Nanometer delivery systems, methods of making and uses thereof

Abstract

The invention provides a nanometer delivery system comprising ferritin, targeting protein and active molecules, and a preparation method and application of the nanometer delivery system. The targeting delivery system and the targeting therapy which are safe, stable, low in toxicity, high in targeting property, multi-specific and convenient in preparation method can be provided.

Description

Nano delivery system, preparation method and use thereof Technical Field

[0001] The present invention belongs to the field of drug delivery systems for disease diagnosis and treatment. Background Art

[0002] Small molecule drugs are widely used in disease treatment, such as tumor chemotherapy. However, the clinical application of small molecule drugs is limited by their water solubility, tissue distribution, cell specificity and toxicity (ZG Chen, Small-molecule delivery by nanoparticles for anticancer therapy. Trends Mol Med 16, 594-602 (2010); LM Dickenstein, P. Garidel, Lipid-based nanoparticle formulations for small molecules and RNA drugs. Expert Opin Drug Deliv 16, 1205-1226 (2019)).

[0003] Since specific molecules are highly expressed on the surface of certain cells, such as tumor cells, antibodies that recognize these molecules can be used to precisely target these cells. This type of antibody-mediated targeted therapy includes CAR T-cell therapy, antibody-drug conjugates (ADCs), bispecific antibodies, and the like. However, the CAR-T cell therapies currently under development are only targeted at hematological tumors, have large side effects, are expensive, take a long time, and are only targeted at specific subjects; ADCs expose cytotoxic drugs to normal cells or tissues, so they are not stable enough and have 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.

[0004] Summary of the Invention

[0005] 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 has a convenient preparation method.

[0006] This application is based on the inventors' unexpected discovery that by linking ferritin nanoparticles to antibodies via sortase A and further encapsulating active molecules, such as small molecule therapeutics, within the nanoparticles, a safe, stable, low-toxic, precisely targeted, and universally applicable targeted delivery system can be readily achieved. Because ferritin nanoparticles are composed of 24 monomers (or units), they can theoretically bind to 24 antibody molecules via sortase A, thereby increasing antibody affinity. Furthermore, multiple different antibodies can be linked to achieve multi-target targeting and multi-specificity.

[0007] Existing studies use ferritin from humans, which can target human transferrin receptor 1 (TfR1). Human TfR1 is expressed at high levels in tumor cells, achieving the purpose of targeted drug delivery. However, human TfR1 is also widely expressed in healthy human tissues, such as bone marrow, lungs, colon and liver, to import iron into cells. Therefore, the use of human ferritin carries the risk of drug accumulation in healthy tissues, as well as the risk of autoimmunity, both of which are 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 ferritin conformation expressed by mammalian cells can be made closer to its natural state while avoiding the above problems, while achieving high yield (>80 mg / L cells).

[0008] On the one hand, a nanodelivery system is provided, which comprises ferritin nanoparticles, a targeting protein and an active molecule, wherein the targeting protein molecule is linked 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.

[0009] In some specific embodiments, the amino acid sequence of ferritin in the nano-delivery system can be as shown in SEQ ID NO.11.

[0010] In the nanodelivery system described herein, 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.

[0011] In the nanodelivery system described herein, the active molecule can be any molecule desired for targeted delivery that can be encapsulated within the ferritin nanoparticles. For example, 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 within the ferritin nanoparticles used herein.

[0012] In the nanodelivery system described herein, the ferritin nanoparticles and the targeting protein are linked by transpeptidase A, thereby forming a linker (N-terminus-targeting molecule-linker-ferritin-C-terminus) shown in SEQ ID NO. 19 (LPXTGGGG, wherein X can be any amino acid) between the two.

[0013] In another aspect, a method of preparing a nanodelivery system is provided, the method comprising:

[0014] 1) encapsulating active molecules in ferritin nanoparticles; and

[0015] 2) Ferritin nanoparticles are linked to targeting proteins through transpeptidase A.

[0016] Herein, steps 1) and 2) may be performed in one reaction or separately in two reactions.

[0017] In some specific embodiments, the method further comprises the step of providing a ferritin unit having SEQ ID NO. 20 (GGGG) linked to its N-terminus and a targeting protein having SEQ ID NO. 21 (LPXTGG) linked to its C-terminus.

[0018] In some specific embodiments, the amino acid sequence of ferritin in the nano-delivery system can be as shown in SEQ ID NO.11.

[0019] In the methods described herein, 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., an IgG antibody) or an antigen-binding fragment thereof. In the case of an antibody molecule, SEQ ID NO. 21 (LPXTGG) can be attached to the C-terminus of its heavy chain.

[0020] In the methods described herein, the active molecule can be any molecule for which targeted delivery is desired, which can be encapsulated within the ferritin nanoparticles. For example, 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.

[0021] In yet another aspect, a modified ferritin is provided, the sequence of which is shown in SEQ ID NO.11.

[0022] In yet another aspect, a polynucleotide encoding a modified ferritin having a sequence as shown in SEQ ID NO. 11 is provided, and its sequence can be, for example, SEQ ID NO. 2.

[0023] In yet another aspect, provided is a use of the modified ferritin protein or a polynucleotide encoding the modified ferritin protein described herein in preparing a nanodelivery system.

[0024] In yet another aspect, provided is a nanodelivery system as described herein for use in treating a tumor.

[0025] In yet another aspect, provided is a method of treating a tumor comprising administering to a subject a nanodelivery system as described herein.

[0026] In some embodiments, tumor-targeting antibodies, anti-tumor small molecule drugs, and nanoparticle technology are combined to produce an antibody-mediated antibody-ferritin nanoparticle with the ability to deliver small molecule drugs in a targeted manner, which can be used as an anti-tumor nanoparticle drug with precise targeting, strong stability, and low toxicity.

[0027] In some embodiments, targeted antibodies for B-cell non-Hodgkin's lymphoma or lymphocytic leukemia (CD19IgG, CD19Fab, CD20IgG, CD22IgG) are coupled to the surface of nanoparticles coated with anti-tumor small molecule drugs through transpeptidase A. The antibodies are used to precisely target tumor cells and mediate the delivery of small molecule drugs, thereby reducing toxicity to normal cells and enhancing stability. They can be used as targeted therapeutic drugs for B-cell non-Hodgkin's lymphoma or lymphocytic leukemia, and have the characteristics of precise targeting, strong stability, and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following description of the embodiments will be made in conjunction with the accompanying drawings so that the above and other aspects and advantages of the present invention will become apparent and easily understood.

[0029] Figure 1: Schematic diagram showing the passive permeation of ferritin at high temperature and the dissociation-reassembly under strong acid and strong base.

[0030] Figure 2: Schematic diagram of the preparation of a nanoparticle delivery system for encapsulating the anti-cancer drug doxorubicin. Under specific temperature and pH conditions, a small molecule drug, such as doxorubicin (DOX), is encapsulated within ferritin nanoparticles. Then, using Sortase A, IgG antibodies or antigen-binding fragments (Fabs) are conjugated to the drug-encapsulated ferritin nanoparticles, forming nanoparticles capable of targeted drug delivery.

[0031] Figure 3: Shows the preparation of ferritin nanoparticles. (A) Schematic diagram of ferritin nanoparticles. Each nanoparticle is composed of 24 ferritin subunits. Sequence ID No. 20 (GGGG) is linked to the N-terminus of ferritin for attachment to a targeting protein molecule via transpeptidase A. (B) HPLC separation of ferritin nanoparticles. The peak indicated by the arrow demonstrates the high purity of the ferritin nanoparticles. (C) Morphology of ferritin nanoparticles under negative staining electron microscopy.

[0032] Figure 4: Ferritin nanoparticles can dissociate or become loose after treatment with strong acidic or alkaline conditions, or at high temperatures, and reassemble or return to their normal state at neutral pH or room temperature. This suggests their potential for encapsulating small molecule drugs. (A) HPLC analysis shows that ferritin nanoparticles dissociate into ferritin monomers after treatment with an acidic solution (pH < 3.0). Subsequently, the solution is replaced with a neutral PBS solution (pH = 7.4), causing the ferritin to reassemble into nanoparticles. (B) HPLC analysis shows that ferritin nanoparticles dissociate into ferritin monomers after treatment with an alkaline solution (pH > 10.0). Subsequently, the solution is replaced with a neutral PBS solution (pH = 7.4), causing the ferritin to reassemble into nanoparticles. (C) HPLC analysis shows that ferritin nanoparticles become loose after treatment with temperatures above 50°C, with larger pores, allowing drug entry. Upon returning to room temperature (20-25°C), the ferritin nanoparticles return to their original state.

[0033] Figure 5: Example of ferritin nanoparticles coated with DOX (Ferritin-Dox). (A) Schematic diagram of ferritin nanoparticles. One nanoparticle is composed of 24 subunits. (B) HPLC separation of ferritin nanoparticles. The peaks indicated by arrows demonstrate the high purity of the ferritin nanoparticles. (C) Morphology of DOX-coated ferritin nanoparticles under negative staining electron microscopy.

[0034] Figure 6: As an example, CD19 IgG-conjugated ferritin (denoted by F) nanoparticles coated with DOX (denoted by D) (abbreviated as CD19 IgG-FD) are shown. (A) Schematic diagram of ferritin nanoparticles conjugated with IgG antibodies and coated with DOX. IgG antibodies are linked to ferritin subunits via transpeptidase A, and up to 24 IgG antibodies can be attached to the surface of each ferritin nanoparticle. (B) HPLC analysis and purification. (C) SDS-PAGE analysis of the purified CD19 IgG-FD product. (D) Negative-stained electron microscopy images of the nanoparticles before DOX coating (left: CD19 IgG-F, before DOX coating; right: CD19 IgG-FD, after DOX coating).

[0035] Figure 7: An example of a CD19 antibody antigen-binding fragment (Fab)-conjugated ferritin nanoparticle coated with DOX (abbreviated as CD19 Fab-FD). (A) Schematic diagram of ferritin nanoparticles coated with DOX, conjugated to an IgG Fab fragment. Fab is linked to ferritin subunits via transpeptidase A, and up to 24 Fabs can be attached to each ferritin nanoparticle surface. (B) HPLC analysis and purification. (C) SDS-PAGE analysis of the purified CD19 Fab-FD product. (D) Negative-stained electron microscopy images of the nanoparticles before DOX coating (left: CD19 Fab-F, before DOX coating; right: CD19 Fab-FD, after DOX coating).

[0036] Figure 8: Test results of ferritin nanoparticles coupled with antibodies after freezing and thawing under different conditions.

[0037] Figure 9: Stability of antibody-conjugated ferritin nanoparticles-DOX tested by SDS-PAGE staining with Coomassie Brilliant Blue. Samples were stored in PBS at 4°C for 1, 2, 3, 5, and 7 weeks, and then SDS-PAGE staining with Coomassie Brilliant Blue was used to determine the stability of the components.

[0038] Figure 10: Stability of unconjugated ferritin nanoparticles-DOX. Samples were stored in PBS at 4°C for 1, 2, 3, 5, and 7 weeks and analyzed by FPLC.

[0039] Figure 11: Stability of ferritin nanoparticles-DOX after antibody conjugation. 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 analyzed by FPLC.

[0040] Figure 12: CD20 IgG-conjugated 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.

[0041] Figure 13: CD22 antibody IgG-conjugated ferritin nanoparticles coated with DOX (abbreviated as CD22IgG-FD). (A) HPLC analysis and purification. (B) SDS-PAGE analysis of the purified CD22 IgG-FD product.

[0042] Figure 14: Antibody-conjugated ferritin nanoparticles-DOX specifically bind to tumor cell lines. Flow cytometry was used to assess the binding of various antibodies and nanoparticle antibodies to Raji (B cells) or K562 (myeloid leukemia) cells. The concentrations of the antibodies and nanoparticle antibodies were 10, 2, and 0.4 μg / mL, respectively. (A) Flow cytometric analysis of antibody binding. (B) Bar graph of the MFI for each group.

[0043] FIG15 : Results showing that antibody-coupled ferritin nanoparticles-DOX can be internalized by tumor cells.

[0044] Figure 16: Antibody-conjugated ferritin nanoparticles-DOX-specific tumor cell killing results. CD19 IgG, CD19IgG-F, CD19IgG–FD, CD19 Fab, CD19Fab-F, CD19Fab–FD, CD20 IgG, CD20IgG-F, CD20IgG–FD, CD22 IgG, CD20IgG-F, and CD22IgG-FD were tested for cell killing using CCK-8 assay at different concentrations.

[0045] Figure 17: Results of in vivo B cell tumor treatment experiments in mice. (A) Mortality of mice after drug treatment in each group; (B) Weight changes of mice after drug treatment in each group; (C) Fluorescence intensity of tumor cells in vivo as shown by in vivo imaging. DETAILED DESCRIPTION

[0046] The nanodelivery system described herein comprises ferritin nanoparticles, a targeting protein, and an active molecule (e.g., a therapeutic agent). The targeting protein molecule is linked to the ferritin unit via SEQ ID NO. 19, thereby being presented on the surface of the ferritin nanoparticle to bind to a target, such as a target cell surface molecule. The active molecule (e.g., a therapeutic agent) is encapsulated within the ferritin nanoparticle. After the targeting protein specifically binds to the cell surface target, the nanoparticle is internalized into the target cell and releases the active molecule (e.g., a therapeutic agent), exerting the desired active effect.

[0047] Ferritin

[0048] 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 transport protein of iron ions in organisms. 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 (2018); Z. Wang et al., Functional ferritin nanoparticles for biomedical applications. Front Chem Sci Eng 11, 633-646 (2017)).

[0049] Herein, 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.

[0050] In some specific embodiments, the ferritin nanoparticles can be linked to 24 identical or different targeting proteins.

[0051] In some specific embodiments, a modified ferritin unit derived from Helicobacter pylori, having a sequence as shown in SEQ ID NO. 11, is provided, wherein the N-glycosylation site N21 is mutated to glutamine (N21Q mutation). The ferritin unit can form spherical particles composed of 24 ferritin subunits.

[0052] To link the ferritin unit to the targeting protein molecule via transpeptidase A, GGGG (SEQ ID NO. 20) can be attached to the N-terminus of the ferritin unit, or the coding sequence for GGGG (SEQ ID NO. 20) can be attached to the 5' end of its coding sequence. Furthermore, LPXTGG (SEQ ID NO. 21) can be attached to the C-terminus of the targeting protein molecule, or the coding sequence for LPXTGG (SEQ ID NO. 21) can be attached to the 3' end of its coding sequence. After an enzymatic reaction using transpeptidase A, the targeting protein molecule and the ferritin unit are linked together via SEQ ID NO. 19, and the targeting protein molecule is attached to the surface of the ferritin nanoparticle.

[0053] In some specific embodiments, ferritin can be produced by a cell line (e.g., 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.

[0054] In some specific embodiments, ferritin nanoparticles are treated at high temperatures (e.g., above 50°C) to loosen their structure, converting the triangular pores into quadrilateral pores, allowing the encapsulated drug to penetrate into the nanoparticles. Subsequently, the nanoparticles are cooled to room temperature, where the pores return to triangular shapes, retaining the drug within the nanoparticle spheres. Ferritin can remain spherical at 80°C, allowing the encapsulated drug to penetrate into the spheres.

[0055] In some specific embodiments, ferritin nanoparticles are treated under strongly acidic (e.g., below pH 3) or strongly alkaline (e.g., above pH 10) conditions to dissociate into ferritin monomers, the drug to be encapsulated is added, and the ferritin nanoparticles are reassembled into spheres under neutral conditions (e.g., around pH 7), while encapsulating the drug inside the spheres.

[0056] In this paper, ferritin can be stored at -20°C to 30°C and repeatedly frozen and thawed without affecting its delivery function.

[0057] As shown in Figure 1, there are mainly two ways for ferritin to encapsulate small molecule drugs. One is passive penetration. The ferritin sphere has a three-fold channel (appearing as triangular pores) on its 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 four-fold larger channel (appearing as square pores). The average diameter of this four-fold channel is 0.9 nm, and thermal fluctuations can also expand the channel diameter to more than 1 nm, providing conditions for the transfer and encapsulation of active molecules (such as therapeutic agents). The other is dissociation-reassembly. Ferritin nanoparticles dissociate into subunits in a strong acid (e.g., below pH 3) or 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 the drug inside the reassembled nanoparticles. See "A critical review of ferritin as a drug nanocarrier: Structure, properties, comparative advantages and challenges", Shuang Yin, Particuology 64 (2022) 65–84.

[0058] Under neutral conditions, the inner cavity of the ferritin sphere is negatively charged, and positively charged metal ions can easily pass through the pores of the ferritin nanoparticles and accumulate inside the sphere.

[0059] In this article, the pore size can be enlarged by high-temperature treatment; further, by adjusting the pH value of the buffer solution, the charge load in the inner cavity of ferritin can be changed, and at the same time, the charge of the drug to be encapsulated can also be changed. The drug can pass through the channel smoothly and enter the cavity by electrostatic drive. Selecting a pH value close to the pKa can promote the deposition of the drug in the inner cavity of the ferritin sphere.

[0060] In this article, the drug can be encapsulated inside the ferritin sphere by adjusting the temperature or pH.

[0061] Transpeptidase A

[0062] Transpeptidase A can carry out a peptide bond acylation reaction to link two peptide or protein molecules in the presence of Ca 2+ and an appropriate pH value (7.0 - 9.0), that is, H2N-GGGG-protein 1 + C-terminal LPXTGG-protein 2 = protein 2-LPXTGGGG-protein 1. 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.

[0063] In the linker LPXTGGGG (SEQ ID NO. 19) formed by transpeptidase A, 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), or F (Phe). In other words, GGGG (SEQ ID NO. 20) is linked to the N-terminus of one of the two peptides or protein molecules to be linked, and LPXTGG (SEQ ID NO. 21) is linked to the C-terminus of the other, and transpeptidase A is used to form the link.

[0064] In some embodiments, to achieve linkage between ferritin and the targeting protein molecule, the sequence GGGG (SEQ ID NO. 20) is linked to the N-terminus of transferrin, and the sequence LPXTGG (SEQ ID NO. 21) is linked to the C-terminus of the targeting protein, where X can be any amino acid, for example, E. In some embodiments, if the targeting molecule is an antibody, such as an IgG antibody, LPXTGG (SEQ ID NO. 21) can be linked to the C-terminus of its heavy chain for linkage to ferritin.

[0065] Active molecule

[0066] Any suitable active molecule (e.g., a therapeutic agent, such as a small molecule drug) can be used herein and encapsulated in ferritin nanoparticles to form a nanodelivery system for targeted delivery.

[0067] In some embodiments, more than one active molecule (e.g., therapeutic agent) can be used simultaneously. In some embodiments, by way of example, suitable active molecules (e.g., therapeutic agents) can be, but are not limited to, anthracyclines such as doxorubicin, daunorubicin, epirubicin, and idarubicin; antimetabolite chemotherapy 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 chemotherapy drugs such as cisplatin; and Borofalan-10B.

[0068] In some embodiments, doxorubicin is encapsulated in ferritin nanoparticles so as to be delivered to the interior of target cells (e.g., tumor cells). In some embodiments, cisplatin is encapsulated in ferritin nanoparticles so as to be delivered to the interior of target cells (e.g., tumor cells).

[0069] Targeting proteins

[0070] A targeted protein is a protein molecule that specifically recognizes a certain molecule or cell-related protein (such as a cell surface molecule) and is usually used as a drug to treat specific diseases.

[0071] The targeting protein used herein has the sequence SEQ ID NO. 21 (LPXTGG) linked to its C-terminus. This is then linked to the sequence SEQ ID NO. 20 (GGGG) at the N-terminus of the ferritin monomer by transpeptidase A, forming a linker SEQ ID NO. 19 (LPXTGGGG), thereby displaying the targeting protein on the surface of ferritin spheres. The targeting protein can be produced in a cell line (e.g., mammalian cells).

[0072] In some specific embodiments, the targeting protein can be an antibody molecule or an antigen-binding fragment thereof. To achieve linkage to ferritin, the linker sequence SEQ ID NO. 21 (LPXTGG) or its coding sequence is linked to the C-terminus of the heavy chain of the antibody molecule or antigen-binding fragment thereof, or the coding sequence of SEQ ID NO. 21 (LPXTGG) is linked to the 3' end of its coding sequence. Any suitable antibody molecule or antigen-binding fragment thereof (e.g., Fab) can be used, such as a monoclonal antibody or antigen-binding fragment thereof that targets CD19, CD20, or CD22.

[0073] In some specific embodiments, 24 identical or different targeting protein molecules can be linked to the ferritin nanoparticle.

[0074] use

[0075] The nano-delivery system involved in this article can target different cells, molecules or environments by connecting different targeting protein molecules and releasing the active molecules (such as therapeutic agents) encapsulated inside, thereby exerting the desired active effect, such as exerting a therapeutic effect on the desired disease.

[0076] The nanodelivery system involved in this article can be used for a variety of purposes, for example, conjugated CD19, CD20 or CD22 antibodies can be used to treat non-Hodgkin's lymphoma or lymphocytic leukemia, and conjugated EGFR or HER2 antibodies can be used to treat non-small cell lung cancer, colorectal cancer, head and neck cancer, breast cancer, etc.

[0077] The nano-delivery system described in this article can provide safe, stable, less toxic, highly targeted, and multi-specific targeted therapies.

[0078] Example

[0079] The scheme of the present invention will be explained below with reference to the examples. Those skilled in the art will appreciate that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature of the art or in accordance with the product or instrument specifications are used. All reagents or instruments are commercially available if their manufacturers are specified.

[0080] The various antibodies or nanoparticle antibodies involved in the examples are abbreviated as follows: CD19 IgG, CD19 IgG 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-F), CD19 IgG coupled to ferritin nanoparticles coated with DOX (CD19Fab-FD), CD20 IgG, 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).

[0081] Example 1. Production and assembly performance testing of ferritin

[0082] The ferritin used in this article is derived from Helicobacter pylori, and its encoding gene was mutated. The mutated coding sequence and amino acid sequence correspond to SEQ ID NO. 2 and SEQ ID NO. 11 shown below, respectively, with the N21Q mutation in the sequence marked in bold and underlined.

[0083] Mammalian cell expression systems are most similar to humans, and therefore their products are closest to authentic in vivo biological activity. We have found that mutating the N21 N-glycosylation site to glutamine (N21Q mutation) results in a loss of N-glycosylation that renders ferritin expressed in mammalian cells conformation closer to its native state while also achieving high yields (>80 mg / L cells).

[0084] 1) Expression and purification of ferritin

[0085] The nucleotide sequence of SEQ ID No. 2 was cloned into the pcDNA3.1 expression vector. After plasmid extraction, the recombinant plasmid was obtained and named pcDNA3.1-Ferritin_Sec. The recombinant plasmid pcDNA3.1-Ferritin_Sec was transfected into 293F cells using polyethyleneimine (PEI). Four days after transfection, the cells were collected by centrifugation. Lysis buffer (150 mM NaCl, 25 mM Tris pH 8, 0.0001% IGEPAL) was added. CA-630, protease inhibitor (Biyuntian Biotechnology, catalog number P1006), after repeated freezing and thawing three times, the supernatant was collected by centrifugation and then purified using a Histrap column (Cytiva, catalog number 29-0510-21) and a molecular sieve gel filtration chromatography column HiLoad 16 / 600 Superdex 200pg (Cytiva, catalog number 28-9893-35).

[0086] 2) Testing the assembly of ferritin nanoparticles

[0087] Ferritin nanoparticles are composed of 24 ferritin subunits. To present targeted protein antibodies on the surface of ferritin nanoparticles, we added a GGGG linker sequence to the N-terminus of each subunit (Figure 3A). Ferritin nanoparticles purified from cell expression were separated by high-performance liquid chromatography, revealing a single peak curve (Figure 3B). Transmission electron microscopy revealed uniform spherical particles after cell expression and purification (Figure 3C). These data demonstrate that ferritin can be efficiently assembled into nanoparticles.

[0088] We tested the ability of ferritin nanoparticles to assemble at different pH and temperatures.

[0089] In a strongly acidic solution with a pH < 3.0, the ferritin nanoparticles dissociated into monomeric ferritin. Subsequently, when the solution was replaced with a neutral PBS solution, the ferritin reassembled into nanoparticles consistent with the original structure (Figure 4A). A strongly alkaline solution with a pH > 10.0 also dissociated the ferritin nanoparticles into monomeric ferritin. After the solution was replaced with a neutral PBS solution, the ferritin reassembled into nanoparticles consistent with the original structure (Figure 4B).

[0090] We placed ferritin nanoparticles at a high temperature greater than 50°C (e.g., 60°C) and found that after the temperature returned to room temperature, the size of the nanoparticles remained the same as before (Figure 4C).

[0091] These results indicate that the ferritin nanoparticles prepared in this paper have good potential as carriers of small molecule drugs.

[0092] 3) Comparison of human ferritin and Helicobacter pylori ferritin

[0093] A comparison of the small molecule drug doxorubicin (DOX) coated with Helicobacter pylori and human ferritin was tested. The amount of DOX coated within the ferritin was calculated by NanoDrop OD480 measurement, and the ferritin concentration was calculated by NanoDrop OD280 measurement (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)).

[0094] Specifically, 1 mg of ferritin nanoparticles was thawed at room temperature, 790 μl of 50 mM Tris-HCl pH 9.0 was added, mixed, and 0.3 mg of Dox was added. The total reaction volume was 1 mL. The mixture was allowed to stand on a 60°C hot plate for 1 hour and covered with tin foil to protect from light. The mixture was transferred to room temperature (20°C-25°C), 1 ml of room temperature dH2O was added, mixed, and transferred to a 3K 15-ml ultrafiltration centrifuge tube (Pall Corporation, catalog number MCP003C46) for concentration and replacement with 10 mM Tris, 500 mM NaCl, pH 8.0. The mixture was concentrated to approximately 0.5 ml, filtered through a 0.2 μm filter membrane, and then tested or aliquoted for storage. The results are shown in Table 1 below.

[0095] Table 1

[0096] It can be seen from the results in Table 1 that the Helicobacter pylori ferritin used in this paper is higher than or equivalent to human ferritin in terms of the number of DOX molecules encapsulated and the ferritin recovery rate, and is a good carrier for small molecule drugs.

[0097] Example 2. Preparation of a nanodelivery system coupled with CD19 antibody and its antigen-binding fragment Fab

[0098] 1) Preparation of transpeptidase A

[0099] For ease of use, we expressed and purified transpeptidase A in-house. However, it will be appreciated that any commercially available transpeptidase A can be used.

[0100] 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). Four days after transfection, the cells were collected by centrifugation. Lysis buffer (150 mM NaCl, 25 mM Tris pH 8, 0.0001% IGEPAL) was added. CA-630, protease inhibitor (Biyuntian Biotechnology, catalog number P1006), after repeated freezing and thawing three times, the supernatant was collected by centrifugation and then purified using a Histrap column (Cytiva, catalog number 29-0510-21) and a molecular sieve gel filtration chromatography column HiLoad 16 / 600 Superdex 200pg (Cytiva, catalog number 28-9893-35).

[0101] 2) Preparation of recombinant antibodies and their antigen-binding fragments

[0102] For ease of use, CD19 IgG antibodies and their antigen-binding fragments were expressed and purified in-house. To facilitate conjugation to ferritin using transpeptidase A, LPETGG was attached to the C-terminus of the heavy chain.

[0103] 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.

[0104] To express CD19 IgG, 293F cells were transfected with the heavy and light chain plasmids pcDNA3.1-CD19IgH and pcDNA3.1-CD19IgK using polyethyleneimine (PEI). Four days after transfection, 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 using a HiLoad 16 / 600 Superdex 200pg molecular sieve gel filtration column (Cytiva, catalog number 28-9893-35).

[0105] Similarly, the 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. Four days after transfection of the cells, the supernatant was centrifuged and purified using affinity resin Protein L (Yisheng Bio, product catalog number 36407ES08). The purified product was then purified using a molecular sieve gel filtration chromatography column HiLoad 16 / 600 Superdex 200pg (Cytiva, product catalog number 28-9893-35).

[0106] 3) Preparation of ferritin nanoparticles coated with small molecule drugs

[0107] Thaw 1 mg of ferritin nanoparticles at room temperature, add 790 μl of 50 mM Tris-HCl, pH 9.0, mix thoroughly, and then add 0.3 mg of Dox. The total reaction volume is 1 mL. Incubate on a 60°C hot plate for 1 hour, covered with tin foil to protect from light. Add 1 ml of room temperature dH2O, mix thoroughly, and transfer to a 3K 15-ml ultrafiltration centrifuge tube (Pall Corporation, catalog number MCP003C46). Concentrate and replace the solution with 10 mM Tris, 500 mM NaCl, pH 8.0. Concentrate to approximately 0.5 ml and filter through a 0.2 μm filter for analysis or storage.

[0108] We encapsulated the small molecule drug DOX into ferritin nanoparticles by treating them at high temperature (60°C for 1 hour) and reassembling them at room temperature (20-25°C) (Figure 5A). High-performance liquid chromatography (HPLC) separation revealed that the peak curve for a single ferritin nanoparticle was identical to that for DOX (Figure 5B). Transmission electron microscopy revealed uniform spherical particles with a darker color within the particles (Figure 5C). These data demonstrate that ferritin nanoparticles can efficiently encapsulate small molecule drugs such as DOX.

[0109] 4) Conjugation of small molecule drug-encapsulated ferritin nanoparticles with antibodies

[0110] A reaction system was set up in a Vivaspin 20 concentrator tube. Ferritin nanoparticles coated with DOX at a final concentration of 120 μM, 120 μM CD19 IgG antibody, and 100 μM transpeptidase A were added, followed by a reaction solution (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). After centrifugation, the reaction system was concentrated to 250 μL and transferred to a 500 μL tube. The reaction was allowed to react overnight on a shaker at room temperature. Purification was then performed using a HiLoad 16 / 600 Superdex 200 pg molecular sieve gel filtration column.

[0111] The CD19 IgG antibody we prepared has an LPETGG tag at the C-terminus of the heavy chain, which can be linked 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 the peak curve of the nanoparticles was completely consistent with the DOX peak curve. In addition, a free, unconjugated IgG peak and a transpeptidase A peak were visible (Figure 6B). SDS-PAGE / Coomassie Brilliant Blue staining showed high purity, containing four bands: ferritin-CD19 IgG heavy chain, CD19 IgG heavy chain, CD19 IgG light chain, and ferritin (Figure 6C). Transmission electron microscopy revealed spherical particles with jagged edges (Figure 6D), indicating that the antibody IgG was displayed on the nanoparticle surface.

[0112] 5) Conjugation of ferritin nanoparticles coated with small molecule drugs and antigen-binding fragment Fab

[0113] Similarly, we attached the CD19 antigen-binding fragment Fab to the surface of ferritin nanoparticles using the catalytic action of transpeptidase A (Figure 7A). The prepared product was separated by high-performance liquid chromatography (HPLC), revealing that the peak curve of the nanoparticles was completely consistent with the DOX peak curve, with an additional peak consisting of unconjugated CD19 Fab and transpeptidase A (Figure 7B). SDS-PAGE / Coomassie Brilliant Blue staining revealed four bands: ferritin-conjugated Fab heavy chain, unconjugated Fab heavy chain, Fab light chain, and unconjugated ferritin (Figure 7C). Transmission electron microscopy revealed spherical particles with jagged edges (Figure 7D), indicating that the Fab was displayed on the nanoparticle surface.

[0114] The above data demonstrates that the antibody-nanoparticle connection is successful and that DOX is successfully encapsulated. The nanoparticles possess the specificity of antibodies, enabling them to target specific targets on the surface of tumor cells and deliver small molecule anti-tumor drugs into tumor cells.

[0115] 6) Temperature stability test of antibody-coupled ferritin nanoparticles coated with small molecule drugs.

[0116] To test the freeze-thaw stability of the antibody-conjugated nanoparticles, we thawed CD19 IgG-ferritin nanoparticles stored at -80°C on ice, at 4°C, and at room temperature. High-performance liquid chromatography (HPLC) separation revealed no visible degradation of the thawed CD19 IgG-ferritin nanoparticles (Figure 8). Subsequently, we stored the samples in PBS at 4°C for 1, 2, 3, 5, and 7 weeks, and then examined their compositional stability by SDS-PAGE staining with Coomassie Brilliant Blue (Figure 9) and HPLC separation (Figure 10). We found that the samples remained stable at 4°C for at least 7 weeks. Similar results were obtained when the solution was switched from PBS to Tris, demonstrating that DOX-coated CD19 IgG-ferritin nanoparticles remain stable at 4°C for at least 7 weeks in either PBS or Tris (Figure 11).

[0117] Example 3: Preparation of Nano-delivery System Conjugated with CD20 Antibody IgG and CD22 Antibody IgG

[0118] 1) Preparation of recombinant antibodies and their antigen-binding fragments

[0119] For ease of use, CD20 IgG and CD22 IgG antibodies were expressed and purified in-house, and both were linked to LPETGG at their C-termini.

[0120] SEQ ID NOs. 6, 7, 8, and 9 were cloned into the pcDNA3.1 expression vector, and recombinant plasmids were obtained by plasmid extraction and named pcDNA3.1-CD20IgH, pcDNA3.1-CD20IgK, pcDNA3.1-CD22IgH, and pcDNA3.1-CD22IgK.

[0121] To express CD20 IgG and CD22 IgG antibodies, 293F cells were transfected with the CD20 heavy and light chain plasmids pcDNA3.1-CD20IgH and pcDNA3.1-CD20IgK, respectively, and the CD22 heavy and light chain plasmids pcDNA3.1-CD22IgH and pcDNA3.1-CD22IgK, respectively, using polyethyleneimine (PEI). Four days after transfection, 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 using a HiLoad 16 / 600 Superdex 200pg molecular sieve gel filtration column (Cytiva, catalog number 28-9893-35).

[0122] 2) Preparation of ferritin nanoparticles coated with small molecule drugs

[0123] Thaw 1 mg of ferritin nanoparticles at room temperature, add 790 μl of 50 mM Tris-HCl, pH 9.0, mix, and then add 0.3 mg of Dox. The total reaction volume is 1 mL. Incubate on a 60°C hot plate for 1 hour, covered with tin foil to protect from light. Add 1 ml of room temperature dH2O, mix, and transfer to a 3K 15-ml ultrafiltration centrifuge tube (Pall Corporation, catalog number MCP003C46). Concentrate and replace the solution with 10 mM Tris, 500 mM NaCl, pH 8.0. Concentrate to approximately 0.5 ml and filter through a 0.2 μm filter for analysis or aliquoting.

[0124] 3) Conjugation of small molecule drug-encapsulated ferritin nanoparticles with antibodies

[0125] A reaction system was set up in a Vivaspin 20 concentrator tube. Ferritin nanoparticles, 120 μM CD20 IgG or CD22 IgG antibody, and 100 μM transpeptidase A were added to a final concentration of 120 μM. A reaction solution (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) was then added. After centrifugation, the reaction system was concentrated to 250 μL and transferred to a 500 μL tube. The tube was shaken and reacted overnight at room temperature. Purification was then performed using a HiLoad 16 / 600 Superdex 200 pg molecular sieve gel filtration column.

[0126] The CD20 IgG and CD22 IgG antibodies we prepared have an LPETGG tag at the C-terminus of their heavy chains, allowing them to be linked to the ferritin nanoparticles containing DOX in the previous step under the catalysis of transpeptidase A. The resulting products were separated by high-performance liquid chromatography (HPLC), revealing a peak profile for the nanoparticles that was identical to the DOX peak profile. In addition, a free, unconjugated IgG peak and a Sortase A peak were visible (Figures 12A and 13A). SDS-PAGE / Coomassie blue staining revealed high purity, with four bands consisting of ferritin-IgG heavy chain, IgG heavy chain, IgG light chain, and ferritin (Figures 12B and 13B).

[0127] Example 4: Surface binding and endocytosis experiments of B cell tumor cell lines

[0128] 1) Cell surface binding assay

[0129] Through the above experiments, we obtained the following antibodies or antibodies with nanoparticles: CD19 IgG, CD19 IgG 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-F), CD19 IgG coupled to ferritin nanoparticles coated with DOX (CD19Fab-FD), CD20 IgG, 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), and CD22 IgG coupled to ferritin nanoparticles coated with DOX (CD22IgG-FD).

[0130] Antibody or nanoparticle antibody samples were diluted to 10, 2, and 0.4 μg / mL, respectively, and mixed with 1x10 5 Raji (B cell line) or K562 (myeloid leukemia) cells were incubated on ice for 30 minutes, washed once with PBS, and then Alexa Fluor 488 anti-human IgG (H+L) (ThermoFisher, Cat. No. A-11013) diluted 1:2000 was added. The cells were incubated on ice for 30 minutes, washed twice with PBS, and fixed with 1% formalin. Flow cytometry was then used to detect the binding of various antibodies and nanoparticle antibodies to Raji or K562 cell lines.

[0131] The results showed that at concentrations of 10, 2, and 0.4 μg / mL, the antibodies all efficiently bound to Raji cells, with no visible binding to K562 cells (Figures 14A and 14B). This is because Raji cells express the corresponding antigens CD19, CD20, and CD22 on their surface, while the control K562 cells do not. Therefore, the DOX-encapsulated nanoparticle antibodies can specifically bind to and target Raji cells.

[0132] 2) Cell endocytosis experiment

[0133] Nanoparticle antibody samples CD19IgG-F, CD19IgG-FD, CD19Fab-F, and CD19Fab-FD were diluted to 10 μg / mL and 1x10 5Raji cells were incubated for 30 minutes or 120 minutes, then fixed with 1% formalin + 1% acetone, washed three times with PBS, and added with 1:1000 diluted Alexa Fluor 488 anti-human IgG (H+L) (ThermoFisher, Cat. No. A-11013). The cells were incubated for 30 minutes, washed twice with PBS, and observed and photographed using a fluorescence confocal microscope.

[0134] Using fluorescence confocal microscopy, we found that CD19IgG-F, CD19IgG-FD, CD19Fab-F, and CD19Fab-FD were mainly bound to the cell surface after 30 minutes of incubation with Raji cells. After 120 minutes of incubation, most of them had been internalized into the cytoplasm ( Figure 15 ).

[0135] Example 5: Specific killing experiment of B cell tumor cell lines

[0136] After determining the specific binding and endocytosis ability of the above antibodies and nanoparticle antibodies, we incubated them with Raji cells and K562 cells at 37°C at different concentrations to test the specific cell killing of nanoparticle antibodies against B cell tumors as anti-tumor drugs.

[0137] Specifically, Raji and K562 cells were plated in 96-well plates with 100 μl of culture medium per well and 3 × 10 4 Cells were plated at 400 μg / ml. Ferritin, ferritin-DOX, antibody, antibody-ferritin, or DOX-coated antibody (FD) were subsequently added, with a maximum total protein concentration of 50 μg / ml and a 5-fold serial dilution. CCK-8 assay (Novagen, Cat. No. A311) was performed after 72 hours of incubation, and OD450 readings were taken 4 hours after addition of the CCK-8 reagent.

[0138] As can be seen from Figure 16, the sample without DOX had no killing effect on Raji cells or K562 cells. Ferritin-DOX had basically the same killing effect on Raji cells and K562 cells. CD19IgG-FD, CD19Fab-FD, CD20IgG-FD, and CD22IgG-FD only had a strong killing effect on Raji cells, and had a weak killing effect on the control K562 cells. Only at the highest concentration did they have a killing effect on K562 cells. The latter reason was the lack of corresponding antigens on the surface of K562 cells. This experiment proves that the nanoparticle antibodies in this patent have specific cell killing effects on B cell tumors.

[0139] Example 6: In vivo B cell tumor treatment experiment

[0140] B-luc-GFP Raji cells were inoculated into female 6-week-old B-NDG mice via the tail vein at a rate of 1 x 10 5 cells / mouse, and then the average luciferase imaging signal in vivo reached 1 x 10 6 During P / S treatment, mice were treated with different treatments (as shown in Table 2 below). Following dosing, mice were weighed daily, and in vivo bioluminescence intensity was measured twice weekly. Based on animal welfare principles, mice were euthanized after a weight loss exceeding 20%. The results are shown in Table 2 below.

[0141] Table 2

[0142] The experimental results, shown in Figure 17, demonstrate that B-luc-GFP Raji cells can form tumors, causing weight loss and death in mice. In vivo imaging revealed significant bioluminescence due to tumor cell proliferation. All mice injected with CD20 IgG-coupled ferritin-DOX (CD20IgG-FD) survived (Figure 17A), showed no weight loss (Figure 17B), and exhibited significantly lower bioluminescence values ​​in vivo than those in other groups (Figure 17C), demonstrating that CD20IgG-FD can effectively treat Raji B cell-derived tumors in mice.

[0143] Table 3 shows the results on day 14 of group-administered therapy. Compared with the control group, the ferritin 100 μg / mouse group and the ferritin DOX 100 μg / mouse group had no inhibitory effect on tumor growth. However, the CD19 IgG-ferritin 100 μg / mouse group, CD19 IgG-FD 100 μg / mouse group, CD19 IgG-FD 50 μg / mouse group, and CD20 IgG-FD 50 μg / mouse group all had significant inhibitory effects on tumor growth (TGI: tumor growth inhibition rate, p < 0.05).

[0144] Table 3. Effects of the test articles on tumor growth in B-NDG mice transplanted with B-luc GFP Raji cells

[0145] Note: a: mean ± standard error;

[0146] b: Statistical comparison of body weight between the drug-treated group and the PBS control group on day 14 of administration using one-way ANOVA analysis. Inter-group comparisons were performed using Dunnett's analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0147] Sequences mentioned in this article

[0148] SEQ ID No. 1: SRTA7_INT <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0149] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.2: Ferritin_SEC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0150] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.3: CD19IgH(Blinatumomab_IgH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0151] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.4:CD19FabH(Blinatumomab_FabH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0152] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.5:CD19IgK(Blinatumomab_IgK_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0153] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.6:CD20IgH(Rituximab_IgH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0154] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.7:CD20IgK(Rituximab_IgK_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0155] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.8: CD22IgH(Inotuzumab_IgH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0156] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.9:CD22IgK(Inotuzumab_IgK_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0157] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.10:SRTA7_INT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0158] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.11: Ferritin_SEC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0159] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.12: CD19IgH(Blinatumomab_IgH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0160] <h2 style=";text-align:left;direction:ltr"> SEQ ID No.13:CD19FabH(Blinatumomab_FabH_LPETGG)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0161] SEQ ID No.14: CD19IgK(Blinatumomab_IgK_LPETGG)

[0162] SEQ ID No.15: CD20IgH(Rituximab_IgH_LPETGG)

[0163] SEQ ID No.16: CD20IgK(Rituximab_IgK_LPETGG)

[0164] SEQ ID No.17: CD22IgH(Inotuzumab_IgH_LPETGG)

[0165] SEQ ID No.18: CD22IgK(Inotuzumab_IgK_LPETGG)

[0166] SEQ ID No.19: Linker sequence formed by transpeptidase

[0167] LPXTGGGG (where X can be any amino acid)

[0168] SEQ ID No. 20: For attachment to the N-terminus of a ferritin monomer

[0169] GGGG

[0170] SEQ ID No.21: C-terminus for linking to a targeting protein molecule

[0171] LPXTGG (where X can be any amino acid)

[0172] Although the present invention has been disclosed with reference to certain embodiments, it is apparent that modifications and variations can be made without departing from the spirit and scope of the present invention as disclosed herein and as provided in the appended claims. In addition, it should be understood that although all examples in the disclosure illustrate embodiments of the present invention, they are provided as non-limiting examples only and, therefore, should not be construed as limiting the various aspects of the invention thus described. The present invention is intended to have the full scope defined by the language of this disclosure, the following claims, and any equivalents thereof. Accordingly, the drawings and detailed description should be regarded as illustrative rather than restrictive.

Claims

1. A nano-delivery system comprising ferritin nanoparticles, a targeting protein and an active molecule, wherein the targeting protein molecule is connected to the surface of the ferritin nanoparticles via SEQ ID NO.19, and the active molecule is encapsulated inside the ferritin nanoparticles.

2. The nanodelivery system of claim 1, wherein the amino acid sequence of ferritin in the ferritin nanoparticles is as shown in SEQ ID NO.

11.

3. The nanodelivery system of claim 1 or 2, wherein the targeting protein is an antibody or an antigen-binding fragment thereof.

4. The nanodelivery system of claim 3, wherein the antibody is an IgG antibody.

5. The nanodelivery system of any one of claims 1 to 4, wherein the connection between the ferritin nanoparticle and the targeting protein is formed by transpeptidase A.

6. A method for preparing a nano-delivery system, the method comprising: 1) Encapsulating active molecules with ferritin nanoparticles; and 2) Ferritin nanoparticles are linked to targeting proteins through transpeptidase A.

7. The method of claim 6, wherein steps 1) and 2) can be carried out in one reaction, or in two reactions respectively.

8. The method of claim 6 or 7, further comprising the step of providing a ferritin unit having SEQ ID NO. 20 (GGGG) linked to the N-terminus and a targeting protein having SEQ ID NO. 21 (LPXTGG) linked to the C-terminus.

9. The method according to any one of claims 6 to 8, wherein the amino acid sequence of the ferritin is as shown in SEQ ID NO.

11.

10. The method according to any one of claims 6 to 9, wherein the targeting protein is an antibody or an antigen-binding fragment thereof, and the ferritin nanoparticles are linked to the C-terminus of the heavy chain of the antibody or the antigen-binding fragment thereof by transpeptidase A.

11. A modified ferritin, the sequence of which is shown in SEQ ID NO.

11.

12. An isolated polynucleotide encoding the ferritin protein as shown in SEQ ID NO.

11.

13. Use of the modified ferritin according to claim 11 or the polynucleotide according to claim 12 in preparing a nano-delivery system.

14. Use of the nano delivery system according to claims 1 to 5 in the preparation of a drug for treating tumors.

Images