereby improving their water solubility and tissue stability (ZG Chen, Small-molecule delivery by nanoparticles for anticancer therapy. Trends Mol Med 16, 594-602 (2010); M. Khoshnejad, H. Parhiz, VV Shuvaev, IJ Dmochowski, VR Muzykantov, Ferritin-based drug delivery systems: Hybrid nanocarriers for vascular immunotargeting. J Control Release 282, 13-24 (2016); Z. Wang et al., Functional ferritin nanoparticles for biomedical applications. Front Chem Sci Eng 11, 633-646(2017)).
  • ferritin can be produced by ferritin encoding genes from different sources including eukaryotes and prokaryotes, and can be wild type or various modified types, which are suitable for production, can encapsulate active molecules (such as therapeutic agents such as small molecule drugs), can be linked to targeting proteins through transpeptidase A, and are suitable for administration to desired subjects, such as cancer patients.
  • active molecules such as therapeutic agents such as small molecule drugs
  • the ferritin nanoparticles may be linked to 24 identical or different targeting proteins.
  • a modified ferritin unit derived from Helicobacter pylori having a sequence as shown in SEQ ID NO. 11 is mutated to glutamine at the N21 N-glycosylation site (N21Q mutation).
  • the ferritin unit can form spherical particles composed of 24 ferritin subunits.
  • GGGG SEQ ID NO.20
  • GGGG SEQ ID NO.20
  • LPXTGG SEQ ID NO.21
  • the targeting protein molecule and the ferritin unit are connected together through SEQ ID NO.19, and the targeting protein molecule is connected to the surface of the ferritin nanoparticle.
  • ferritin can be produced by a cell line (eg, a mammalian cell line) or synthesized.
  • the targeting protein is linked to the surface of the ferritin nanosphere particles by transpeptidase A and can specifically bind to the target molecule.
  • the ferritin nanoparticles are treated at high temperature (e.g., above 50°C) to loosen their structure and change the pores from triangular to quadrilateral, so that the drug to be encapsulated can penetrate into the interior of the nanoparticles, and then transferred to room temperature, the pores return to triangular shape, thereby retaining the drug inside the nanoparticle sphere.
  • Ferritin can remain as a sphere at 80°C, and the drug to be encapsulated can penetrate into the sphere.
  • ferritin nanoparticles are treated under strongly acidic (e.g., below pH 3) or strongly alkaline (e.g., above pH 10) conditions to dissociate them into ferritin monomers, the drug to be encapsulated is added, and they are reassembled into spheres under neutral conditions (e.g., around pH 7), while encapsulating the drug inside the spheres.
  • strongly acidic e.g., below pH 3
  • strongly alkaline e.g., above pH 10
  • ferritin can be stored at -20°C to 30°C and repeatedly frozen and thawed without affecting its delivery function.
  • ferritin encapsulates small molecule drugs mainly in two ways.
  • One is passive penetration.
  • Ferritin spheres have a 3-fold channel (triangular pores in appearance) on the surface at room temperature (e.g., 20°C-25°C); when treated at high temperature, such as 50°C, the channel can be transformed into a 4-fold larger channel (quadrangular pores in appearance).
  • the average diameter of the 4-fold channel is 0.9nm. Thermal fluctuations can also expand the channel diameter to more than 1nm, providing conditions for the transfer and encapsulation of active molecules (e.g., therapeutic agents).
  • the other is dissociation-reassembly.
  • Ferritin nanoparticles dissociate into subunits in a strong acid (e.g., below pH 3) or a strong base (e.g., above pH 10) environment, and then reassemble when the pH returns to neutral (e.g., around pH 7) or when 3.0 ⁇ pH ⁇ 10.0, thereby encapsulating drugs inside the reassembled nanoparticles.
  • a strong acid e.g., below pH 3
  • a strong base e.g., above pH 10
  • the pH returns to neutral e.g., around pH 7
  • 3.0 ⁇ pH ⁇ 10.0 3.0 ⁇ pH ⁇ 10.0
  • ferritin spheres Under neutral conditions, the inner cavity of ferritin spheres is negatively charged, and positively charged metal ions can easily pass through the pores of ferritin nanoparticles and accumulate inside the spheres.
  • the pore size can be enlarged by high temperature treatment; further, the charge load of the ferritin cavity can be changed by adjusting the pH value of the buffer solution, and the charge of the drug to be encapsulated can also be changed, so that the drug can pass through the channel smoothly and enter the cavity through electrostatic drive. Selecting a pH value close to pKa can promote the deposition of drugs in the lumen of ferritin spheres.
  • the drug can be encapsulated inside the ferritin spheres by adjusting the temperature or pH.
  • Transpeptidase A is widely used in the field of genetic engineering to precisely insert or bind non-natural molecules, such as fluorescein or amide, into proteins to change the physical or chemical properties of proteins.
  • X can be any amino acid, such as M (Met), A (Ala), V (Val), L (Leu), I (Ile), C (Cys), S (Ser), T (Thr), N (Asn), Q (Gln), D (Asp), E (Glu), H (His), K (Lys), R (Arg), G (Gly), P (Pro), Trp (W), Y (Tyr)
  • GGGG SEQ ID NO. 20
  • LPXTGG SEQ ID NO. 21
  • the sequence GGGG (SEQ ID NO.20) is connected to the N-terminus of transferrin, and the sequence LPXTGG (SEQ ID NO.21) is connected to the C-terminus of the targeting protein, wherein X can be any amino acid, for example, E.
  • the targeting molecule is an antibody, such as an IgG antibody
  • LPXTGG (SEQ ID NO.21) can be connected to the C-terminus of its heavy chain for connection with ferritin.
  • Any suitable active molecule e.g., therapeutic agent, such as small molecule drug
  • a suitable active molecule e.g., therapeutic agent, such as small molecule drug
  • ferritin nanoparticles can be used herein and encapsulated in ferritin nanoparticles to form a nano-delivery system for targeted delivery.
  • active molecules may be used simultaneously.
  • suitable active molecules may be, but are not limited to, anthracyclines such as doxorubicin, daunorubicin, epirubicin, and idarubicin; antimetabolite chemotherapeutic agents such as 5-fluorouracil, methotrexate, capecitabine, azacitidine, acivicin; B-cell lymphoma-2 (Bcl-2) inhibitors such as venetoclax, ABT-737, or navitoclax; tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib, afatinib, dacomitinib, lapatinib, and ametinib; platinum chemotherapeutic agents such as cisplatin; Borofalan-10B.
  • anthracyclines such as doxorubicin, daunorubicin, epirubicin, and idarubicin
  • doxorubicin is encapsulated in ferritin nanoparticles so that it is delivered to the inside of target cells (e.g., tumor cells).
  • cisplatin is encapsulated in ferritin nanoparticles so that it is delivered to the inside of target cells (e.g., tumor cells).
  • Targeted proteins are protein molecules that specifically recognize certain molecules or cell-related proteins (such as cell surface molecules) and are usually used as drugs to treat specific diseases.
  • the targeting protein used in this article has a sequence of SEQ ID NO.21 (LPXTGG) connected to its C-terminus, and then connected by transpeptidase A with SEQ ID NO.20 (GGGG) at the N-terminus of the ferritin monomer protein to form a linker SEQ ID NO.19 (LPXTGGGG), thereby displaying the targeting protein on the surface of the ferritin sphere.
  • the targeting protein can be expressed by cell lines (such as mammalian cells) produce.
  • the targeting protein can be an antibody molecule or an antigen-binding fragment thereof.
  • the C-terminal connection sequence SEQ ID NO.21 (LPXTGG) or its coding sequence of the heavy chain of the antibody molecule or its antigen-binding fragment is connected, or the coding sequence of the connection sequence SEQ ID NO.21 (LPXTGG) is connected at the 3' end of its coding sequence.
  • Any suitable antibody molecule or antigen-binding fragment thereof e.g., Fab
  • Fab monoclonal antibody or antigen-binding fragment thereof targeting CD19, CD20 or CD22.
  • 24 identical or different targeting protein molecules can be linked to the ferritin nanoparticle.
  • the nano-delivery system involved in this article can target different cells, molecules or environments by connecting different targeting protein molecules, and release the active molecules (such as therapeutic agents) encapsulated inside, so as to exert the desired active effect, such as exerting a therapeutic effect on the desired disease.
  • active molecules such as therapeutic agents
  • the nanodelivery system involved in this article can be used for a variety of purposes, for example, coupling with CD19, CD20 or CD22 antibodies can be used to treat non-Hodgkin's lymphoma or lymphocytic leukemia, and coupling with EGFR or HER2 antibodies can be used to treat non-small cell lung cancer, colorectal cancer, head and neck cancer, breast cancer, etc.
  • the nano-delivery system described in this article can provide safe, stable, less toxic, highly targeted, and multi-specific targeted therapies.
  • Protein nanoparticles (CD20IgG) Protein nanoparticles (CD20I
  • the ferritin used in this article is from Helicobacter pylori, and its coding gene is mutated.
  • the mutated coding sequence and amino acid sequence correspond to SEQ ID NO.2 and SEQ ID NO.11 shown below, respectively, and the N21Q site mutation in the sequence is marked with a bold underline.
  • the mammalian cell expression system is the closest to humans, so its products are closest to the real biological activity in vivo.
  • N21Q mutation the mutation of the N-glycosylation site N21 to glutamine
  • the loss of N-glycosylation make the ferritin conformation expressed by mammalian cells closer to its natural state, while achieving high yield (>80mg/L cells).
  • the nucleotide sequence of SEQ ID No.2 was cloned into the pcDNA3.1 expression vector, and the recombinant plasmid was obtained by plasmid extraction and named pcDNA3.1-Ferritin_Sec.
  • the recombinant plasmid pcDNA3.1-Ferritin_Sec was transfected into 293F cells using polyethyleneimine (PEI). After 4 days of transfection, the cells were collected by centrifugation.
  • PEI polyethyleneimine
  • Ferritin nanoparticles are composed of 24 ferritin subunits.
  • a GGGG linker sequence to the N-terminus of each subunit.
  • the purified ferritin nanoparticles expressed in cells were separated by high-performance liquid chromatography, and a single peak curve was observed (Figure 3B).
  • the purified ferritin nanoparticles expressed in cells showed uniform spherical particles under a transmission electron microscope ( Figure 3C). The above data prove that ferritin can be efficiently assembled into nanoparticles.
  • ferritin nanoparticles prepared in this paper have good potential as carriers of small molecule drugs.
  • the small molecule drug doxorubicin (DOX) coated with Helicobacter pylori and human ferritin was tested.
  • the amount of DOX coated in ferritin was calculated by NanoDrop measurement of OD480, and the ferritin concentration was calculated by NanoDrop measurement of OD280 (Zhang J., Cheng D., He J. et al. Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery. Nat Protoc 16, 4878–4896 (2021)).
  • transpeptidase A was expressed and purified in-house in the laboratory. However, it will be appreciated that any commercially available transpeptidase A may be used.
  • SEQ ID No.1 was cloned into the pcDNA3.1 expression vector, and the recombinant plasmid was obtained by plasmid extraction and named pcDNA3.1-SrtA7_Int.
  • the recombinant plasmid pcDNA3.1-SrtA7_Int was transfected into 293F cells using polyethyleneimine (PEI). After 4 days of transfection, the cells were collected by centrifugation.
  • PEI polyethyleneimine
  • CD19 IgG antibody and its antigen-binding fragment were expressed and purified in-house.
  • LPETGG was linked to the C-terminus of its heavy chain.
  • SEQ ID NOs.3, 4, and 5 were cloned into the pcDNA3.1 expression vector, and recombinant plasmids were obtained by plasmid extraction and named pcDNA3.1-CD19IgH, pcDNA3.1-CD19FabH, and pcDNA3.1-CD19IgK.
  • the heavy chain and light chain plasmids pcDNA3.1-CD19IgH and pcDNA3.1-CD19IgK of CD19 antibody were transfected into 293F cells using polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the supernatant was centrifuged and purified using affinity resin Antibody column package, modern Protein A (Cytiva, catalog number 29497628).
  • the purified product was then purified again using molecular sieve gel filtration chromatography column HiLoad 16/600 Superdex 200pg (Cytiva, catalog number 28-9893-35).
  • CD19 antibody Fab fragment plasmids pcDNA3.1-CD19FabH and pcDNA3.1-CD19IgK were transfected into 293F cells using polyethyleneimine (PEI) to prepare the antigen-binding fragment Fab of the CD19 antibody.
  • PEI polyethyleneimine
  • the supernatant was centrifuged and purified using affinity resin Protein L (Yishen Biotechnology, product catalog number 36407ES08). The purified product was then purified again using a molecular sieve gel filtration chromatography column HiLoad 16/600 Superdex 200pg (Cytiva, product catalog number 28-9893-35).
  • the reaction system was set up in a Vivaspin 20 concentrator tube, and ferritin nanoparticles with a final concentration of 120 ⁇ M coated with DOX, 120 ⁇ M CD19 IgG antibody and 100 ⁇ M transpeptidase A were added, and a reaction solution (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) was added. After centrifugation, the reaction system was concentrated to 250 ⁇ L and transferred to a 500 ⁇ L tube, which was placed on a shaker to react at room temperature overnight. It was then purified using a molecular sieve gel filtration chromatography column HiLoad 16/600 Superdex 200 pg.
  • the CD19 IgG antibody we prepared has an LPETGG tag at the C-terminus of the heavy chain, which can be connected to the ferritin nanoparticles containing DOX in the previous step under the catalysis of transpeptidase A ( Figure 6A).
  • the prepared product was separated by high-performance liquid chromatography, and it can be seen that the peak curve of the nanoparticles is completely consistent with the DOX peak curve.
  • a free, uncoupled IgG peak and a transpeptidase A peak can be seen ( Figure 6B).
  • the above data prove that the antibody-nanoparticle connection is successful and DOX is successfully encapsulated.
  • the nanoparticle has the specificity of the antibody, so it can target specific targets on the surface of tumor cells and deliver small molecule anti-tumor drugs into tumor cells.
  • CD20 IgG and CD22 IgG antibodies were expressed and purified in-house in the laboratory, and LPETGG was linked to their C-termini.
  • SEQ ID NOs. 6, 7, 8, and 9 were cloned into the pcDNA3.1 expression vector, and recombinant plasmids were obtained after plasmid extraction and named pcDNA3.1-CD20IgH, pcDNA3.1-CD20IgK, pcDNA3.1-CD22IgH, and pcDNA3.1-CD22IgK.
  • the heavy chain and light chain plasmids pcDNA3.1-CD20IgH and pcDNA3.1-CD20IgK of CD20 antibody, and the heavy chain and light chain plasmids pcDNA3.1-CD22IgH and pcDNA3.1-CD22IgK of CD22 antibody were transfected into 293F cells using polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the supernatant was centrifuged and purified using affinity resin Antibody column package modern Protein A (Cytiva, product catalog number 29497628).
  • the purified product was then purified again using molecular sieve gel filtration chromatography column HiLoad 16/600 Superdex 200pg (Cytiva, product catalog number 28-9893-35).
  • the reaction system was set up in a Vivaspin 20 concentrator tube, and ferritin nanoparticles with a final concentration of 120 ⁇ M, 120 ⁇ M CD20 IgG or CD22 IgG antibody and 100 ⁇ M transferase A were added, and a reaction solution (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) was added. After centrifugation, the reaction system was concentrated to 250 ⁇ L and transferred to a 500 ⁇ L tube, which was placed on a shaker and reacted at room temperature overnight. It was then purified using a molecular sieve gel filtration chromatography column HiLoad 16/600 Superdex 200 pg.
  • the CD20 IgG and CD22 IgG antibodies we prepared have LPETGG tags at the C-terminus of the heavy chain, which can be connected to the ferritin nanoparticles containing DOX in the previous step under the catalysis of transpeptidase A.
  • the prepared products were separated by high-performance liquid chromatography, and it can be seen that the peak curve of the nanoparticles is completely consistent with the DOX peak curve.
  • a free, uncoupled IgG peak and a Sortase A peak can be seen ( Figure 12A, Figure 13A).
  • CD19 IgG, CD19IgG coupled to ferritin nanoparticles CD19IgG-F
  • CD19 IgG coupled to ferritin nanoparticles coated with DOX CD19IgG-FD
  • CD19 Fab, CD19 Fab coupled to ferritin nanoparticles CD19Fab coupled to ferritin nanoparticles coated with DOX
  • CD20IgG, CD20 IgG coupled to ferritin nanoparticles CD20 IgG coupled to ferritin nanoparticles (CD20IgG-F), CD20 IgG coupled to ferritin nanoparticles coated with DOX (CD20IgG-FD)
  • CD22 IgG, CD22 IgG coupled to ferritin nanoparticles CD20IgG-F
  • CD22 IgG coupled to ferritin nanoparticles coated with DOX CD22IgG-FD
  • the antibody or nanoparticle antibody samples were diluted to 10, 2, and 0.4 ⁇ g/mL, respectively, and incubated with 1x10 5 Raji (B cell line) or K562 cells (myeloid leukemia cells) on ice for 30 minutes, then washed once with PBS, and added with 1:2000 diluted Alexa Fluor 488 anti-human IgG (H+L) (ThermoFisher, Cat. No. A-11013), and the cells were incubated on ice for 30 minutes, washed twice with PBS, and fixed with 1% formalin. We then used flow cytometry to detect the binding of the above antibodies and nanoparticle antibodies to Raji cell lines or K562 cell lines.
  • Nanoparticle antibody samples CD19IgG-F, CD19IgG-FD, CD19Fab-F, and CD19Fab-FD were diluted to 10 ⁇ g/mL and added to 1x10 5 Raji cells and incubated for 30 minutes or 120 minutes, followed by fixation with 1% formalin + 1% acetone, washing three times with PBS, adding 1:1000 diluted Alexa Fluor 488 anti-human IgG (H+L) (ThermoFisher, catalog number A-11013), incubating for 30 minutes, washing twice with PBS, and observing and photographing with a fluorescence confocal microscope.
  • CD19IgG-F, CD19IgG-FD, CD19Fab-F, and CD19Fab-FD were mainly bound to the cell surface after 30 minutes of incubation with Raji cells, and most of them had been internalized into the cytoplasm after 120 minutes of incubation (Figure 15).
  • Raji and K562 cells were plated in 96-well plates, 100 ⁇ l of culture medium/well, 3 x 10 4 cells/ml.
  • Ferritin, ferritin-DOX, antibody, antibody-ferritin, or antibody (FD) encapsulated with DOX were subsequently added, with a maximum total protein concentration of 50 ⁇ g/ml and 5-fold gradient dilution.
  • CCK-8 detection (Novozyme, Catalog No. A311) was performed after 72 hours of culture, and the OD450 reading time was 4 hours after the addition of CCK-8 reagent.
  • B-luc-GFP Raji cells were inoculated into female 6-week-old B-NDG mice through the tail vein, 1 x 10 5 cells/mouse, and then different treatments were performed when the average imaging signal of luciferase in vivo imaging reached 1 x 10 6 P/S (as shown in Table 2 below).
  • the body weight was weighed every day after administration, and the bioluminescence intensity of in vivo imaging was measured twice a week. Based on the principle of animal welfare, the mice were euthanized after the weight loss exceeded 20%. The results are shown in Table 2 below.
  • Table 3 shows the results of group administration on day 14.
  • ferritin 100 ⁇ g/ The CD19 IgG-ferritin 100 ⁇ g/group, CD19 IgG-FD 100 ⁇ g/group, CD19 IgG-FD 50 ⁇ g/group, and CD20 IgG-FD 50 ⁇ g/group had no inhibitory effect on tumor growth; the CD19 IgG-ferritin 100 ⁇ g/group, CD19 IgG-FD 100 ⁇ g/group, CD19 IgG-FD 50 ⁇ g/group all had significant inhibitory effects on tumor growth (TGI: tumor growth inhibition rate, p ⁇ 0.05).
  • SEQ ID No.21 used to connect to the C-terminus of the targeting protein molecule
  • LPXTGG (where X can be any amino acid)

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Abstract

提供一种包含铁蛋白、靶向性蛋白和活性分子的纳米递送系统，其制备方法及用途。可以提供安全、稳定、毒性小、靶向性强、多特异性、制备方法便捷的靶向递送系统和靶向疗法。

Description

纳米递送系统、其制备方法和用途 技术领域
本发明属于用于疾病诊断和治疗的药物递送系统领域。
背景技术
小分子药物被广泛应用于疾病治疗，例如肿瘤化疗中。然而，小分子药物的临床应用受到其水溶性、组织分布、细胞特异性以及毒性的限制(Z.G.Chen,Small-moleculedelivery by nanoparticles for anticancer therapy.Trends Mol Med 16,594-602(2010)；L.M.Ickenstein,P.Garidel,Lipid-based nanoparticle formulations for small molecules and RNA drugs.Expert Opin Drug Deliv 16,1205-1226(2019))。
由于特定分子在某些细胞例如肿瘤细胞表面高表达，因此可以利用识别该分子的抗体来精确靶向这些细胞。这种抗体介导的靶向治疗方法包括CAR T-细胞疗法、抗体偶联药物(ADC)、双特异性抗体等。然而，目前开发的CAR-T细胞疗法仅针对血液系统肿瘤、副作用大、价格昂贵、花费时间长且仅针对特定受试者；ADC将细胞毒性药物暴露于正常细胞或组织，因此稳定性不足，且副作用较大；双特异抗体可能引起一些不良反应且存在抗体稳定性等问题。因此，仍然需要安全、便捷、稳定、毒性小、靶向性强且普适的靶向递送系统和靶向疗法。
发明内容
本发明的目的是提供安全、稳定、毒性小、靶向性强、多特异性、制备方法便捷的靶向递送系统和靶向疗法。
本申请基于发明人的一个意外发现，即通过转肽酶A(sortase A)将铁蛋白(Ferritin)纳米颗粒和抗体连接起来并且在纳米颗粒中进一步包裹活性分子如小分子治疗剂，可以便捷地获得安全、稳定性强、毒性小、靶向性精确、且普适的靶向递送系统。由于铁蛋白纳米颗粒由24个单体(或单元)组成，理论上能通过转肽酶A结合24个抗体分子，因此能提高抗体的亲和力；另外也可以连接多种不同的抗体，从而实现对多个靶点的靶向性和多特异性。
现有的研究使用来自人的铁蛋白，它能靶向人转铁蛋白受体1(TfR1)。人TfR1在肿瘤细胞以高水平表达，达到药物靶向递送的目的。但是，人TfR1也在健康人体组织中普遍表达，如骨髓、肺、结肠和肝脏，以将铁输入细胞。因此，使用人铁蛋白存在药物在健康组织中累积的风险，同时还存在自身免疫的风险，这都是不希望的。发明人意外地发现，通过将来自幽门螺杆菌(Helicobactor pylori)的铁蛋白编码基因进行修饰并在哺乳动物细胞(例如CHO细胞)中表达，可以在避免上述问题的同时使得哺乳动物细胞表达的铁蛋白构象更接近其天然状态，同时实现了高产(>80mg/L细胞)。
一方面，提供了纳米递送系统，其包含铁蛋白纳米颗粒、靶向性蛋白和活性分子，所述靶向性蛋白分子通过SEQ ID NO.19(LPXTGGGG，其中X可以为任何氨基酸)连接在铁蛋白纳米颗粒表面，所述活性分子包裹在铁蛋白纳米颗粒内部。
在一些具体的实施方案中，所述的纳米递送系统中铁蛋白的氨基酸序列可以如SEQ ID NO.11所示。
在本文所述的纳米递送系统中，所述的靶向性蛋白可以是任何具有靶向性(例如靶向特定细胞，例如靶向肿瘤细胞)的蛋白，例如抗体分子(例如IgG抗体)或其抗原结合片段。
在本文所述的纳米递送系统中，其中所述活性分子可以为任何期望靶向递送的分子，其能够包裹在铁蛋白纳米颗粒内部。例如，合适的活性分子可以是任何合适的治疗剂，例如小分子药物，例如抗肿瘤药物，只要其能够被包裹在本文使用的铁蛋白纳米颗粒内部。
在本文所述的纳米递送系统中，所述铁蛋白纳米颗粒和靶向性蛋白是通过转肽酶A连接的，从而在两者之间形成了SEQ ID NO.19(LPXTGGGG，其中X可以为任何氨基酸)所示的接头(N末端-靶向性分子-接头-铁蛋白-C末端)。
另一方面，提供了制备纳米递送系统的方法，所述方法包括：
1)使铁蛋白纳米颗粒包裹活性分子；和
2)通过转肽酶A使铁蛋白纳米颗粒连接靶向性蛋白。
在本文中，步骤1)和2)可以在一个反应中进行，或者分别在两个反应中进行。
在一些具体的实施方案中，所述方法还包括提供N末端连接SEQ ID NO.20(GGGG)的铁蛋白单元和C末端连接有SEQ ID NO.21(LPXTGG)的靶向性蛋白的步骤。
在一些具体的实施方案中，所述的纳米递送系统中铁蛋白的氨基酸序列可以如SEQ ID NO.11所示。
在本文所述的方法中，所述的纳米递送系统中的靶向性蛋白可以是任何具有靶向性(例如靶向特定细胞，例如靶向肿瘤细胞)的蛋白，例如抗体分子(例如IgG抗体)或其抗原结合片段。在抗体分子的情况下，SEQ ID NO.21(LPXTGG)可以连接在其重链的C末端。
在本文所述的方法中，其中所述活性分子可以为任何期望靶向递送的分子，其能够包裹在铁蛋白纳米颗粒内部。例如，合适的活性分子可以是任何合适的治疗剂，例如小分子药物，例如抗肿瘤药物，只要其能够被本文使用的铁蛋白纳米颗粒包裹。
在再一方面，提供了修饰的铁蛋白，其序列如SEQ ID NO.11所示。
在再一方面，提供了编码序列如SEQ ID NO.11所示的修饰的铁蛋白的多核苷酸，其序列可以为例如SEQ ID NO.2。
在再一方面，提供了本文所述的修饰的铁蛋白或编码修饰的铁蛋白的多核苷酸在制备纳米递送系统中的用途。
在再一方面，提供了本文所述的纳米递送系统，用于治疗肿瘤。
在再一方面，提供了治疗肿瘤的方法，包括向受试者使用本文所述的纳米递送系统。
在一些实施方案中，将肿瘤靶向抗体、抗肿瘤小分子药物以及纳米颗粒技术结合，产生了一种抗体介导的、具有靶向输送小分子药物作用的抗体-铁蛋白纳米颗粒，可以用作靶向性精确、稳定性强、毒性小的抗肿瘤纳米颗粒药物。
在一些实施方案中，将B细胞非霍奇金淋巴瘤或淋巴细胞白血病的靶向抗体(CD19IgG、CD19Fab、CD20IgG、CD22IgG)通过转肽酶A偶联在包裹了抗肿瘤小分子药物的纳米颗粒表面，利用抗体精确靶向定位肿瘤细胞，介导小分子药物的输送，从而减少对正常细胞的毒性并增强稳定性，其可作为B细胞非霍奇金淋巴瘤或淋巴细胞白血病的靶向治疗药物，具有靶向性精 确、稳定性强、毒性小等特点。
附图说明
以下将结合附图对实施例进行描述，从而使本发明的上述和其他方面和优点变得明显且容易理解。
图1：显示铁蛋白在高温下的被动渗透和强酸、强碱下的解离-重组装的示意图。
图2：作为示例显示了包裹抗肿瘤药物多柔比星的纳米颗粒递送系统的制备流程图。在特定温度或pH值条件下，将小分子药物如多柔比星(Doxorubicin，DOX)包裹入铁蛋白纳米颗粒。然后利用转肽酶A(Sortase A)，将IgG抗体或抗原结合片段Fab与包裹药物的铁蛋白纳米颗粒偶联起来，形成具有靶向输送药物作用的纳米颗粒。
图3：显示了铁蛋白纳米颗粒的制备。(A)铁蛋白纳米颗粒示意图。一个纳米颗粒由24个铁蛋白亚单位构成，铁蛋白的N端连接SEQ ID NO.20(GGGG)，用于通过转肽酶A连接靶向性蛋白分子。(B)高效液相色谱仪分离铁蛋白纳米颗粒。箭头所示峰图证明铁蛋白纳米颗粒具有较高纯度。(C)负染电镜下铁蛋白纳米颗粒的形态。
图4：铁蛋白纳米颗粒经过强酸性、强碱性或高温条件处理可以解离或变松散，并在PH中性或常温条件重新组装或恢复常态。这提示其包裹小分子药物的潜力。(A)高效液相色谱仪分析显示，铁蛋白纳米颗粒经过酸性pH<3.0的溶液处理，解离成铁蛋白单体。随后将溶液换成中性(pH＝7.4)的PBS溶液，铁蛋白重新组装成纳米颗粒。(B)高效液相色谱仪分析显示，铁蛋白纳米颗粒经过碱性pH>10.0的溶液处理，解离成铁蛋白单体。随后将溶液换成中性(pH＝7.4)的PBS溶液，铁蛋白重新组装成纳米颗粒。(C)高效液相色谱仪分析显示，铁蛋白纳米颗粒在大于50℃高温处理后呈松散状态，孔径变大，从而能够允许药物进入)，再将温度恢复至常温(20-25℃)，铁蛋白纳米颗粒可恢复原有颗粒状态。
图5：作为示例显示了铁蛋白纳米颗粒包被DOX(Ferritin-Dox)。(A)铁蛋白纳米颗粒示意图。一个纳米颗粒由24个亚单位构成。(B)高效液相色谱仪分离铁蛋白纳米颗粒。箭头所示峰图证明铁蛋白纳米颗粒具有较高纯 度。(C)负染电镜下包裹DOX的铁蛋白纳米颗粒的形态。
图6：作为示例显示了CD19 IgG抗体偶联的铁蛋白(表示为F)纳米颗粒包被DOX(表示为D)(简写为CD19 IgG-FD)。(A)IgG抗体偶联包被DOX的铁蛋白纳米颗粒示意图。IgG抗体通过转肽酶A酶与铁蛋白亚单位连接，每个铁蛋白纳米颗粒表面可以最多连接24个IgG抗体。(B)高效液相色谱仪分析与纯化。(C)SDS-PAGE分析纯化后的CD19 IgG-FD产物。(D)负染电镜下包被DOX之前(左图：CD19 IgG-F，包裹DOX之前；右图：CD19 IgG-FD，包裹DOX之后)的纳米颗粒形态。
图7：作为示例显示了CD19抗体的抗原结合片段(Fab)偶联的铁蛋白纳米颗粒包被DOX(简写为CD19 Fab-FD)。(A)抗体IgG的Fab片段偶联包被DOX的铁蛋白纳米颗粒示意图。Fab通过转肽酶A与铁蛋白亚单位连接，每个铁蛋白纳米颗粒表面可以最多连接24个Fab。(B)高效液相色谱仪分析与纯化。(C)SDS-PAGE分析纯化后的CD19 Fab-FD产物。(D)负染电镜下包被DOX之前(左图：CD19 Fab-F，包裹DOX之前；右图：CD19 Fab-FD，包裹DOX之后)的纳米颗粒形态。
图8：偶联抗体后铁蛋白纳米颗粒冻存后，在不同条件下解冻的测试结果。
图9：SDS-PAGE考马斯亮蓝染色测试抗体偶联铁蛋白纳米颗粒-DOX的稳定性。样品保存于PBS，4℃存放1、2、3、5、7周后用SDS-PAGE考马斯亮蓝染色检测其成分稳定性。
图10：未偶联抗体的铁蛋白纳米颗粒-DOX的稳定性。样品保存于PBS，4℃存放1、2、3、5、7周后用FPLC检测。
图11：偶联抗体后铁蛋白纳米颗粒-DOX的稳定性。样品保存于PBS或Tris缓冲液(10mM Tris pH8,500mM NaCl)，4℃存放1、2、3、5、7周后用FPLC检测。
图12：CD20 IgG抗体偶联的铁蛋白纳米颗粒包被DOX(简写为CD19IgG-FD)。(A)高效液相色谱仪分析与纯化。(B)SDS-PAGE分析纯化后的CD20 IgG-FD产物。
图13：CD22抗体IgG偶联的铁蛋白纳米颗粒包被DOX(简写为CD22IgG-FD)。(A)高效液相色谱仪分析与纯化。(B)SDS-PAGE分析纯化后 的CD22 IgG-FD产物。
图14：抗体偶联铁蛋白纳米颗粒-DOX特异性结合肿瘤细胞系。流式细胞术检测各种抗体与纳米颗粒抗体与Raji细胞系(B细胞)或K562细胞系(骨髓性白血病细胞)的结合。各种抗体与纳米颗粒抗体浓度分别为10、2、0.4μg/mL。(A)抗体结合的流式细胞术检测。(B)各组MFI的柱状图。
图15：抗体偶联铁蛋白纳米颗粒-DOX能够被肿瘤细胞内吞的结果。
图16：抗体偶联铁蛋白纳米颗粒-DOX的肿瘤药细胞特异性杀伤结果。CD19 IgG、CD19IgG-F、CD19IgG–FD、CD19 Fab、CD19Fab-F、CD19Fab–FD、CD20 IgG、CD20IgG-F、CD20IgG–FD、CD22 IgG、CD20IgG-F、CD22IgG-FD在不同浓度下，通过CCK-8测试细胞杀伤。
图17：小鼠体内B细胞肿瘤治疗实验的结果。(A)各组药物治疗后小鼠死亡率；(B)各组药物治疗后小鼠的体重变化：(C)活体成像显示的肿瘤细胞在体内的荧光强度。
具体实施方式
本文涉及的纳米递送系统包含铁蛋白纳米颗粒、靶向性蛋白和活性分子(例如治疗剂)，所述靶向性蛋白分子通过SEQ ID NO.19与铁蛋白单元连接，从而呈现在铁蛋白纳米颗粒的表面，以与靶标结合，所述靶标是例如靶细胞表面分子；所述活性分子(例如治疗剂)被包裹在铁蛋白纳米颗粒内部。在靶向性蛋白与细胞表面靶标特异性结合后，所述纳米颗粒被内吞到靶细胞内并释放活性分子(例如治疗剂)，发挥期望的活性作用。
铁蛋白
铁蛋白(Ferritin)纳米颗粒，是由24个铁蛋白亚单元组成的直径约12-20nm的球形颗粒，是生物体内铁离子的主要储存蛋白和运载蛋白。铁蛋白可以高效包裹小分子抗肿瘤药物，从而提高其水溶性与组织稳定性(Z.G.Chen,Small-moleculedelivery by nanoparticles for anticancer therapy.Trends Mol Med 16,594-602(2010)；M.Khoshnejad,H.Parhiz,V.V.Shuvaev,I.J.Dmochowski,V.R.Muzykantov,Ferritin-based drug delivery systems:Hybrid nanocarriers for vascular immunotargeting.J Control Release 282,13-24(2018)；Z.Wang et al.,Functional ferritin nanoparticles for biomedical applications.Front  Chem Sci Eng 11,633-646(2017))。
在本文中，铁蛋白可以由包括真核生物和原核生物在内的不同来源的铁蛋白编码基因产生，可以为野生型或者各种修饰类型，其适合生产、能够包裹活性分子(例如小分子药物等治疗剂)、能够通过转肽酶A与靶向性蛋白连接并且适合施用于期望的受试者，例如癌症患者。
在一些具体的实施方案中，所述铁蛋白纳米颗粒可以连接有24个相同或不同的靶向性蛋白。
在一些具体的实施方案中，涉及一种序列如SEQ ID NO.11所示的源自幽门螺杆菌(Helicobactor pylori)的修饰的铁蛋白单元，其在N21这个N-糖基化位点被突变成谷氨酰胺(N21Q突变)。所述铁蛋白单元可以形成由24个铁蛋白亚单元组成的球形颗粒。
为了通过转肽酶A与靶向性蛋白分子连接，可以在铁蛋白单元的N端连接GGGG(SEQ ID NO.20)或者在其编码序列的5’端连接GGGG(SEQ ID NO.20)的编码序列，并且可以在靶向性蛋白分子的C末端连接LPXTGG(SEQ ID NO.21)或者在其编码序列的3’端连接LPXTGG(SEQ ID NO.21)的编码序列。在利用转肽酶A进行酶反应后，靶向性蛋白分子与铁蛋白单元通过SEQ ID NO.19连接在一起，靶向性蛋白分子被连接到铁蛋白纳米颗粒表面。
在一些具体的实施方案中，铁蛋白可以通过细胞系(例如哺乳动物细胞系)产生或者合成产生。靶向性蛋白通过转肽酶A被连接在铁蛋白纳米球颗粒的表面，可以与靶标分子特异性结合。
在一些具体的实施例中，将铁蛋白纳米颗粒在高温(例如50℃以上)处理，使其结构变得松散，孔隙由三角形变为四边形，从而使待包裹的药物可通过渗透进入纳米颗粒内部，随后将其转移至室温下，空隙恢复三角形从而将药物保持在纳米颗粒球体内部。铁蛋白可以在80℃依然保持为球体，待包裹的药物可渗透进入球体内部。
在一些具体的实施方案中，将铁蛋白纳米颗粒在强酸性(例如pH3以下)或强碱性(例如pH10以上)条件下处理，使其解离为铁蛋白单体，加入待包裹的药物，并在中性(例如pH7左右)条件重新组装为球体，同时将药物包裹在球体内部。
在本文中，铁蛋白可以储藏在-20℃至30℃，反复冻融而其递送功能不受影响。
如图1所示，铁蛋白包裹小分子药物主要通过两种方式。一种是被动渗透，铁蛋白球体在常温状态(例如20℃-25℃)下表面具有一个3倍通道(外观呈三角形孔隙)；在高温，如50℃处理时，通道可转变为一个4倍大通道(外观呈四角形孔隙)，该4倍通道的平均直径为0.9nm，热波动还可以使通道直径扩展到1nm以上，为活性分子(例如治疗剂)的转移和包裹提供条件。另一种是解离-重组装，铁蛋白纳米颗粒在强酸(例如pH3以下)或强碱(例如pH10以上)环境中解离为亚单位，随后在pH恢复中性(例如pH7左右)或在3.0<pH<10.0时重组装，由此可将药物包裹在重组装的纳米颗粒内部。参见A critical review of ferritin as a drug nanocarrier:Structure,properties,comparative advantages and challenges”,Shuang Yin,Particuology 64(2022)65–84。
在中性条件下，铁蛋白的球体内腔带负电荷，带正电荷的金属离子很容易通过铁蛋白纳米颗粒的孔隙并积聚在球体内部。
在本文中，可以通过高温处理，使孔隙孔径变大；进一步地，可以通过调整缓冲液pH值，改变铁蛋白内腔电荷的载量，同时还可以改变待包裹药物电荷，通过静电驱动使药物得以顺利通过通道，进入腔体。选择接近pKa的pH值，可以促进药物在铁蛋白球体内腔沉积。
在本文中，可以通过调节温度或pH，以使药物包裹在铁蛋白球体内部。
转肽酶A
转肽酶A可以在存在Ca2+和适宜的pH值(7.0-9.0)条件下，发生肽键酰化反应来连接两个肽或蛋白质分子，即H2N-GGGG-蛋白1+C端LPXTGG-蛋白2＝蛋白2-LPXTGGGG-蛋白1。转肽酶A被广泛应用于基因工程领域，用于在蛋白质中精确地插入或绑定非天然分子，例如荧光素或酰胺，以改变蛋白质的物理或化学性质。
在转肽酶A所形成的连接子LPXTGGGG(SEQ ID NO.19)中，X可以是任何氨基酸，例如M(Met)、A(Ala)、V(Val)、L(Leu)、I(Ile)、C(Cys)、S(Ser)、T(Thr)、N(Asn)、Q(Gln)、D(Asp)、E(Glu)、H(His)、K(Lys)、R(Arg)、G(Gly)、P(Pro)、Trp(W)、Y(Tyr) 或F(Phe)。换句话来说，在待连接的两个肽或蛋白质分子中的一个的N端连接GGGG(SEQ ID NO.20)，在另一个的C端连接LPXTGG(SEQ ID NO.21)，通过转肽酶A，即可形成连接。
在一些实施方案中，为了实现铁蛋白和靶向性蛋白分子之间的连接，在转铁蛋白的N端连接序列GGGG(SEQ ID NO.20)，在靶向性蛋白的C末端连接序列LPXTGG(SEQ ID NO.21)，其中X可以为任何氨基酸，例如为E。在一些实施方案中，如果靶向性分子为抗体，例如IgG抗体，则可以在其重链C端连接LPXTGG(SEQ ID NO.21)，从而用于与铁铁蛋白进行连接。
活性分子
本文可以使用任何合适的活性分子，(例如治疗剂，如小分子药物)，将其包裹在铁蛋白纳米颗粒中，形成纳米递送系统，进行靶向递送。
在一些实施方案中，可以同时使用一种以上活性分子(例如治疗剂)。在一些实施方案中，作为举例，合适的活性分子(例如治疗剂)可以是，但不限于蒽环类药物，例如多柔比星、柔红霉素、表柔比星和伊达比星；抗代谢化疗剂，例如5-氟尿嘧啶、甲氨蝶呤、卡培他滨、阿扎胞苷、阿西维辛；B细胞淋巴瘤-2(Bcl-2)抑制剂，例如维奈托克(venetoclax)、ABT-737或navitoclax；酪氨酸激酶抑制剂，例如吉非替尼、厄洛替尼、埃克替尼、阿法替尼、达克替尼、拉帕替尼和阿美替尼；铂类化疗药，例如顺铂；Borofalan-10B。
在一些实施方案中，将多柔比星包裹在铁蛋白纳米颗粒中，从而将其递送至靶细胞(例如肿瘤细胞)内部。在一些实施方案中，将顺铂包裹在铁蛋白纳米颗粒中，从而将其递送至靶细胞(例如肿瘤细胞)内部。
靶向性蛋白
靶向性蛋白是一种特异性识别某种分子或细胞相关蛋白质(例如细胞表面分子)的蛋白分子，通常被用作治疗特定疾病的药物。
本文使用的靶向性蛋白在其C端连接有序列SEQ ID NO.21(LPXTGG)，从而通过转肽酶A与铁蛋白单体蛋白N末端的SEQ ID NO.20(GGGG)反应而连接，形成连接子SEQ ID NO.19(LPXTGGGG)，由此将靶向性蛋白展示在铁蛋白球体表面。靶向性蛋白可以通过细胞系(例如哺乳动物细胞) 产生。
在一些具体的实施方案中，靶向性蛋白可以是抗体分子或其抗原结合片段。为了实现与铁蛋白的连接，在抗体分子或其抗原结合片段的重链的C末端连接序列SEQ ID NO.21(LPXTGG)或其编码序列或在其编码序列的3’端连接连接序列SEQ ID NO.21(LPXTGG)的编码序列。可以使用任何合适的抗体分子或其抗原结合片段(例如Fab)，例如靶向CD19、CD20或CD22的单克隆抗体或其抗原结合片段。
在一些具体的实施方案中，所述铁蛋白纳米颗粒上可以连接24个相同或不同的靶向性蛋白分子。
用途
本文涉及的纳米递送系统可以通过连接不同的靶向性蛋白分子，靶向不同的细胞、分子或环境，并释放内部包裹的活性分子(例如治疗剂)，从而发挥期望的活性作用，例如针对期望的疾病发挥治疗作用。
本文涉及的纳米递送系统可用于多种用途，例如偶联CD19、CD20或CD22抗体可用于治疗非霍奇金淋巴瘤或淋巴细胞白血病，偶联EGFR或HER2抗体可用于治疗非小细胞肺癌、结直肠癌、头颈部癌、乳腺癌等。
利用本文的纳米递送系统，可以提供安全、稳定、毒性小、靶向性强、多特异性的靶向疗法。
实施例
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅仅用于说明本发明，而不应视为限定发明的范围。实施例中未注明具体技术或条件的，按照本领域的文献所描述的技术或条件或者按照产品、仪器说明书进行。所有试剂或仪器注明生产商者，均可以市购。
实施例中涉及的各种抗体或纳米颗粒抗体简写如下：CD19 IgG，CD19IgG偶联铁蛋白纳米颗粒(CD19IgG-F)，CD19 IgG偶联铁蛋白纳米颗粒包裹DOX(CD19IgG-FD)，CD19 Fab，CD19 Fab偶联铁蛋白纳米颗粒(CD19Fab-F)，CD19 IgG偶联铁蛋白纳米颗粒包裹DOX(CD19Fab-FD)，CD20 IgG，CD20 IgG偶联铁蛋白纳米颗粒(CD20IgG-F)，CD20 IgG偶联铁蛋白纳米颗粒包裹DOX(CD20IgG-FD)，CD22 IgG，CD22 IgG偶联铁 蛋白纳米颗粒(CD20IgG-F)，CD22 IgG偶联铁蛋白纳米颗粒包裹DOX(CD22IgG-FD)。
实施例1.铁蛋白的生产和组装性能测试
本文使用的铁蛋白来自幽门螺杆菌(Helicobactor pylori)，对其编码基因进行突变。突变后的编码序列和氨基酸序列分别对应于以下显示的SEQ ID NO.2和SEQ ID NO.11，序列中N21Q位点突变用粗体下划线标记。
哺乳动物细胞表达系统最接近人类，因而其产物最为接近体内真实的生物学活性。我们发现将N21这个N-糖基化位点突变成谷氨酰胺(N21Q突变)，这种N-糖基化的缺失使得哺乳动物细胞表达的铁蛋白构象更接近其天然状态，同时实现了高产(>80mg/L细胞)。
1)铁蛋白的表达和纯化
将SEQ ID No.2的核苷酸序列克隆在pcDNA3.1表达载体上，经过质粒抽提获取重组质粒，命名为pcDNA3.1-Ferritin_Sec。将重组质粒pcDNA3.1-Ferritin_Sec利用聚乙烯亚胺(PEI)转染293F细胞。转染细胞4天后，离心收集细胞。加入裂解液(150mM NaCl，25mM Tris pH＝8，0.0001％IGEPALCA-630，蛋白酶抑制剂(碧云天生物，货号P1006))，反复冻融3次后，离心取上清，随后利用His标签亲和柱Histrap Column(Cytiva，产品目录号29-0510-21)和分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg(Cytiva，产品目录号28-9893-35)进行纯化。
2)测试铁蛋白纳米颗粒的组装
铁蛋白纳米颗粒由24个铁蛋白亚单位组成。为了将靶向性蛋白抗体呈递在铁蛋白纳米颗粒表面，我们将每个亚单位的N端加上GGGG连接子序列(图3A)。细胞表达纯化后的铁蛋白纳米颗粒通过高效液相色谱分离，可见单一的峰曲线(图3B)。细胞表达纯化后的铁蛋白纳米颗粒在投射电镜下呈现均匀的球形颗粒(图3C)。以上数据证明铁蛋白可高效组装成为纳米颗粒。
我们测试了不同pH及温度下，铁蛋白纳米颗粒组装的能力。
在pH<3.0的强酸性溶液中，铁蛋白纳米颗粒解离为单体铁蛋白，随后我们将其溶液重新换成中性的PBS溶液，铁蛋白重新组装成与此前一致的纳米颗粒(图4A)。pH>10.0的强碱性溶液同样可以使铁蛋白纳米颗粒解离为单体铁蛋白，溶液换成中性的PBS溶液之后，铁蛋白重新组装成与此前一致 的纳米颗粒(图4B)。
我们将铁蛋白纳米颗粒置于大于50℃(例如60℃)的高温下，发现温度恢复常温后，纳米颗粒的大小依然与此前一致(图4C)。
这些结果表明，本文的铁蛋白纳米颗粒具有良好的潜力作为小分子药物的载体。
3)来自人的铁蛋白和幽门螺杆菌的铁蛋白的比较
测试了幽门螺杆菌与人的铁蛋白包被小分子药物多柔比星(DOX)的对比。包被在铁蛋白内的DOX的量由NanoDrop测量OD480计算得出，铁蛋白浓度由NanoDrop测量OD280计算得出(Zhang J.,Cheng D.,He J.et al.Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery.Nat Protoc 16,4878–4896(2021))。
具体地，将1mg铁蛋白纳米颗粒室温解冻，加入790μl 50mM Tris-HCl pH9.0，混匀，加入0.3mg Dox。总反应体积1mL。60℃热孔板上静置1小时，用锡纸盖住避光。转入室温(20℃-25℃)加入1ml室温dH2O，混匀后转入3K 15-ml超滤离心管(Pall Corporation，产品目录号MCP003C46)浓缩并将溶液置换为10mM Tris，500mM NaCl，pH8.0。浓缩至约0.5ml，0.2μm滤膜过滤后检测或分装保存。结果如下表1。
表1
由表1的结果可以看出，本文使用的幽门螺杆菌铁蛋白在包裹DOX分子的数量和铁蛋白回收率上均高于人铁蛋白或与之相当，是小分子药物的良好载体。
实施例2.偶联CD19抗体和其抗原结合片段Fab的纳米递送系统的制备
1)转肽酶A的制备
为了方便使用，实验室内部表达并纯化了转肽酶A。但可以理解，可以使用任何市售的转肽酶A。
将SEQ ID No.1克隆在pcDNA3.1表达载体上，经过质粒抽提获取重组质粒，命名为pcDNA3.1-SrtA7_Int。将重组质粒pcDNA3.1-SrtA7_Int利用聚乙烯亚胺(PEI)转染293F细胞。转染细胞4天后，离心收集细胞。加入裂解液(150mM NaCl，25mM Tris pH＝8，0.0001％ IGEPALCA-630，蛋白酶抑制剂(碧云天生物，货号P1006))，反复冻融3次后，离心取上清，随后利用His标签亲和柱Histrap Column(Cytiva，产品目录号29-0510-21)和分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg(Cytiva，产品目录号28-9893-35)进行纯化。
2)重组抗体及其抗原结合片段的制备
为了方便使用，实验室内部表达并纯化了CD19 IgG抗体及其抗原结合片段。为了利用转肽酶A与铁蛋白连接，在其重链C端连接LPETGG。
将SEQ ID NOs.3、4、5克隆在pcDNA3.1表达载体上，经过质粒抽提获取重组质粒，命名为pcDNA3.1-CD19IgH、pcDNA3.1-CD19FabH和pcDNA3.1-CD19IgK。
为表达CD19 IgG抗体，将CD19抗体的重链和轻链质粒pcDNA3.1-CD19IgH和pcDNA3.1-CD19IgK利用聚乙烯亚胺(PEI)转染293F细胞。转染细胞4天后，离心取上清，利用亲和树脂Antibody column package，modern Protein A(Cytiva，产品目录号29497628)进行纯化。纯化后的产物再利用分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg(Cytiva，产品目录号28-9893-35)进行二次纯化。
同样地，将CD19抗体Fab片段质粒pcDNA3.1-CD19FabH和pcDNA3.1-CD19IgK利用聚乙烯亚胺(PEI)转染293F细胞，制备获取CD19抗体的抗原结合片段Fab。转染细胞4天后，离心取上清，利用亲和树脂Protein L(翊圣生物，产品目录号36407ES08)进行纯化。纯化后的产物再利用分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg(Cytiva，产品目录号28-9893-35)进行二次纯化。
3)包被小分子药物的铁蛋白纳米颗粒的制备
将1mg铁蛋白纳米颗粒室温解冻，加入790μl 50mM Tris-HCl pH9.0， 混匀，加入0.3mg Dox。总反应体积1mL。60℃热孔板上静置1小时，用锡纸盖住避光。加入1ml室温dH2O，混匀后转入3K 15-ml超滤离心管(Pall Corporation，产品目录号MCP003C46)浓缩并将溶液置换为10mM Tris，500mM NaCl，pH8.0。浓缩至约0.5ml，0.2μm滤膜过滤后检测或分装保存。
我们通过将铁蛋白纳米颗粒高温处理(60℃1小时)、重组装(室温20-25℃)，将小分子药物DOX包裹进纳米颗粒(图5A)。通过高效液相色谱分离，可见单一的铁蛋白纳米颗粒峰曲线与DOX峰曲线完全一致(图5B)。包裹DOX后的铁蛋白纳米颗粒在投射电镜下呈现均匀的球形颗粒，颗粒内颜色较深(图5C)。以上数据证明铁蛋白纳米颗粒可高效包裹DOX等小分子药物。
4)包被小分子药物的铁蛋白纳米颗粒与抗体的偶联
在Vivaspin 20浓缩管中设置反应体系，加入包裹有DOX的终浓度为120μM的铁蛋白纳米颗粒，120μM的CD19 IgG抗体和100μM的转肽酶A，并加入反应溶液(50mM Tris，150mM NaCl，5mM CaCl2，pH7.5)。经过离心，将反应体系浓缩至250μL并转移至500μL管中，置于摇床上室温反应过夜。随后利用分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg进行纯化。
我们所制备的CD19 IgG抗体在重链C端有LPETGG标签，可以在转肽酶A的催化作用下，与上一步包含有DOX的铁蛋白纳米颗粒进行连接(图6A)。制备完成后的产物通过高效液相色谱分离，可见纳米颗粒的峰曲线与DOX峰曲线完全一致，另外可见一个游离的、未偶联的IgG峰和一个转肽酶A峰(图6B)。在SDS-PAGE/考马斯亮蓝染色中可见其纯度较高，含有铁蛋白-CD19 IgG重链、CD19 IgG重链、CD19 IgG轻链与铁蛋白四个条带(图6C)。投射电镜下呈现周围带有锯齿状的球形颗粒(图6D)，提示抗体IgG已经展示在了纳米颗粒表面。
5)包被小分子药物的铁蛋白纳米颗粒与抗原结合片段Fab的偶联
同样地，我们将CD19抗原结合片段Fab也通过转肽酶A酶的催化作用连接在铁蛋白纳米颗粒表面(图7A)。制备完成后的产物通过高效液相色谱分离，可见纳米颗粒的峰曲线与DOX峰曲线完全一致，另外可见一个未偶联的CD19 Fab和转肽酶A组成的峰(图7B)。在SDS-PAGE/考马斯亮蓝染色中可见其组成部分是铁蛋白偶联的Fab重链、未偶联的Fab重链、Fab轻 链与未偶联的铁蛋白四个条带(图7C)。透射电镜下呈现周围带有锯齿状的球形颗粒(图7D)，提示Fab已经展示在了纳米颗粒表面。
以上数据证明，该抗体-纳米颗粒连接成功并有DOX成功包裹在内。该纳米颗粒具有抗体的特异性，从而能够针对肿瘤细胞表面的特定靶点，将小分子抗肿瘤药物输送到肿瘤细胞中。
6)包被小分子药物的抗体偶联铁蛋白纳米颗粒的温度稳定性测试。
为测试抗体偶联的纳米颗粒的冻融稳定性，我们将储存在-80℃的CD19IgG-铁蛋白纳米颗粒分别置于冰上、4℃和室温解冻，通过高效液相色谱分离，发现融化后的CD19 IgG-铁蛋白纳米颗粒未出现可见的降解(图8)。随后，我们将样品保存于4℃PBS溶液中存放1、2、3、5、7周后用SDS-PAGE考马斯亮蓝染色(图9)和高效液相色谱分离(图10)检测其成分稳定性，发现其可在4℃存放至少7周保持稳定。溶液由PBS换成Tris所得结果一样，证明包被了DOX的CD19 IgG-铁蛋白纳米颗粒可在4℃在PBS或Tris中存放至少7周保持稳定(图11)。
实施例3：偶联CD20抗体IgG、CD22抗体IgG的纳米递送系统的制备
1)重组抗体及其抗原结合片段的制备
为了方便使用，实验室内部表达并纯化了CD20 IgG和CD22 IgG抗体，在其C端均连接LPETGG。
将SEQ ID NOs.6、7、8、9克隆在pcDNA3.1表达载体上，经过质粒抽提获取重组质粒，命名为pcDNA3.1-CD20IgH、pcDNA3.1-CD20IgK、pcDNA3.1-CD22IgH和pcDNA3.1-CD22IgK。
为表达CD20 IgG和CD22 IgG抗体，将CD20抗体的重链和轻链质粒pcDNA3.1-CD20IgH和pcDNA3.1-CD20IgK，以及CD22抗体的重链和轻链质粒pcDNA3.1-CD22IgH和pcDNA3.1-CD22IgK分别利用聚乙烯亚胺(PEI)转染293F细胞。转染细胞4天后，离心取上清，利用亲和树脂Antibody column package modern Protein A(Cytiva，产品目录号29497628)进行纯化。纯化后的产物再利用分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg(Cytiva，产品目录号28-9893-35)进行二次纯化。
2)包被小分子药物的铁蛋白纳米颗粒的制备
将1mg铁蛋白纳米颗粒室温解冻，加入790μl 50mM Tris-HCI，pH9.0，混匀，加入0.3mg Dox。总反应体积1mL。60℃热孔板上静置1小时，用锡纸盖住避光。加入1ml室温dH2O，混匀后转入3K 15-ml超滤离心管(Pall Corporation，产品目录号MCP003C46)浓缩并将溶液置换为10mM Tris，500mM NaCl，pH8.0。浓缩至约0.5ml，0.2μm滤膜过滤后检测或分装保存。
3)包被小分子药物的铁蛋白纳米颗粒与抗体的偶联
在Vivaspin 20浓缩管中设置反应体系，加入终浓度为120μM的铁蛋白纳米颗粒，120μM的CD20 IgG或CD22 IgG抗体和100μM的转肽酶A，并加入反应溶液(50mM Tris，150mM NaCl，5mM CaCl2，pH7.5)。经过离心，将反应体系浓缩至250μL并转移至500μL管中，置于摇床上室温反应过夜。随后利用分子筛凝胶过滤层析柱HiLoad 16/600 Superdex 200pg进行纯化。
我们所制备的CD20 IgG和CD22 IgG抗体在重链C端有LPETGG标签，可以在转肽酶A的催化作用下，与上一步包含有DOX的铁蛋白纳米颗粒进行连接。制备完成后的产物通过高效液相色谱分离，可见纳米颗粒的峰曲线与DOX峰曲线完全一致，另外可见一个游离的、未偶联的IgG峰和一个Sortase A峰(图12A，图13A)。在SDS-PAGE/考马斯亮蓝染色中可见其纯度较高，含有铁蛋白-IgG重链、IgG重链、IgG轻链与铁蛋白四个条带(图12B，图13B)。
实施例4：B细胞肿瘤细胞系的表面结合与内吞实验
1)细胞表面结合实验
我们通过上述实验获得了如下抗体或与纳米颗粒抗体：CD19 IgG，CD19IgG偶联铁蛋白纳米颗粒(CD19IgG-F)，CD19 IgG偶联铁蛋白纳米颗粒包裹DOX(CD19IgG-FD)，CD19 Fab，CD19 Fab偶联铁蛋白纳米颗粒(CD19Fab-F)，CD19 IgG偶联铁蛋白纳米颗粒包裹DOX(CD19Fab-FD)，CD20 IgG，CD20 IgG偶联铁蛋白纳米颗粒(CD20IgG-F)，CD20 IgG偶联铁蛋白纳米颗粒包裹DOX(CD20IgG-FD)，CD22 IgG，CD22 IgG偶联铁蛋白纳米颗粒(CD20IgG-F)，CD22 IgG偶联铁蛋白纳米颗粒包裹DOX(CD22IgG-FD)。
分别将抗体或纳米颗粒抗体样品稀释至10、2、0.4μg/mL，与1x105 Raji(B细胞系)或K562细胞(骨髓性白血病细胞)在冰上孵育30分钟，随后用PBS洗一遍，加入1：2000稀释的Alexa Fluor 488 anti-human IgG(H+L)(ThermoFisher，货号A-11013)，细胞在冰上孵育30分钟，PBS洗两遍后用1％福尔马林固定。随后我们用流式细胞术检测以上各种抗体与纳米颗粒抗体与Raji细胞系或K562细胞系的结合。
结果显示，在浓度分别为10、2、0.4μg/mL时，上述抗体均可高效结合Raji细胞，对K562细胞无可见结合(图14A，图14B)，原因是Raji细胞表面表达相应抗原CD19、CD20和CD22，而作为对照的K562细胞则不表达。因此，包裹DOX的纳米颗粒抗体可以特异性结合和靶向Raji细胞。
2)细胞内吞实验
纳米颗粒抗体样品CD19IgG-F、CD19IgG-FD、CD19Fab-F、CD19Fab-FD稀释至10μg/mL，加入1x105 Raji细胞，孵育30分钟或120分钟，随后用1％福尔马林+1％丙酮固定，PBS洗三遍，加入1：1000稀释的Alexa Fluor 488 anti-human IgG(H+L)(ThermoFisher，货号A-11013)，孵育30分钟，PBS洗两遍后用荧光共聚焦显微镜观察拍照。
通过荧光共聚焦显微镜，我们发现CD19IgG-F、CD19IgG-FD、CD19Fab-F、CD19Fab-FD在与Raji细胞孵育30min后主要结合在细胞表面，在孵育120min后，已经大部分被内吞进入细胞质以内(图15)。
实施例5：B细胞肿瘤细胞系的特异性杀伤实验
在确定上述抗体与纳米颗粒抗体的特异性结合和内吞能力之后，我们在不同浓度下分别与Raji细胞和K562细胞在37℃孵育，测试纳米颗粒抗体作为抗肿瘤药物对B细胞肿瘤的特异性细胞杀伤。
具体地，将Raji和K562细胞铺96孔板，100μl培养基/孔，3 x 104细胞/ml。随后加入铁蛋白、铁蛋白-DOX、抗体、抗体-铁蛋白或包裹了DOX的抗体(FD)，最高总蛋白浓度为50μg/ml，5倍梯度稀释。培养72小时后进行CCK-8检测(诺唯赞，货号A311)，OD450读数时间为加入CCK-8试剂后的4小时。
由图16可以看出，不带有DOX的样品对Raji细胞和K562细胞均无杀 伤。Ferritin-DOX对Raji细胞和K562细胞的杀伤作用基本一致。CD19IgG-FD、CD19Fab-FD、CD20IgG-FD和CD22IgG-FD仅对Raji细胞杀伤作用较强，对对照K562细胞的杀伤作用弱，仅在最高浓度下才对K562细胞产生杀伤效果，后者的原因是K562细胞表面缺乏相应抗原。该实验证明本专利中的纳米颗粒抗体对B细胞肿瘤有特异性细胞杀伤。
实施例6：体内B细胞肿瘤治疗实验
将B-luc-GFP Raji细胞通过尾静脉接种雌性6周龄B-NDG小鼠，1 x 105细胞/小鼠，随后在活体成像荧光素酶平均成像信号达1 x 106P/S时，分别进行不同处理(如下表2所示)。给药后每天称体重，每周测2次活体成像生物发光强度。基于动物福利的原则，小鼠体重减少超过20％之后对小鼠进行安乐死。结果如下表2所示。
表2
实验结果如图17所示，显示B-luc-GFP Raji细胞能够形成肿瘤，造成小鼠体重下降并导致死亡，活体成像可见明显的肿瘤细胞增殖导致的生物发光。偶联CD20 IgG的铁蛋白-DOX(CD20IgG-FD)注射的小鼠全部存活(图17A)，未出现体重减轻(图17B)，且活体成像生物发光值较其他组显著变低(图17C)，证明CD20IgG-FD可以有效治疗Raji B细胞在小鼠体内形成的肿瘤。
表3显示了分组给药第14天的结果。与对照组相比，铁蛋白100μg/只 组、铁蛋白DOX 100μg/只组对肿瘤生长均无抑制作用；CD19 IgG-铁蛋白100μg/只组、CD19 IgG-FD 100μg/只组、CD19 IgG-FD 50μg/只组、CD20 IgG-FD 50μg/只组对肿瘤生长均具有显著抑制作用(TGI：肿瘤生长抑制率，p＜0.05)。
表3.受试品对B-luc GFP Raji细胞移植B-NDG小鼠肿瘤生长的影响
注：a：平均数±标准误；
b：给药组体重与PBS对照组体重在分组给药第14天统计学比较，使用One-way ANOVA分析，组间比较使用Dunnett分析。*p＜0.05，**p＜0.01，***p＜0.001，****p＜0.0001。
本文中涉及的序列
SEQ ID No.1：SRTA7_INT
SEQ ID No.2：Ferritin_SEC
SEQ ID No.3：CD19IgH(Blinatumomab_IgH_LPETGG)
SEQ ID No.4：CD19FabH(Blinatumomab_FabH_LPETGG)
SEQ ID No.5：CD19IgK(Blinatumomab_IgK_LPETGG)

SEQ ID No.6：CD20IgH(Rituximab_IgH_LPETGG)
SEQ ID No.7：CD20IgK(Rituximab_IgK_LPETGG)
SEQ ID No.8：CD22IgH(Inotuzumab_IgH_LPETGG)

SEQ ID No.9：CD22IgK(Inotuzumab_IgK_LPETGG)
SEQ ID No.10：SRTA7_INT
SEQ ID No.11：Ferritin_SEC
SEQ ID No.12：CD19IgH(Blinatumomab_IgH_LPETGG)

SEQ ID No.13：CD19FabH(Blinatumomab_FabH_LPETGG)
SEQ ID No.14：CD19IgK(Blinatumomab_IgK_LPETGG)
SEQ ID No.15：CD20IgH(Rituximab_IgH_LPETGG)

SEQ ID No.16：CD20IgK(Rituximab_IgK_LPETGG)
SEQ ID No.17：CD22IgH(Inotuzumab_IgH_LPETGG)
SEQ ID No.18：CD22IgK(Inotuzumab_IgK_LPETGG)
SEQ ID No.19：转肽酶所形成的连接子序列
LPXTGGGG(其中X可以为任何氨基酸)
SEQ ID No.20：用于连接至铁蛋白单体的N末端
GGGG
SEQ ID No.21：用于连接至靶向性蛋白分子的C末端
LPXTGG(其中X可以为任何氨基酸)
尽管已参考某些实施方案公开本发明，但显而易见的是，可以在不偏离如本文中公开和如随附权利要求书提供的本发明的精神和范围的情况下作出修改和变化。此外，应理解，虽然公开中的所有实施例说明了本发明的实施方案，但它们仅是作为非限制性实施例而提供，因此不应视为限制由此说明的本发明的各个方面。本发明意图具有由本公开、以下权利要求书的语言及其任何等效物界定的全部范围。因此，附图和详述应被视为是说明性的而不是限制性的。

Claims (14)

  1. 纳米递送系统，其包含铁蛋白纳米颗粒、靶向性蛋白和活性分子，所述靶向性蛋白分子通过SEQ ID NO.19连接在铁蛋白纳米颗粒表面，所述活性分子包裹在铁蛋白纳米颗粒内部。
  2. 如权利要求1所述的纳米递送系统，其中所述铁蛋白纳米颗粒中铁蛋白的氨基酸序列如SEQ ID NO.11所示。
  3. 如权利要求1或2所述的纳米递送系统，其中所述靶向性蛋白是抗体或其抗原结合片段。
  4. 如权利要求3所述的纳米递送系统，其中所述抗体是IgG抗体。
  5. 如权利要求1-4任一项所述的纳米递送系统，其中所述铁蛋白纳米颗粒和靶向性蛋白间的连接是通过转肽酶A形成的。
  6. 制备纳米递送系统的方法，所述方法包括：
    1)使铁蛋白纳米颗粒包裹活性分子；和
    2)通过转肽酶A使铁蛋白纳米颗粒连接靶向性蛋白。
  7. 如权利要求6所述的方法，其中步骤1)和2)可以在一个反应中进行，或者分别在两个反应中进行。
  8. 如权利要求6或7所述的方法，其中还包括提供N末端连接SEQ ID NO.20(GGGG)的铁蛋白单元和C末端连接有SEQ ID NO.21(LPXTGG)的靶向性蛋白的步骤。
  9. 如权利要求6-8任一项所述的方法，其中所述铁蛋白的氨基酸序列如SEQ ID NO.11所示。
  10. 如权利要求6-9任一项所述的方法，其中所述靶向性蛋白是抗体或其抗原结合片段，通过转肽酶A使铁蛋白纳米颗粒连接所述抗体或其抗原结合片段的重链C末端。
  11. 一种修饰的铁蛋白，其序列如SEQ ID NO.11所示。
  12. 一种分离的多核苷酸，其编码序列如SEQ ID NO.11所示的铁蛋白。
  13. 权利要求11所述的修饰的铁蛋白或权利要求12所述的多核苷酸在制备纳米递送系统中的用途。
  14. 权利要求1至5所述的纳米递送系统在制备用于治疗肿瘤的药物中 的用途。
PCT/CN2024/100135 2023-07-03 2024-06-19 纳米递送系统、其制备方法和用途 WO2025007748A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106237341A (zh) * 2016-07-12 2016-12-21 浙江大学 一种抗体偶联药物及其制备方法和应用
CN109069611A (zh) * 2016-03-29 2018-12-21 美国政府(由卫生和人类服务部的部长所代表) 取代修饰的融合前rsv f蛋白及其用途
CN109512799A (zh) * 2019-01-17 2019-03-26 中国科学院生物物理研究所 一种装载抗肿瘤药物的纳米药物载体、其制备方法及应用
CN112076315A (zh) * 2020-08-25 2020-12-15 中国农业科学院生物技术研究所 新冠病毒s蛋白和铁蛋白亚基融合的纳米抗原颗粒、新冠疫苗及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109069611A (zh) * 2016-03-29 2018-12-21 美国政府(由卫生和人类服务部的部长所代表) 取代修饰的融合前rsv f蛋白及其用途
CN106237341A (zh) * 2016-07-12 2016-12-21 浙江大学 一种抗体偶联药物及其制备方法和应用
CN109512799A (zh) * 2019-01-17 2019-03-26 中国科学院生物物理研究所 一种装载抗肿瘤药物的纳米药物载体、其制备方法及应用
CN112076315A (zh) * 2020-08-25 2020-12-15 中国农业科学院生物技术研究所 新冠病毒s蛋白和铁蛋白亚基融合的纳米抗原颗粒、新冠疫苗及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 31 August 2021 (2021-08-31), "ferritin [Helicobacter pylori]", XP093259431, Database accession no. WP_000949190.1 *

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