Cell-targeted active ingredient delivery systems

Abstract

The present invention relates to an isolated cell-targeted delivery system comprising CD45 + leukocytes, in said cells comprising a complex of one or more iron binding proteins and an active ingredient, the present invention also relates to a method for preparing such an isolated cell-targeted delivery system, and the use of the system for therapy, in particular for the treatment of cancer.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on June 22, 2016, with application number 201680047507.X and invention title "Cell-Targeted Active Ingredient Delivery System". Technical Field

[0002] This invention relates to a segregated cell-targeted delivery system containing CD45. + Leukocytes, containing a complex of one or more iron-binding proteins and active ingredients, the present invention also relates to a method for preparing such an isolated cell-targeted delivery system, and the use of the system for treatment, particularly for the treatment of cancer. Background Technology

[0003] Current imaging tools are capable of detecting large metastatic tumors (greater than 0.5 cm to 1 cm in size). However, they rarely detect the early spread of metastatic tumor cells. Human metastatic tumors smaller than 0.5 cm are avascular and therefore lack adequate blood and oxygen supply. This means that it is impossible to deliver contrast agents through the bloodstream to label these metastatic tumors and to image them. The presence of hypoxia is a common feature of micrometastases, where the hypoxic portion can be as high as 90%, with almost no blood perfusion (Li et al., 2012, Journal of Solid Tumours, 2(2): 28-33). Therefore, severe hypoxia is considered a general feature of micrometastases.

[0004] Targeting one or more micrometastases hidden within large groups of normal cells presents unique challenges because access to micrometastases is hindered by several biological barriers, poor blood supply, and the small size of micrometastases and their dispersion to organs present other obstacles.

[0005] For the same reasons, the treatment of micrometastases is often challenging. Although the solid tumors from which micrometastases originate usually respond well to conventional treatment, they often regrow at the site of the primary tumor or at the site of the metastasis. This poses a serious problem in clinical oncology (Muthana et al., 2012, Cancer Res; 73(2); 490-495). This is related to the microenvironmental characteristics of solid tumors that limit drug penetration, thus exposing the tumor to concentrations below the effective concentration of the drug (Hobbs et al., 1998, Proc Natl Acad Sci USA: 4607-4612). This is caused by an inadequate vascular system, which leads to high heterogeneity of cancer cells in the mass, low oxygen tension (hypoxia), low pH, and low glucose concentration (Kizaka-Kondoh et al., 2003, Cancer Sci 94(12): 1021-1028). Furthermore, in some areas, rapid tumor cell proliferation can outpace the rate of new blood vessel growth, promoting the formation of hypoxic regions (Lewis and Murdoch, 2005, Am J Pathol 167(3): 627-635). This abnormal vascular structure leading to tumor hypoxia and its subsequent impaired function are associated with a more malignant phenotype and lower survival rates in patients with solid tumors, and with treatment failure due to reduced drug uptake and hypoxia-induced alterations in cancer cells (Sun et al., 2012, Clin Cancer Res 18(3): 758-770; Sullivan et al., 2008 MolCancer Ther 7(7): 1961-1973; Kizaka-Kondoh et al., 2003, Cancer Sci 94(12): 1021-1028). In addition, chemotherapy or radiotherapy can cause additional formation of large areas of tumor hypoxia, making tumor treatment even more difficult. The fact that the effectiveness of anti-cancer treatments is limited by the presence of hypoxic tumor cells has led to the introduction of various treatment methods aimed at overcoming this problem.

[0006] The inventor has discovered CD45 + Leukocytes, particularly activated macrophages, can take up active ingredients complexed in vitro with one or more iron-binding proteins and deliver these complexes in vivo to cells, preferably to tumor cells. Based on this achievement, the inventors have overcome one or more of the aforementioned problems of the prior art. Therefore, the targeted delivery system of the present invention particularly provides one or more of the following advantages: (i) specific delivery of one or more active ingredients to cells that attract the aforementioned CD45. +(ii) delivery to diseased cells, preferably to tissues containing leukocytes; (iii) protection of the active ingredient from inactivation in the bloodstream or clearance from the body; (iv) delivery of the active ingredient, preferably to cells in poorly vascularized or non-vascularized disease areas, such as metastases, large tumors, rheumatic lesions, avascular wounds, or hypoxic areas in the skin; (v) reduction of the toxicity of the active ingredient; (v) delivery of an active ingredient with poor pharmacokinetics; (vi) reduction of drug side effects due to targeted drug delivery; (vii) higher therapeutic efficacy at lower doses due to targeted delivery; and / or (viii) administration of a drug linked to an iron-binding protein loaded on CD45. + Therefore, the risk of local tissue damage at the drug application site is lower due to the presence of white blood cells (Pérez-Herrero E, Fernández-Medarde A. 2015, Eur J Pharm Biopharm 93:52-79). Summary of the Invention

[0007] In a first aspect, the present invention relates to a segregated targeted delivery system comprising CD45 + White blood cells, CD45 + Leukocytes are preferably capable of internalizing iron-binding proteins, and the cells contain a complex of one or more iron-binding proteins and one or more active ingredients.

[0008] In a second aspect, the present invention relates to a method for preparing the separated targeted delivery system of the first aspect of the present invention, comprising the following steps:

[0009] a) Provides a preferred purified iron-binding protein;

[0010] b) Covalently or non-covalently linking the active ingredient to iron-binding proteins and / or encapsulating the active ingredient in iron-binding proteins;

[0011] c) Provide CD45 + White blood cells; and

[0012] d) Culturing CD45 in the presence of the complex of the iron-binding protein and the active ingredient generated in step b) + White blood cells, up to CD45 + The leukocytes are at least partially loaded with the complex of the iron-binding protein and the active ingredient produced in step b).

[0013] In a third aspect, the present invention relates to a segregated targeted delivery system of the first aspect of the invention, which is used as a drug.

[0014] In a fourth aspect, the present invention relates to pharmaceutical compositions comprising the isolated targeted delivery system of the present invention and pharmaceutically acceptable carriers and / or suitable excipients.

[0015] In a fifth aspect, the present invention relates to a segregated targeted delivery system of the first aspect of the invention for the prevention / treatment of tumors, inflammatory diseases or ischemic areas, wherein the tumors are preferably solid tumors, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer or tumors with hypoxic areas, particularly in skin wounds or after organ infarction (heart) or retinal ischemia. Attached Figure Description

[0016] Figure 1Group (A) shows the minimum active fragment of the common amino acid sequence in the mammalian ferritin H chain and two full-length common sequences based on several mammalian ferritin H chains (see SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 7, respectively), as well as the minimum and full-length amino acid sequences of the mouse ferritin H chain (SEQ ID NO: 3 and SEQ ID NO: 4) and the minimum and full-length amino acid sequences of the human ferritin H chain (SEQ ID NO: 5 and SEQ ID NO: 6). Group (B) shows the minimum active fragment of the common amino acid sequence in the mammalian ferritin L chain and two full-length common sequences based on several mammalian ferritin L chains (see SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 14, respectively), as well as the minimum and full-length amino acid sequences of the mouse ferritin L chain (SEQ ID NO: 10 and SEQ ID NO: 11) and the minimum and full-length amino acid sequences of the human ferritin L chain (SEQ ID NO: 12 and SEQ ID NO: 13). Group (C) shows the minimum active fragment of the common amino acid sequence in the mammalian hemoglobin α chain and a full-length common sequence based on several mammalian hemoglobin α chains (see SEQ ID NO: 15 and SEQ ID NO: 16, respectively), as well as the minimum amino acid sequence and full-length amino acid sequence of the human hemoglobin α chain (SEQ ID NO: 17 and SEQ ID NO: 18). Group (D) shows the minimum active fragment of the common amino acid sequence in the mammalian hemoglobin β chain and a full-length common sequence based on several mammalian hemoglobin β chains (see SEQ ID NO: 19 and SEQ ID NO: 20, respectively), as well as the minimum amino acid sequence and full-length amino acid sequence of the human hemoglobin β chain (SEQ ID NO: 21 and SEQ ID NO: 22). Group (E) shows the N-terminal and C-terminal minimal active fragments of the common amino acid sequence in mammalian transferrin (SEQ ID NO: 23 and SEQ ID NO: 24) and the full-length common sequence based on several mammalian transferrins (SEQ ID NO: 25), as well as the N-terminal and C-terminal minimal active fragments of human transferrin (SEQ ID NO: 26 and SEQ ID NO: 27) and the full-length amino acid sequence of human transferrin (SEQ ID NO: 28). In the common sequence, X represents a variable position and represents any native amino acid. Preferably, in each case, X is independent of the other X and represents an amino acid present in the human protein.

[0017] Figure 2 : Macrophages within a mouse tumor mass (TRITC-stained before injection, loaded with FITC-modified ferritin).

[0018] Figure 3 Confocal microscopy images of mouse tumor tissue injected with breast cancer cells and intravenously administered with macrophages loaded with FITC-ferritin (asterisk) – clearly showing the spread of ferritin-FITC throughout the tumor mass, not only in macrophages but also in cancer cells.

[0019] Figure 4 : A snapshot of one channel (in the original green channel of the image converted to grayscale) recorded using confocal microscopy of macrophages (indicated by *, loaded with FITC-ferritin) and cancer cells (indicated by arrows, stained with red markers, and therefore not observed in the green channel before ferritin uptake) sampled in vitro at the starting time point (A) and after sufficient time for ferritin-filled cancer cells (B). FITC-ferritin is dynamically transported into cancer cells (first accumulating in vesicles; then diffusing throughout the cytoplasm, as shown in this image (cells appear in the green channel).

[0020] Figure 5 : Shows the survival rate of mice that received placebo and those that received macrophages loaded with ferritin-conjugated melphalan and ferritin-conjugated chlorambucil.

[0021] Figure 6 : This shows tumor cell apoptosis induced by treatment with cyclophosphamide and treatment with cyclophosphamide (administered at the same dose) encapsulated in ferritin loaded into macrophages.

[0022] Figure 7 : MRI images of mouse mammary tumors. Mice were treated with macrophages loaded with ferritin Ft (intravenous injection) (at time point 0 h). An increase in the diameter of blood vessels (arrows) filled with injected macrophages (significantly reduced T2-signal) was then observed, followed by macrophage diffusion into the tissue (dot pattern; arrows). These changes were observed in all mice examined (at the same time points).

[0023] Figure 8 The images show ferritin, hemoglobin, and transferrin taken up by macrophages, ferritin and hemoglobin taken up by monocytes, and ferritin taken up by lymphocytes and granulocytes.

[0024] Figure 9 This indicates the stability of ferritin stored in macrophages.

[0025] Figure 10 This study showed the metastasis of ferritin, hemoglobin, and transferrin from macrophages to different cancer cells.

[0026] Figure 11This indicates the transfer of ferritin from macrophages to cancer cells and non-cancer cells.

[0027] Figure 12 This study demonstrates the metastasis of ferritin encapsulated with hypoxia-activated prodrugs from macrophages to cancer cells.

[0028] Figure 13 This study showed apoptosis in cancer cells that received ferritin encapsulated with different anticancer agents or soluble ferritin with the same agent from co-cultured macrophages.

[0029] Figure 14 This study showed the metastasis of ferritin, hemoglobin, and transferrin from monocytes to different cancer cells.

[0030] Figure 15 This indicates the metastasis of ferritin and hemoglobin from granulocytes to different cancer cells.

[0031] Figure 16 This indicates the metastasis of ferritin and hemoglobin from lymphocytes to different cancer cells.

[0032] Figure 17 The image shows a tumor from a two-photon microscope, which was received by mice that had received pre-labeled macrophages containing ferritin-FITC (before administration).

[0033] Figure 18 : Whole-body imaging of mice that received intravenous labeled macrophages, showing the accumulation of macrophages at tumor sites and their distribution in other organs.

[0034] Figure 19 : Shows macrophage migration to hypoxic tissue, cross-section of tumor from mice, mice administered pre-labeled macrophages intravenously, hypoxic areas of the tumor visualized using the hypoxia marker pimonidazolone.

[0035] Figure 20 : This demonstrates the localization of FITC-loaded ferritin (round objects) vesicles within the tumor microenvironment. FITC-loaded ferritin-containing macrophages were administered intravenously to mice.

[0036] Figure 21 : This image shows the signal recorded by PET in a whole-body analysis of mice with metastatic 4T1 cancer. Mice received intravenous administration of macrophages loaded with 18F-FDG. Increased signal accumulation was observed in the lungs of mice with micrometastases (confirmed by pathological examination). Mice receiving unstained 18F-FDG or without 4T1 cancer showed lower PET signal. Detailed Implementation

[0037] Before the following detailed description of the invention, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these methods, schemes, and reagents can vary. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0038] The elements of the invention will now be described. These elements are listed with particular embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the invention solely to the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with a number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all elements described herein should be considered as disclosed in the description of this application.

[0039] Several references are cited throughout this specification. Each reference cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether preceding or following, is incorporated herein by reference in its entirety. Nothing herein is to be construed as an admission that the invention has no rights prior to this disclosure due to prior invention.

[0040] definition

[0041] In practice of this invention, conventional methods of chemical, biochemical and recombinant DNA techniques as described in the literature in the art have been used unless otherwise stated (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

[0042] Throughout this specification and the following claims, unless the context requires, the word "comprising" and its variations shall be understood to mean comprising the described whole or step or group of wholes or groups of steps, but not excluding any other whole or step or group of wholes or groups of steps. Indicators without quantifiers, as used in this specification and the appended claims, include plural indicators unless expressly stated otherwise.

[0043] The term "targeted drug delivery" refers to the delivery of an active ingredient to a patient, resulting in an increased concentration of the active ingredient in a specific area of ​​the body compared to other areas of the patient's body. Preferably, the relative concentrations are compared between the diseased area of ​​the body and other areas of the body with similar circulatory pathways. In a preferred embodiment, preferably 2 to 24 hours after application of the targeted delivery system of the invention, the concentration of the active ingredient in a given number of cells or a given volume of biopsy tissue from the diseased area is at least 10% higher than the concentration of the active ingredient in the same number of cells or volume of biopsy tissue from a non-diseased area. More preferably, preferably 2 to 24 hours after application of the targeted delivery system of the invention, the concentration of the active ingredient in the diseased area of ​​the patient's body is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, more preferably at least 1000% higher than the concentration of the active ingredient in the non-diseased area of ​​the body. When assessing systemic distribution, it is preferable to deliver at least 5%, preferably at least 10%, and more preferably at least 15% of the active ingredient applied to the patient to the affected area of ​​the body. Targeted delivery of the active ingredient limits its potential harmful effects on the affected area of ​​the body.

[0044] The terms “targeted drug delivery system” or “targeted delivery system” are used synonymously in this application and refer to a system capable of delivering an active ingredient to a target region, i.e., a system capable of targeted delivery, wherein the target region is preferably located within a patient.

[0045] The terms "active ingredient" or "medicine" are used synonymously in the context of this invention and refer to any compound, i.e., a prodrug, that alters or modulates cellular activity or can be activated to alter or modulate cellular activity, preferably altering or modulating cellular activity in a patient. Examples of such active ingredients include so-called "small molecules" and peptides. The term "small molecule" is used in the context of this invention to refer to hydrocarbons or pharmaceutically active radioisotopes with a molecular weight of less than 1500 g / mol. Preferably, medicines that may be used include anticancer drugs, pharmaceutically active radioisotopes, or ferrohydrate.

[0046] As used in the context of this invention, the term "prodrug" refers to any active ingredient that, upon administration, is metabolized or otherwise converted into a biologically active or more active component (or drug) with respect to at least one property. A prodrug is chemically modified in a manner that renders it less active or inactive relative to the drug, but the chemical modification results in the production of the corresponding drug through metabolism or other biological processes after administration of the prodrug to a patient. For example, a prodrug may have altered metabolic stability or delivery properties, fewer side effects or lower toxicity, or improved taste relative to the active drug (see, for example, Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388–392, incorporated herein by reference). Prodrugs can be synthesized using reactants other than the corresponding drug.

[0047] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and are understood to refer to polymers or oligomeric macromolecules formed from nucleotide monomers. A nucleotide monomer consists of a base, a pentose sugar (e.g., but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, polynucleotides are formed via phosphodiester bonds between the individual nucleotide monomers. Nucleic acid molecules referred to in the context of this invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be, for example, chemically synthesized, such as by the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). An “aptamer” is a nucleic acid that binds to a polypeptide with high affinity. Aptamers can be isolated from a large number of different single-stranded RNA molecules by selection methods, such as SELEmir146-a (see, for example, Jayaseena (1999) Clin. Chem. 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5582981). Aptamers can also be isolated in their mirror form, for example, as L-ribonucleotides for synthesis and selection (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The form isolated in this way has the advantage of not being degraded by naturally occurring ribonucleases, and therefore has greater stability.

[0048] The terms "peptide" or "polypeptide" are used interchangeably in the context of this invention, referring to a chain of at least two amino acids linked by peptide bonds. Therefore, in the context of this invention, the term "peptide" also refers to an amino acid chain having more than 50, more than 100, or more than 150 amino acids.

[0049] As used in the context of this invention, the term "antibody" refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are generally used interchangeably. Antibodies are protein molecules produced by plasma cells that are used by the immune system to recognize and neutralize foreign objects, such as bacteria and viruses. Antibodies recognize a unique portion of a foreign target, namely its antigen.

[0050] As used herein, the term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen. Examples of binding fragments included in the term "antibody fragment" include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), and V... H Structural domain, V L Domains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin neoantigen receptor), bi-scFv, bispecific T-cell adaptor (BITE), dual-affinity retargeting (DART) molecules, triple bodies, biantibodies, single-chain biantibodies, alternative scaffold proteins and their fusion proteins.

[0051] As used in this specification, the term "biantibody" refers to a fusion protein or divalent antibody that can bind to different antigens. A biantibody consists of two single-chain proteins containing antibody fragments, or variable fragments. Biantibodies contain the same polypeptide chain (V... H -V L or V L -V H ) connected to the light chain variable structural domain (V L The heavy chain variable structural domain (V) H By using a short peptide linking two variable domains, the domains pair with complementary domains on another chain, creating two antigen-binding sites. Dual antibodies can target the same (monospecific) or different (bispecific) antigens.

[0052] "Single-domain antibodies" refer to antibody fragments composed of a single monomeric variable domain of the antibody. Simply put, they contain only the variable heavy chain region of a single heavy chain antibody produced by camelids or cartilaginous fish. Due to their diverse origins, they are also known as VHH or VNAR (variable neoantigen receptor) fragments. Alternatively, single-domain antibodies can be obtained by using genetic engineering to monomerize the variable domain of a conventional mouse or human antibody. They have a molecular weight of approximately 12 to 15 kDa, making them the smallest antibody fragments capable of recognizing antigens. Other examples include nanobodies.

[0053] As used in the context of this specification, the term "antibody mimic" refers to a compound that can specifically bind to an antigen in a manner similar to that of an antibody, but is structurally unrelated to an antibody. Typically, an antibody mimic is a synthetic peptide or protein with a molar mass of about 3 to 20 kDa that contains one, two, or more exposed domains that specifically bind to antigens. Examples include, in particular, LACI-D1 (a lipoprotein-associated coagulation inhibitor); affilins, such as human-γ-B crystals or human ubiquitin; cysteine ​​protease inhibitors; Sac7D from *Sulfolobus acidocaldarius*; lipid carriers and anticalin derived from lipid carriers; DARPin (designed ankyrin repeat domain); the SH3 domain of Fyn; the Kunits domain of protease inhibitors; monoclonal antibodies, such as the tenth type III domain of fibronectin; adnectin:knottin (cysteine ​​knottin); atrimer; evibody, for example, CTLA4-based binders; and affibody, such as those from *Staphylococcus aureus*. Armadillo proteins include: protein A with a triple helix of the Z-domain; transbody proteins, such as human transferrin; tetralectins, such as monomeric or trimeric human C-type lectin domains; microantibodies, such as trypsin inhibitor II; affilin; and armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody aptamers, but are not artificial antibodies, antibody fragments, or fusion proteins composed of these. Their common advantages over antibodies are better solubility, tissue penetration, heat and enzyme stability, and relatively low production costs.

[0054] Throughout this specification, the term "sequence identity" is used regarding peptide and nucleotide sequence alignment. When aligning two sequences and no reference sequence is specified for calculating the percentage of sequence identity, the longer of the two sequences is used to calculate sequence identity unless otherwise specified. If a reference sequence is specified, sequence identity is determined based on the full length of the reference sequence shown in the SEQ ID. For example, a 200-amino acid peptide sequence may show a maximum percentage of sequence identity of 66.6% (200 / 300) compared to a reference 300-amino acid long peptide sequence, while a 150-amino acid long sequence may show a maximum percentage of sequence identity of 50% (150 / 300). If 15 of these 150 amino acids differ from the corresponding amino acids in the 300-amino acid long reference sequence, the sequence identity level is reduced to 45%. The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined through sequence alignment. This alignment can be performed using several algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), which can be found, for example, at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html. The preferred parameters to use are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The sequence identity level (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Use the BLASTP program for BLAST protein searches, with a score of 50 and a word length of 3.To obtain gap alignments for alignment purposes, Gapped BLAST, as described by Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, was used. When using the BLAST and Gapped BLAST procedures, the default parameters for each procedure were used. Sequence matching analysis could be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: I54-162) or Markov random fields. Structure-based alignments of multiple protein sequences and / or structures can also be performed using information retrieved from sequence databases, available homologues with 3D structures, and user-defined constraints (Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of distantly related proteins; Bioinformatics 2007, 23:802-808; 3DCoffee@igs: a webserver for combining sequences and structures into a multiple sequence alignment; Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1; 32: W37-40). When percentages of sequence identity are mentioned in this application, unless otherwise specified, these percentages are calculated relative to the full length of the longer sequence.

[0055] In the context of this invention, the term "leukocyte" refers to cells involved in the immune system that protects the body from infectious diseases and foreign invaders. All leukocytes are produced and derived from pluripotent cells in the bone marrow, which are called hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic systems. All leukocytes have a nucleus, which distinguishes them from other blood cells, anucleated red blood cells (RBCs), and platelets. Leukocytes can be classified in a standard manner. They are classified into two pairs of broad categories based on their structure (granulocytes or agranulocytic leukocytes) or cell lineage (bone marrow cells or lymphocytes). These broadest categories can be further divided into five main types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytes. They can be further classified into subtypes; for example, B cells, T cells, and NK cells are found among lymphocytes. Granulocytes are distinguished from agranulocytic leukocytes by the shape of their nuclei (lobed versus round, i.e., segmented versus mononuclear) and their cytoplasmic granules (present or absent, or more precisely, visible or invisible under a light microscope). Another dichotomy is by lineage: bone marrow cells (neutrophils, monocytes, eosinophils, and basophils) are distinguished from lymphoid cells (lymphocytes) by hematopoietic lineage (cell differentiation lineage).

[0056] This invention has observed CD45 + Expression is characteristic of leukocytes and is suitable for use in the targeted delivery system of this invention, especially due to CD45. + Leukocytes are attracted to specific tissues and cells within the body, and complexes capable of delivering one or more iron-binding proteins and one or more active ingredients to or from cells. Those skilled in the art should understand that, apart from clonal CD45... + Outside of white blood cells, CD45 + Leukocytes are a mixed population of different leukocytes that express CD45. + Common properties of surface antigens. Therefore, throughout the instructions, different groups of CD45... + The cellular subsets within leukocytes are characterized by other functional and / or structural features. The term "CD45" + "This indicates that most or almost all cells in the cell population express CD45." +Surface antigens. Regarding this and other cell surface antigens, the term "expression" refers to the production of a surface antigen within the cell and its detectable exposure on the cell surface. The expression level of a surface antigen detectably exposed on the cell surface, and consequently the quantity, can vary considerably among different leukocytes. Generally, a cell is considered positive for a cell surface antigen if at least 5, preferably at least 10 copies, of the surface antigen are detectably exposed on the cell surface, i.e., denoted as "expression". + Those skilled in the art are well aware of how to detect, quantify, and select cells that are positive (or negative) for a given cell surface antigen. Preferred methods include fluorescence-activated cell sorting (FACS). In this technique, fluorescently labeled antibodies are used to bind to the cell surface antigen of a cell population, the cells are then separated into individual cells, and characterized as positive or negative for a given cell surface antigen based on the fluorescence intensity measured on the individual cells. In some embodiments of the invention, it is indicated that the expression of a given protein is high or low. This means that the protein is detectably expressed in both cases, i.e., "..." + However, these proteins are expressed at different levels. High expression and low expression represent different absolute quantities of the protein per cell for different proteins. Therefore, if there are more than 500 detectable copies of a protein per cell, it can be considered that the given protein is expressed at a high level; if there are 1 to 50 detectable copies per cell, it can be considered that the given protein is expressed at a low level. Similarly, if another protein has more than 5000 detectable copies, it can be considered that it is expressed at a high level; if there are 1 to 500 detectable copies per cell, it can be considered that it is expressed at a low level. How to use flow cytometry and Becton Dickinson Quantibrite TM Quantifying the amount of proteins expressed or produced in cells using beading (see, for example, Pannu, KK, 2001, Cytometry. 2001 Dec 1; 45(4): 250-8) or mass spectrometry (see, for example, Milo, R., 2013, Bioessays, 35(12): 1050-1055) is well known in the art. For the purposes of this invention, the term “high expression” of a given protein means that detectable expression of that protein is present in healthy CD45 cells. + The highest expression level found in leukocyte populations is at least 70% of the copy number per cell. The term "low expression" for a given protein refers to detectable expression of that protein at levels found in healthy CD45 cells. + The highest expression level found in a leukocyte population, i.e., 30% or less of the protein copy number per cell. Preferably, the "highest expression level" is determined by the level of healthy CD45 in different subjects. +The average of the highest expression levels found in leukocytes. In some embodiments, a preferred cell subpopulation is characterized by "producing" a given protein. This is understood to mean that the protein does not necessarily have to be detectable on the cell surface, but can be present only inside the cell. Those skilled in the art are well aware of how to detect and / or quantify proteins produced intracellularly and / or select cells that produce such proteins.

[0057] In the context of this invention, the term "differentiated monocytes" refers to monocytes that differentiate from directed precursors called macrophage-DC precursors (MDPs), which are primarily found in the bone marrow (but can also be found in the spleen), and differentiate into dendritic cells or macrophages. In mice, they consist of two main subsets: (i) those with high expression of CX3CR1, CCR2, and Ly6C. - Low expression of CD11b + Cells, and (ii) cells with low expression of CX3CR1, CCR2 and Ly6C + High expression of CD11b + Cells. After leaving the bone marrow, mouse Ly6C + Monocytes differentiate into Ly6C in circulation. - Monocytes. Similarly, in human monocyte differentiation, it is believed that CD14 is present in the peripheral blood circulation. ++ Typical monocytes leave the bone marrow and differentiate into CD14. ++ CD16 + Intermediate monocytes, which continue to differentiate into CD14 cells. + CD16 ++ Atypical mononuclear cells (Yang et al., 2014; Biomark Res 2(1) doi.10.1186 / 2050-7771-2-1).

[0058] Macrophages are specialized phagocytic cells and antigen-presenting cells (APCs) found in tissues, differentiated from circulating peripheral blood mononuclear cells (PBMs). In the context of this invention, the term "activated macrophage" refers to any polarized macrophage. Macrophage activation is typically achieved through culture with interleukins, cytokines, and / or growth factors. In particular, IL-4 and M-CSF can be used as activators. Activated macrophages of different phenotypes are classified into M1-macrophages, typically activated macrophages (CAMs), and M2-macrophages, alternatively activated macrophages (AAMs). Typically activated M1-macrophages comprise immune effector cells with an acute inflammatory phenotype. These cells are highly aggressive against bacteria and produce large amounts of lymphokines (Murray and Wynn, 2011, J Leukoc Biol, 89(4): 557-63). Alternatively activated anti-inflammatory M2-macrophages can be divided into at least three subgroups. These subtypes have a variety of different functions, including immune regulation, maintenance of tolerance, and tissue repair / wound healing. In the context of this invention, the term "M1 inducer" refers to a compound that guides PBM differentiation into M1 macrophages. In the context of this invention, the term "M2 inducer" refers to a compound that guides PBM differentiation into M2 macrophages. Those skilled in the art know many ways to promote differentiation into M1 or M2 macrophages.

[0059] The term "phagocytosis by macrophages" refers to the process by which macrophages engulf solid particles to form internal vesicles called phagosomes.

[0060] The term "iron-binding protein" as used refers to proteins that are non-covalently bound to iron ions. Examples of such proteins include ferritin, hemoglobin, transferrin, and lactoferrin. Iron-binding proteins bind via cell surface receptors, which facilitate the internalization of these proteins into the cell.

[0061] In a first aspect, the present invention relates to a segregated targeted delivery system comprising CD45 + Leukocytes, preferably capable of internalizing iron-binding proteins, comprising a complex of one or more iron-binding proteins and one or more active ingredients.

[0062] Given CD45 + The ability of leukocytes or cell populations to internalize iron-binding proteins depends on the expression of receptors involved in this internalization process. Receptors that lead to ferritin internalization include, for example, TfR, CXCR4, CD163, and TIM-2. Those skilled in the art are well aware of how to measure the uptake of iron-binding proteins, and preferred methods for measuring uptake are described in the following Examples section. The inventors also note that CD45... +Leukocyte subsets tend to internalize one iron-binding protein relative to another, thus achieving higher complex concentrations and / or exhibiting less leakage of the complex from the cell. This CD45 is described in more detail below. + Leukocyte subsets.

[0063] In the context of this invention, the phrase "a complex of one or more iron-binding proteins and an active ingredient" refers to a composition in which one or more active ingredient molecules are covalently or non-covalently bound to one or more iron-binding proteins. The covalent or non-covalent binding between one or more iron-binding proteins and one or more active ingredients can be direct or indirect. In the latter case, the active ingredient is linked to the iron-binding protein via a linker or spacer. Linkers or spacers are known to those skilled in the art, for example, polyalanine, polyglycine, carbohydrates, (CH2). n Groups or peptide linkers. Therefore, those skilled in the art will be able to select appropriate linkers or spacers according to the corresponding application. If the iron-binding protein forms a cage, such as ferritin, the term "complex" also includes an enclosure of the active ingredient within the cage, even if there is no covalent or non-covalent bond between the protein and the active compound. When the iron-binding protein is intracellularly converted, the formation of the complex allows the active ingredient to be transported into the cell. Therefore, it is preferred that the active ingredient binds to the iron-binding protein in a manner that does not interfere with the transport mechanism. This can be readily tested by those skilled in the art using uptake assays known in the art, and is described in the Examples section below. Preferably, the complex is sufficiently stable to be retained during transport from the cell to the target region in vivo. Therefore, it is preferred to deliver the complex rather than the active ingredient alone to the cells or cells in the target region. This property also reduces the effect of the active ingredient on CD45. + Potentially harmful effects on leukocytes, such as cytotoxicity. If the active ingredient is covalently coupled to an iron-binding protein, this coupling is preferably via amino acid residues known to be located in the surface region, which do not involve binding to cellular receptors required for cellular uptake of the iron-binding protein. The iron-binding protein used in the present invention can form stable, non-covalently bound complexes with a variety of active ingredients.

[0064] CD45 + The leukocytes are derived from the patient to be treated, in which case the cells loading the complex are autologous to the patient. It is also anticipated that the patient is of MHC type prior to the targeted delivery treatment of the present invention, and that the leukocyte type used for a given patient is an MHC-matched type. In both preferred embodiments, CD45... + White blood cells are primary cells or derived from primary cells through a small number of differentiation steps. Alternatively, CD45... +White blood cells can be derived from immortalized cells, but unconverted CD45 cells are preferred. + Leukocyte cell lines. Therefore, used for CD45. + The blood used for macrophage isolation, preferably for leukocyte separation, is obtained from the patient to be treated or a healthy donor. Alternatively, blood can be obtained from a blood bank. Umbilical cord blood is also considered in this study.

[0065] The inventors noted that it can be made by CD34 + CD45 produced by hematopoietic progenitor cells + Leukocyte subsets are particularly well-suited for targeted delivery of active ingredients. Therefore, leukocytes produced from CD34+ hematopoietic progenitor cells are preferred for generating targeted delivery systems. Technicians are well aware of how to select CD34+ cells. + Hematopoietic precursor cells and how to differentiate these cells into white blood cells.

[0066] Preferably, CD45 + Leukocytes are selected from monocytes, differentiated monocytes, lymphocytes, and granulocytes. Preferred subpopulations of these general categories of leukocytes are defined below by structural parameters, such as the presence or absence of a given protein, its functional properties, and / or the manner of its generation / differentiation. As summarized above, if not every cell in the cell population possesses a specific property, the targeted delivery system of the present invention will still provide the advantages summarized above, provided that the majority of cells in the population possess that property. Therefore, the properties of a preferred cell for the targeted delivery system of the present invention are described below. Those skilled in the art will appreciate that the pharmaceutical compositions of the present invention will comprise millions of cells, and not every cell in this population will possess the functional and / or structural properties summarized herein, but the pharmaceutical composition can still be used to treat diseases if the majority of cells possess the corresponding functional and / or structural properties.

[0067] The inventor has realized that CD45 + Subpopulations of leukocytes possess particularly advantageous properties, especially including the efficiency and / or amount of overall complex uptake, the ability to retain the complex within the cell (i.e., avoiding leakage and targeted release of the active ingredient), and the efficiency of uptake of specific iron-binding proteins and / or targeting of specific tissues or cells, thereby making them suitable for treating or improving specific diseases. The inventors have observed, for example, CD45... + Leukocytes expressing one or more of the following antigens—CD204, CD206, CD200R, and CCR2—prefer ferritin over other iron-binding proteins. Therefore, if the iron-binding protein in the complex is ferritin, CD45 expressing one or more of the following antigens is preferred. + White blood cells: CD204, CD206, CD200R, CCR2. Therefore, in a preferred embodiment of the present invention...

[0068] (i) Monocytes are CD11b + Mononuclear cells, preferably selected from CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CD14 + CD16 + Monocytes, CD11b + CD14 + MHCII + Monocytes, CD11b + CD14 + CD115 + Monocytes, CD11b + CD114 + Monocytes, CD11b + CD116 + Monocytes, CD11b + CCR1 + Monocytes, CD11b + CCR2 + Monocytes, CD11b + CX3CR + Monocytes, CD11b + CXR4 + Monocytes, CD11b + CXR6 + Monocytes and CD11b + CD14 + CD33 + Mononuclear cells;

[0069] (ii) Differentiated monocytes are selected from macrophages, activated macrophages, preferably CD11b + Macrophages, preferably CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, CD11b + CD68 + Macrophages, preferably CD11b + CD86 +M1 macrophages, preferably those that produce inducible nitric oxide synthase (iNOS) and / or secrete interleukin-12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + Major Histocompatibility Complex II + (MHCII + (Low or high expression) M2 macrophages, CD11b + CD200R + M2 macrophages, CD11b + CD163 + M2 macrophages, or activated macrophages that produce and / or secrete arginase-1 and / or interleukin-10 (IL-10); or dendritic cells, preferably expressing CD11bCD11c, CD11bCD80, CD11cCD80, CD11cCD86, CD11c MHCII, and CD11cCD123, preferably differentiated monocytes that do not express lectin-like oxidized low-density lipoprotein receptor-1 (Lox1). + CXC chemokine receptor type 7 (CXCR7) + ) and nuclear factor (erythrocyte-derived 2)-like 2 (NRF2) + Foam cells are a type of macrophage that aggregate on fatty deposits on the walls of blood vessels. On the vessel walls, they take up low-density lipoprotein and convert it into lipid-laden material, giving them a foamy appearance. These cells secrete various substances involved in plaque growth, and their death promotes inflammation, leading to cardiovascular disease.

[0070] (iii) Monocytes or activated monocytes expressing at least one chemokine receptor, preferably selected from CCR1, CCR2, CXR4, and CXR6, or monocytes or activated monocytes expressing at least one growth factor receptor, preferably selected from macrophage colony-stimulating factor receptor (CD115), granulocyte colony-stimulating factor receptor (CD114), and granulocyte-macrophage colony-stimulating factor receptor (composed of CD116 and CD131); monocytes with these characteristics are particularly suitable for the treatment of inflammatory conditions and cancer;

[0071] (iv) Lymphocytes are selected from CD3. + and CD4 + or CD8 + T lymphocytes or CD19 + CD20 + CD21 + CD19 + CD20 + CD19 + CD21 + CD20 + CD21 + Or CD19 + CD20 + CD21 + B lymphocytes; or

[0072] (v) Granulocytes are selected from neutrophils, preferably CD66b. + Neutrophils, eosinophils, and basophils, preferably CD193. + Eosinophils.

[0073] In a preferred embodiment of the targeted delivery system of the present invention, activated macrophages:

[0074] (i) Can be produced in vitro by culturing monocytes or macrophages or their precursors with factors capable of altering expression markers on macrophages, preferably

[0075] (a) Produced by in vitro culture with at least one M1 inducer,

[0076] (b) Produced by in vitro culture with at least one M2 inducer,

[0077] (c) or produced in vitro with factors capable of altering the ability of macrophages to secrete cytokines, preferably IL-10 and IL-12, chemokines, and / or produce iNOS, arginase, or other immunomodulatory enzymes; examples of such factors include: activated platelets, IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, thermally activated gamma globulin, glucocorticoids, tumor growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2), IL-6, macrophage colony-stimulating factor (M-CSF), peroxisome proliferator-activated receptor gamma (PPARγ) agonists, leukocyte suppressors, etc. Influences on the expression of monocytes include LIF (ligand-inhibiting factor), adenosine, worm and fungal infections, lipopolysaccharide (LPS), interferon-γ (INF-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and viral and bacterial infections. In this regard, it was observed that monocytes activated with M1 inducers, particularly LPS, induced iNOS expression; monocytes activated with M1 inducers, particularly LPS, induced arginase-1 non-expression; monocytes activated with M2 inducers, particularly IL-4, induced arginase-1 expression; and monocytes activated with M2 inducers, particularly IL-4, induced iNOS non-expression.

[0078] (ii) Characterized by the expression of at least one of the following antigens: high expression of CD64, CD86, CD16, CD32, MHCII, and / or the production of iNOS and / or IL-12;

[0079] (iii) It can be produced in vitro by monocytes or macrophages with factors that can induce macrophage phagocytosis, such as IL-18, opsonins (e.g., complement-derived proteins, such as iC3b, immunoglobulin G), calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), viral infection and / or bacterial infection;

[0080] (iv) Characterized by the expression of at least one of the following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC motif chemokine receptor 4 (CXCR4), CD163 and / or T cell immunoglobulin domain and mucin domain 2 (TIM-2), and / or showing low expression of MHCII; activated macrophages with these properties are particularly well-suited to containing complexes containing ferritin as an iron-binding protein;

[0081] (v) Possesses the ability to devour; and / or

[0082] (vi) It is capable of secreting cytokines or producing inducible nitric oxide synthase (iNOS) (or other pro-inflammatory compounds), arginase or other immunosuppressive / anti-inflammatory compounds, wherein the cytokines are preferably IL-12 or IL-10.

[0083] In a preferred embodiment of the targeted delivery system of the present invention, the M1 inducer for differentiating macrophages into M1 macrophages is selected from: lipopolysaccharide (LPS), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and viral and bacterial infections; the M2 inducer for differentiating macrophages into M2 macrophages is selected from: IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, thermally activated γ-globulin, glucocorticoids, tumor growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2), IL-6, macrophage colony-stimulating factor (M-CSF), peroxisome proliferator-activated receptor γ (PPARγ) agonist, leukocyte inhibitory factor (LIF), adenosine, worm and fungal infections.

[0084] The inventors unexpectedly discovered that complexes loaded with both M1 and M2 macrophages are suitable for targeted delivery of active agents to hypoxic tissues, preferably tumors or their metastases. Generally, 3% to 5% of administered M1 macrophages are observed to be located at the tumor site, while approximately 35% of M2 macrophages show tumor-specific targeting. However, this general advantage of M2 macrophages is offset when using complexes containing hemoglobin and the drug, as a considerable amount of the complex can be loaded onto M1 macrophages rather than M2 macrophages. This specificity typically makes M2 macrophages more suitable for treating tumors and diseases characterized by hypoxic tissues.

[0085] In a preferred embodiment of the targeted delivery system of the present invention, monocytes:

[0086] (i) Can be generated by CD34 + Production of hematopoietic precursor cells;

[0087] (ii) It can be produced by in vitro culture of mononuclear cells with at least one inducer, preferably M1 or M2 inducer, more preferably at least one M2 inducer;

[0088] (iii) Characterized by the expression of at least one of the following antigens: TfR, CD163, TIM-2, CD14, CD16, CD33 and / or CD115;

[0089] (iv) Characterized by the expression of at least one of the following antigens: TfR, CD163, TIM-2, CXCR4, CD14 and / or CD16; and / or

[0090] (v) It has the ability to devour.

[0091] In this embodiment of the targeted delivery system of the present invention, the M1 inducer for differentiating monocytes is selected from LPS, IFN-γ, GM-CSF, or viral or bacterial infection, or the M2 inducer for differentiating monocytes is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, thermally activated γ-globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor (LIF), cancer conditioned medium, cancer cells, adenosine, and worm or fungal infection.

[0092] The inventors unexpectedly discovered that monocytes are well-suited for targeted delivery of active agents to hypoxic tissues, preferably tumors or their metastases, and that monocytes treated with M2 activator are even more suitable for targeted delivery of active agents to hypoxic tissues, preferably tumors or their metastases. This specificity makes monocytes treated with M2 activator more suitable for treating tumors and hypoxic sites.

[0093] In a preferred embodiment of the targeted delivery system of the present invention, lymphocytes:

[0094] (i) Can be obtained from blood, spleen or bone marrow, or can be obtained from CD34 + Precursor cell generation, as known to the craft and described, for example, in Lewisor and Kim, 2010, J Vis Exp 40:2017; Tassone and Fidler, 2012, Methods in Molecular Biology 882:351-357; Kouro et al., 2005, Current Protocols in Immunology, 66:F22F.1:22F.1.1-22F.1.9;

[0095] (ii) are immune-active lymphocytes;

[0096] (iii) Expressing antigen-specific T cell receptors; and / or

[0097] (iv) Characterized by the expression of at least one of the following antigens: (a) CD3 and CD4 or CD8, or (b) CD19, CD20, CD21, CD19 CD20, CD19 CD21, CD20 CD21, or CD19 CD20 CD21 antigen, and preferably capable of producing immunoglobulins.

[0098] In a preferred embodiment of the targeted delivery system of the present invention, granulocytes:

[0099] (i) It can be obtained from blood, spleen or bone marrow, or from CD34. + Precursor cell generation, as described in, for example, Kuhs et al., 2015, Curr Protoc Immunol 111: 7.23-1-7.23.16; Coquery et al., 2012, Cytometry A81(9): 806-814; Swemydas and Lionakis 2013, J Vis Exp 77: 50586;

[0100] (ii) Characterized by the expression of at least one of CD66b and / or CD193;

[0101] (iii) is a segmented leukocyte, characterized by the presence of granules in its cytoplasm; and / or

[0102] (iii) Characterized by the expression of at least one of the following substances: TfR, CD163, TIM-2 and / or CXCR4.

[0103] In a preferred embodiment of the targeted delivery system of the present invention, the iron-binding protein is selected from ferritin, preferably heavy (H) ferritin, light (L) ferritin and / or mitochondrial ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemoglobin-binding protein, transferrin; and lactoferrin. The terms ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemoglobin-binding protein, transferrin; and lactoferrin include structural variants of naturally occurring proteins, and therefore relate to proteins having at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and more preferably at least 100% of the iron-binding capacity of the corresponding wild-type protein. The iron-binding protein used in this invention is preferably derived from mammals, more preferably from mice, rats, dogs, or apes, especially from chimpanzees or humans, and most preferably from humans. Figure 1 The concordant sequence of the preferred iron-binding protein used in the context of this invention is shown. Preferred structural variants are based on... Figure 1 The sequence is shown. The residues labeled with X vary in different mammalian ferritins, transferrins, and hemoglobins. Changes in these residues are not critical to the protein's ability to bind iron ions. Therefore, it is preferred that amino acid mutations or deletions affect one or more of the residues labeled with X.

[0104] Plasma proteins have long been dedicated carriers for the delivery of active pharmaceutical ingredients in cancer therapy. Therefore, albumin, as the most abundant plasma protein, is currently used in therapeutic regimens for the delivery of taxane molecules and doxorubicin derivatives (Larsen MT et al., 2016, Mol Cell Ther 27; 4:3).

[0105] Human transferrin and ferritin have been considered as effective carriers for delivering small molecules or toxin-conjugates to cancer cells specifically targeting them. To date, despite considerable efforts, no transferrin or ferritin drug complex has been successfully used in clinical practice (Luck AN et al., 2013, Adv Drug Deliv Rev 65(8): 1012-9).

[0106] Ferritin possesses a cage-like structure and the ability to bind iron, making it suitable for encapsulating drugs within its lumen. Ferritin is not abundant in plasma, but it can be readily produced in high yields as a recombinant protein in common protein expression vectors such as *Escherichia coli* cells. The H-chain or L-chain of ferritin encodes a small protein (21 kDa for the H-chain and 19 kDa for the L-chain) capable of assembling 24-mers into a cage-like structure defining a lumen with a diameter of 8 nm. The inventors note that, in the context of this invention, H-ferritin homopolymers represent a preferred protein construct for specifically delivering encapsulated drugs to TfR-expressing CD45 cells. + White blood cells. In addition, H-ferritin targets the active ingredients of the complex to the cell nucleus (thus directly delivering DNA-binding proteins to the cell nucleus).

[0107] Purified transferrin can be effectively bound to some anticancer drugs via appropriately released covalent linkers within the cell (Beyer U et al., 1998, J Med Chem 41(15): 2701-2708). In the case of transferrin, only the lysine groups on the protein surface can be used for covalent linking.

[0108] Hemoglobin has been considered a potential drug carrier in the past due to its multifunctionality in binding to pharmaceutical chemistry, its abundance in blood, and its relative stability (Somatogen, 1993, WO1993008842 A1). However, the lack of receptor-targeting properties has hindered its biomedical applications beyond blood substitutes or anti-salting agents. In fact, Hb can only be recognized by the CD163 (haptoglobin / hemoglobin receptor) epitope on leukocytes, particularly by CD163 epitopes derived from monocytes and macrophages. The CD45-based epitope described in this application... + Leukocyte-mediated protein delivery, particularly macrophage-based protein delivery, allows the central portion of hemoglobin to move as a target-specific drug carrier. Hemoglobin can be readily covalently linked to suitable drug conjugates, accommodating hydrophobic drug molecules within the heme-binding pouch, or even transporting small cytotoxic molecules bound to heme iron. Hb can be readily modified by selectively linking suitable drug conjugates to the β93-cysteine ​​residue, the only titratable cysteine ​​residue on the protein surface. Maleimide-functionalized drugs, such as the tubulin inhibitor monomethylauristatin (MMAE) or the DNA cross-linking drug pyrrolobenzodiazepine dimer (PBD), are prime examples of highly potent cytotoxic agents that readily and specifically attach to the associated β93-cysteine ​​residue.

[0109] Alternatively, lysine residues on the surface of Hb (at least 10 titratable lysine residues per Hb tetramer) can readily bind drugs via cleavable succinimide linkers. Hemoglobin also offers the unique ability to release non-covalently bound heme groups at acidic pH. As demonstrated in the case of paclitaxel, the resulting Apo-hemoglobin is able to contain several hydrophobic molecules within an empty heme pouch (Meng Z et al., 2015 J Pharm Sci 104(3):1045-55).

[0110] Regardless of the conjugation / adsorption / binding method, this invention demonstrates that hemoglobin (Hb), transferrin (Tf), and ferritin are dedicated drug carriers once loaded into suitable cellular systems with tumor-targeting properties, such as activated macrophages. The simple, rapid, inexpensive, and safe purification procedures for these proteins also provide significant added value.

[0111] Based on the sequences of mammalian H-ferritin, L-ferritin, hemoglobin α-chain, hemoglobin β-chain, and transferrin, a common sequence was determined for each of these proteins. These are shown... Figure 1 The sequences (SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 25, respectively) are shown in the diagram. Based on this, and on the deletion and structural analysis disclosed in the prior art, it was determined that H-ferritin, L-ferritin, hemoglobin α chain, and hemoglobin β chain are sufficient to be detected by CD45. + The smallest fragments taken up by leukocytes. These are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 (H-type ferritin); SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 (L-type ferritin); SEQ ID NO: 15 and SEQ ID NO: 17 (hemoglobin α chain); and SEQ ID NO: 19 and SEQ ID NO: 21 (hemoglobin β chain). If contained in a polypeptide and located between 100 to 450 amino acids, preferably between 150 to 400 amino acids, more preferably between 200 to 350 amino acids, and even more preferably between 250 to 320 amino acids, then transferrin contains an N-terminal domain and a C-terminal domain, which are important for binding iron and being taken up by CD45. +Leukocyte uptake is essential. The N-terminal domain contains amino acids 1 to 82 of the full-length concordant transferrin (SEQ ID NO: 25) or full-length human transferrin (SEQ ID NO: 28). The C-terminal domain contains amino acids 396 to 510 of the full-length concordant transferrin (SEQ ID NO: 25) or full-length human transferrin (SEQ ID NO: 28). In each case, X shown in the concordant sequence, which independently represents any amino acid, and characterizes an amino acid that is not retained or only poorly retained in mammalian H-type ferritin, which can be mutated without or with little impairment of the iron-binding properties of the corresponding iron-binding protein. Preferably, X in each case has the meaning of the corresponding amino acid of the human iron-binding protein to which X is compared. This information can be obtained, for example, from Figure 1 The results show common sequence alignments of human proteins, and in some cases, common sequence alignments of mouse proteins.

[0112] The preferred H-type ferritin comprises the amino acid sequence shown in SEQ ID NO: 1 and its variants or is composed of the amino acid sequence shown in SEQ ID NO: 1 and its variants, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, said variant has H-type ferritin composed of the amino acid sequence according to SEQ ID NO: 1, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages. In SEQ ID NO: 1, the X at position 5 may or may not be present; if present, it represents any amino acid, preferably Ile; the X at position 6 represents any amino acid, preferably Asn; the X at position 14 represents any amino acid, preferably Tyr; the X at position 24 represents any amino acid, preferably Tyr or Cys; the X at position 66 represents any amino acid, preferably Phe; the X at position 68 represents any amino acid, preferably Gln; the X at position 75 represents any amino acid, preferably Arg or Cys; the X at position 90 represents any amino acid, preferably His; the X at position 94 represents any amino acid, preferably Ser or Asn; the X at position 120 may or may not be present; if present, it represents any amino acid, preferably His or Tyr, more preferably His; the X at position 123 represents any amino acid, preferably Asn or Ser, more preferably Asn; and the X at position 128 represents any amino acid, preferably Ala or Ser, more preferably Ala.

[0113] In a preferred embodiment, the H-type ferritin comprises or is composed of the murine ferritin according to SEQ ID NO: 3. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises H-type ferritin composed of the amino acid sequence according to SEQ ID NO: 3, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0114] In a preferred embodiment, H-ferritin comprises or is composed of human ferritin according to SEQ ID NO: 5. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises H-ferritin composed of the amino acid sequence according to SEQ ID NO: 5, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0115] In a preferred embodiment, the H-type ferritin comprises or is composed of a mammalian common sequence obtained by comparing the full-length H-type ferritin according to SEQ ID NO: 2 or SEQ ID NO: 7, preferably SEQ ID NO: 2. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises H-type ferritin composed of the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 7, preferably SEQ ID NO: 2, which is CD45 +The uptake capacity of leukocytes preferably M2 macrophages is at least 70%. In SEQ ID NO: 2, X at position 6 can be any naturally occurring amino acid, preferably Pro; X ​​at position 14 can be any naturally occurring amino acid, preferably His; X at position 16 can be any naturally occurring amino acid, preferably Asp; X at position 21 may or may not be present, and if present, it represents any amino acid, preferably Ile; X at position 22 represents any amino acid, preferably Asn; X at position 30 can be any naturally occurring amino acid, preferably Tyr; X at position 40 can be any naturally occurring amino acid, preferably Tyr or Cys, more preferably Tyr; X at position 82 can be any naturally occurring amino acid, preferably Phe; X at position 84 can be any naturally occurring amino acid, preferably Gln; X at position 91 can be any naturally occurring amino acid, preferably Arg or Cys, more preferably Cys; X at position 106 can be any naturally occurring amino acid, preferably His; X at position 110 can be any... The amino acid is naturally occurring, preferably Asn or Ser, more preferably Asn. The X at position 137 can be any naturally occurring amino acid, preferably His or Tyr, more preferably His. The X at position 140 can be any naturally occurring amino acid, preferably Asn or Ser, more preferably Asn. The X at position 145 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala. The X at position 164 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ser. The X at position 166 can be any naturally occurring amino acid, preferably Met or Leu, more preferably Leu. The X at position 178 can be any naturally occurring amino acid, preferably Asp or His, more preferably Asp. The X at position 181 is absent or is any naturally occurring amino acid, preferably Asn. The X at position 182 is absent or is any naturally occurring amino acid, preferably Glu. The X at position 183 is absent or is any naturally occurring amino acid, preferably Ser.In SEQ ID NO: 7, X at position 6 can be any naturally occurring amino acid, preferably Pro; X ​​at position 14 can be any naturally occurring amino acid, preferably His; X at position 16 can be any naturally occurring amino acid, preferably Asp; X at position 21 may or may not be present, and if present, it represents any amino acid, preferably Ile; X at position 29 can be any naturally occurring amino acid, preferably Tyr; X at position 81 can be any naturally occurring amino acid, preferably Phe; X at position 83 can be any naturally occurring amino acid, preferably Gln; X at position 105 can be any naturally occurring amino acid, preferably His; X at position 144 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala; X at position 180 is absent or is any naturally occurring amino acid, preferably Asn; X at position 181 is absent or is any naturally occurring amino acid, preferably Glu; X at position 182 is absent or is any naturally occurring amino acid, preferably Ser.

[0116] In a preferred embodiment, the H-type ferritin comprises or is composed of the full-length murine ferritin according to the preferred SEQ ID NO: 4. Therefore, the preferred structural variant has at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises murine H-type ferritin composed of the amino acid sequence according to SEQ ID NO: 4, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0117] In a preferred embodiment, the H-type ferritin comprises or is composed of the human full-length ferritin according to the preferred SEQ ID NO: 6. Thus, the preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it has human H-type ferritin composed of the amino acid sequence according to SEQ ID NO: 6, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0118] The preferred L-ferritin comprises the amino acid sequence shown in SEQ ID NO: 8 and its variants or consists of the amino acid sequence shown in SEQ ID NO: 8 and its variants, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it has L-ferritin composed of the amino acid sequence according to SEQ ID NO: 8 and CD45 +The uptake capacity of leukocytes preferably M2 macrophages is at least 70%. In SEQ ID NO: 8, X at position 5 can be any naturally occurring amino acid, preferably Asp or Glu, more preferably Asp; X at position 12 can be any naturally occurring amino acid, preferably Arg or Ser, more preferably Ser; X at position 17 can be any naturally occurring amino acid, preferably Ser or Arg, more preferably Ser; X at position 19 can be any naturally occurring amino acid, preferably Arg or Gln, more preferably Gln; X at position 29 can be any naturally occurring amino acid, preferably Phe; X at position 30 can be any naturally occurring amino acid, preferably Tyr or Phe, more preferably Tyr; X at position 42... X can be any naturally occurring amino acid, preferably Ser or Gly, more preferably Ser. X at position 56 can be any naturally occurring amino acid, preferably Ala or Tyr, more preferably Tyr. X at position 61 can be any naturally occurring amino acid, preferably Glu or Lys, more preferably Lys. X at position 62 can be any naturally occurring amino acid, preferably Met or Phe, more preferably Met. X at position 65 can be any naturally occurring amino acid, preferably Asp or Gln, more preferably Gln. X at position 75 can be any naturally occurring amino acid, preferably Ile or Val, more preferably Ile. X at position 76 can... The X at position 79 can be any naturally occurring amino acid, preferably Lys or Gln, more preferably Lys. The X at position 80 can be any naturally occurring amino acid, preferably Glu or Gln, more preferably Gln. The X at position 87 can be any naturally occurring amino acid, preferably Pro or Gln, more preferably Pro. The X at position 88 can be any naturally occurring amino acid, preferably Glu or Asp, more preferably Asp. The X at position 91 can be any naturally occurring amino acid, preferably Glu or Lys, more preferably Lys. The X at position 94 can be any... The X at position 96 can be any naturally occurring amino acid, preferably Met or Leu, more preferably Leu; the X at position 99 can be any naturally occurring amino acid, preferably Lys or Asn, more preferably Lys; the X at position 115 can be any naturally occurring amino acid, preferably Thr or Ala, more preferably Thr; the X at position 119 can be any naturally occurring amino acid, preferably Leu; the X at position 125 can be any naturally occurring amino acid, preferably Ser or Thr, more preferably Thr; and the X at position 127 can be any naturally occurring amino acid.Preferably Tyr or Phe, more preferably Phe; the X at position 130 can be any naturally occurring amino acid, preferably Lys or Glu, more preferably Glu; the X at position 140 can be any naturally occurring amino acid, preferably Asp or Asn, more preferably Asp; the X at position 146 can be any naturally occurring amino acid, preferably Arg or His, more preferably His; the X at position 148 can be any naturally occurring amino acid, preferably Leu or Val, more preferably Leu.

[0119] In a preferred embodiment, the L-ferritin comprises or is composed of the murine L-ferritin according to SEQ ID NO: 10. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises L-ferritin composed of the amino acid sequence according to SEQ ID NO: 10, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0120] In a preferred embodiment, L-ferritin comprises or is composed of human ferritin according to SEQ ID NO: 12. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, L-ferritin having the amino acid sequence according to SEQ ID NO: 12 is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0121] In a preferred embodiment, the L-ferritin comprises or is composed of a mammalian common sequence obtained by comparing the full-length H-ferritin according to SEQ ID NO: 9 or SEQ ID NO: 14, preferably SEQ ID NO: 9. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises L-ferritin composed of the amino acid sequence according to SEQ ID NO: 9 or SEQ ID NO: 14, preferably SEQ ID NO: 9, CD45 +The leukocytes preferably have at least 70% of the ability to be taken up by M2 macrophages. In SEQ ID NO: 9, X at position 12 can be any naturally occurring amino acid, preferably Asp or Glu, more preferably Asp; X at position 19 can be any naturally occurring amino acid, preferably Ser or Arg, more preferably Ser; X at position 24 can be any naturally occurring amino acid, preferably Ser or Arg, more preferably Ser; X at position 26 can be any naturally occurring amino acid, preferably Arg or Gln, more preferably Gln; X at position 36 can be any naturally occurring amino acid, preferably Phe; X at position 37 can be any naturally occurring amino acid, preferably Tyr or Phe, more preferably Tyr; X at position 47... The X at position 63 can be any naturally occurring amino acid, preferably Ser or Gly, more preferably Ser; the X at position 68 can be any naturally occurring amino acid, preferably Glu or Lys, more preferably Lys; the X at position 69 can be any naturally occurring amino acid, preferably Met or Phe, more preferably Met; the X at position 72 can be any naturally occurring amino acid, preferably Asp or Gln, more preferably Gln; the X at position 82 can be any naturally occurring amino acid, preferably Ile or Val, more preferably Ile; the X at position 83... X can be any naturally occurring amino acid, preferably Lys or Gln, more preferably Lys; X at position 86 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala; X at position 87 can be any naturally occurring amino acid, preferably Glu or Gln, more preferably Gln; X at position 94 can be any naturally occurring amino acid, preferably Pro or Gln, more preferably Pro; X ​​at position 95 can be any naturally occurring amino acid, preferably Glu or Asp, more preferably Asp; X at position 98 can be any naturally occurring amino acid, preferably Glu or Lys, more preferably Lys; X at position 101... X can be any naturally occurring amino acid, preferably Met or Leu, more preferably Leu. X at position 103 can be any naturally occurring amino acid, preferably Met or Leu, more preferably Met. X at position 106 can be any naturally occurring amino acid, preferably Lys or Asn, more preferably Lys. X can be any naturally occurring amino acid. X at position 126 can be any naturally occurring amino acid, preferably Leu. X at position 132 can be any naturally occurring amino acid, preferably Ser or Thr, more preferably Thr. X at position 134 can be any naturally occurring amino acid, preferably Tyr or Phe, more preferably Phe.X at position 137 can be any naturally occurring amino acid, preferably Lys or Glu, more preferably Glu; X at position 147 can be any naturally occurring amino acid, preferably Asp or Asn, more preferably Asp; X at position 153 can be any naturally occurring amino acid, preferably Arg or His, more preferably His; X at position 155 can be any naturally occurring amino acid, preferably Leu or Val, more preferably Leu; X at position 161 can be absent or any naturally occurring amino acid, preferably Ala; X at position 163 can be absent or any naturally occurring amino acid, preferably Thr; X at position 166 can be absent or any naturally occurring amino acid, preferably Pro; X ​​at position 168 can be any naturally occurring amino acid, preferably Gly or Ala, more preferably Ala. In SEQ ID NO: 14, X at position 36 can be any naturally occurring amino acid, preferably Phe; X at position 37 can be any naturally occurring amino acid, preferably Tyr or Phe, more preferably Tyr; X at position 94 can be any naturally occurring amino acid, preferably Pro or Gln, more preferably Pro; X ​​at position 126 can be any naturally occurring amino acid, preferably Leu; X at position 137 can be any naturally occurring amino acid, preferably Lys or Glu, more preferably Glu; X at position 147 can be any naturally occurring amino acid, preferably Asp or Asn, more preferably Asp; X can be any naturally occurring amino acid; X at position 163 can be absent or any naturally occurring amino acid, preferably Thr; X at position 166 can be absent or any naturally occurring amino acid, preferably Pro; X ​​at position 168 can be any naturally occurring amino acid, preferably Gly or Ala, more preferably Ala.

[0122] In a preferred embodiment, the L-ferritin comprises or is composed of the full-length murine ferritin according to the preferred SEQ ID NO: 11. Therefore, the preferred structural variant has at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises murine L-ferritin composed of the amino acid sequence according to SEQ ID NO: 11, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0123] In a preferred embodiment, L-ferritin comprises or is composed of human full-length ferritin according to the preferred SEQ ID NO: 13. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises human L-ferritin composed of the amino acid sequence according to SEQ ID NO: 13, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0124] In a preferred embodiment, the α-hemoglobin comprises or is constituted by the minimum mammalian common sequence obtained by comparing the full-length α-hemoglobin according to SEQ ID NO: 15. It preferably comprises or is constituted by the amino acid sequence shown in SEQ ID NO: 15 and its variants, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it has an α-hemoglobin composed of the amino acid sequence according to SEQ ID NO: 15 that is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0125] In a preferred embodiment, the α-hemoglobin comprises or is composed of the minimum human amino acid sequence obtained from or constituted by human α-hemoglobin according to SEQ ID NO: 17. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises α-hemoglobin composed of the amino acid sequence according to SEQ ID NO: 17, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0126] In a preferred embodiment, the α-hemoglobin comprises or is composed of a mammalian common sequence obtained by comparing the full-length α-hemoglobin according to SEQ ID NO: 16. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, the α-hemoglobin having the amino acid sequence according to SEQ ID NO: 16 is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0127] In a preferred embodiment, the α-hemoglobin comprises or is composed of the full-length human α-hemoglobin according to SEQ ID NO: 18. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises the full-length human α-hemoglobin composed of the amino acid sequence according to SEQ ID NO: 18, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0128] In a preferred embodiment, the α-hemoglobin comprises or consists of a minimal mammalian common sequence obtained by comparing the full-length β-hemoglobin according to SEQ ID NO: 19 and variants thereof, said variant having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises β-hemoglobin consisting of the amino acid sequence according to SEQ ID NO: 19, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0129] In a preferred embodiment, the α-hemoglobin comprises or is composed of the minimum human amino acid sequence obtained from or constituted by human β-hemoglobin according to SEQ ID NO: 21. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises β-hemoglobin composed of the amino acid sequence according to SEQ ID NO: 21, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0130] In a preferred embodiment, the β-hemoglobin comprises or is constituted by a mammalian common sequence obtained by comparing the full-length β-hemoglobin according to SEQ ID NO: 20. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises β-hemoglobin consisting of the amino acid sequence according to SEQ ID NO: 20, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0131] In a preferred embodiment, the β-hemoglobin comprises or is composed of human full-length β-hemoglobin according to SEQ ID NO: 22. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises human full-length β-hemoglobin composed of the amino acid sequence according to SEQ ID NO: 22, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0132] In a preferred embodiment, the transferrin comprises or is constituted by a mammalian common sequence obtained by comparing with the full-length α-hemoglobin according to SEQ ID NO: 25. Therefore, particularly, the preferred transferrin comprises or is constituted by the amino acid sequence shown in SEQ ID NO: 25 and its variants, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it has a transferrin composed of the amino acid sequence according to SEQ ID NO: 25 that is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0133] In a preferred embodiment, the transferrin comprises or is composed of human transferrin according to SEQ ID NO: 28. Therefore, preferred structural variants have at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises transferrin consisting of the amino acid sequence according to SEQ ID NO: 28, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0134] The iron-binding properties of transferrin depend on the N-terminal and C-terminal domains. Therefore, in a preferred embodiment, the transferrin used in this invention comprises at least the N-terminal domain according to SEQ ID NO: 23 and the C-terminal domain according to SEQ ID NO: 24. Preferred transferrins comprise proteins containing the amino acid sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 and their variants, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it has a transferrin composed of the amino acid sequences according to SEQ ID NO: 23 and SEQ ID NO: 24 that is CD45-bound. + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages. SEQ ID NO: 23 or SEQ ID NO: 24 represents a common sequence of mammalian transferrin.

[0135] Therefore, in a preferred embodiment, the transferrin used in this invention comprises at least an N-terminal domain according to SEQ ID NO: 26 and a C-terminal domain according to SEQ ID NO: 27. Preferred transferrins comprise proteins comprising the amino acid sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27 and variants thereof, said variants having at least 70% amino acid identity, more preferably at least 75% amino acid identity, more preferably at least 80% amino acid identity, more preferably at least 85% amino acid identity, more preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity, and in each case, it comprises transferrin consisting of the amino acid sequences according to SEQ ID NO: 26 and SEQ ID NO: 27 respectively, and is CD45-bound. + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0136] Regardless of the given amino acid sequence, preferred ferritins also include proteins that conform to a 24-mer subunit assembly of a four-helix bundle protein module, which fall within a given sequence alignment of a distant relative protein defined by a 3D structure-based alignment.

[0137] The inventors unexpectedly observed that lymphocytes and M2 macrophages were better at taking up complexes containing one or more ferritins and one or more active ingredients, M1 macrophages were better at taking up complexes containing one or more hemoglobins and one or more active ingredients, and macrophages were better at taking up complexes containing one or more transferrins and one or more active ingredients. Therefore, based on CD45... + Tissue and cellular tropism of white blood cells: monocytes, M1 and M2 macrophages, granulocytes and lymphocytes; if the tropism of M2 macrophages, lymphocytes or monocytes is desired, then the above-mentioned complex containing one or more ferritins and one or more active ingredients is used to load M2 macrophages, lymphocytes or monocytes; if the tropism of M1 macrophages is desired, then the complex containing one or more hemoglobins and one or more active ingredients is used to load M1 macrophages.

[0138] In a preferred embodiment of the targeted delivery system of the present invention, the active ingredient is selected from proteins, nucleic acids, non-protein, non-nucleic acid compounds with a molecular weight of less than 1.5 kD, more preferably less than 1 kD, preferably anticancer drugs, especially drugs that inhibit cell growth, cytotoxic drugs and their prodrugs; anti-atherosclerotic drugs; and anti-inflammatory drugs; and photosensitizing compounds; viruses, especially oncolytic viruses; radioactive isotopes that emit α or β rays, and also emit γ rays in a cell-damaging amount, preferably selected from lutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, cesium-131, palladium-103, radium-233, iodine-125 and boron-10, or α rays in a cell-damaging amount, preferably selected from actinium-225, bismuth-213, lead-212 and polonium-212.

[0139] Preferred anticancer drugs are selected from apoptosis / autophagy or necrosis inducing agents. These agents can be any drug that can effectively induce apoptosis / autophagy or necrosis even in cells with abnormal cell proliferation. These drugs are preferably used in the form of a complex with one or more ferritins.

[0140] Preferred anticancer drugs are selected from apoptosis-inducing drugs, alkylating agents, antimetabolites, antibiotics, epothilone, nuclear receptor agonists and antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, cell cycle-dependent protein kinase (CDK) inhibitors, cyclooxygenase and / or lipoxygenase inhibitors, biologically derived fatty acids, biologically derived fatty acid derivatives, including prostaglandins and leukotrienes, protein kinase inhibitors, protein phosphatase inhibitors, lipid kinase inhibitors, platinum coordination complexes, ethyleneimine, methylmelamine, triazine, vinca alkaloids, pyrimidine analogs, purine analogs, alkyl sulfonates, folic acid analogs, anthrone, substituted urea, and methylhydrazine derivatives, enediyne antibiotics, microtubule polymerization inhibitors, such as maytansinoid or oliquistatin derivatives, immune checkpoint inhibitors, and inhibitors of tumor-specific proteins or markers, preferably Rho-GDP-degradation inhibitors, more preferably Grp94.

[0141] Other preferred anticancer drugs are selected from sulfadiazine, arubicin, ambazone, aminoglutethimide, L-asparaginase, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, leucovorin, carboplatin, capecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, dauphine, daunorubicin, dibromopropamidine, diethylstilbestrol, docetaxel, doxorubicin, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogens, ethinylestradiol, etoposide, flavin, fluorouracil, and fludaradoxime. Labin, Fluorouracil, Flumethasone, Flutamifluthrin, Furazolidone, Gemcitabine, Gonadotropin-releasing hormone analogs, Hexamethylmelamine, Hydroxyurea, Hydroxymethylfurantoin, Hydroxyprogesterone hexanoate, Hydroxyurea, Idarubicin, Iodidine, Ifosfamide, Interferon Alpha, Irinotecan, Leuprorelin, Lomustine, Letopecan, Sulfamethoxazole Sulfate, Diethylamine Dichloromethamphetamine, Medroxyprogesterone Acetate, Melphalan, Miphalin, Mercaptopurine, Methotrexate, Metronidazole, Mitomycin C, Mitodazole, Mitotane, Mitoxantrone, Photomycin, Nafidic Acid, Nifuratel, Nifurofenazide, Furazolidone, Nifuratel Nitrofurantoin, Nitrosine, Nitrofurantoin, Nitrogen Mustard, Oleomucin, Oxyquinacrine, Pentamidine, Pentostatin, Phenylephrine, Phthasulfathiazole, Piperbromide, Prednimustine, Prednisone, Preussin, Procarbazine, Pyrimethamine, Raltitrexed, Rapamycin, Rofecoxib, Rosiglitazone, Sulfasalazine, Acriflavin, Semustine, Streptozotocin, Sulfaamide, Sulfaacetyl, Sulfachlorpyridazine, Sulfamethoxazole, Sulfapentine, Sulfadisoxine, Sulfadiazole, Sulfaisoxazole, Sulfaguanidine, Sulfaguanoxazole, Sulfamethoxazole, Compound Sulfamethoxazole, Sulfamethoxypyrimidine, Sulfamethoxypyridazine The following are the ingredients: sulfadiazine, sulfadimidine, sulfamethoxazole, sulfadiazine, sulfamethoxazole, sulfadiazine, sulfadiazine, astrone, tamoxifen, paclitaxel, teniposide, tertiposide, testosterone lactone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triamine, trimethoprim, trimethoprim, trefocillin, UCN-01, vincristine, vinblastine, vinorelbine, vinorelbine, and zorubicin, preferably selected from olritamine, banoxantrone, bendamustine, chlorambucil, chaliceamycin, cyclophosphamide, dynemycin A, maytansine, melphalan, mertansine, and neocazinostatin, most preferably banoxantrone, bendamustine, chlorambucil, cyclophosphamide, pyrrolobenzodiazepine, and melphalan.

[0142] Particularly preferred are the anticancer drugs, which are proliferation-inhibiting proteins, nucleic acids, siRNAs, or DNases. The proliferation-inhibiting proteins are preferably cell cycle inhibitors, antibodies, or antibody-like substances. The antibodies or antibody-like substances specifically bind to targets on or within cells of target tissues that regulate the disease state of cells. The targets are preferably proliferation-promoting proteins. The nucleic acids preferably encode proliferation-inhibiting proteins, antibodies, or antibody-like substances. The antibodies or antibody-like substances specifically bind to targets on or within cells of target tissues that regulate the disease state of cells. The targets are preferably proliferation-promoting proteins.

[0143] Preferred examples of antibodies used in the context of this invention are single-chain antibodies, antibody fragments, nanobodies, light or heavy chains, variable light or heavy chains, or biantibodies. Preferred antibody fragments include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single-domain antibodies (sdAb), single-chain variable fragments (scFv), variable fragments (Fv), and V... H Structural domain, V L Domains, single-domain antibodies, nanobodies, IgNAR (immunoglobulin neoantigen receptor), dual scFv, bispecific T cell adaptor (BITE), dual-affinity retargeting (DART) molecules, triantibodies, biantibodies, single-chain biantibodies, and their fusion proteins.

[0144] If the active ingredient is a nucleic acid, it is preferably a miRNA, siRNA, DNase, or nucleic acid encoding a drug-active protein, such as an antibody, antibody mimic, cytokine, prodrug converting enzyme, etc.

[0145] As outlined above, the targeted delivery system of the present invention is particularly suitable for delivering active ingredients to hypoxic regions. Using an active ingredient activated under hypoxic conditions further increases the specificity of the active ingredient's targeting and / or further reduces its side effects. Therefore, in a particularly preferred embodiment, the active ingredient is a hypoxic-activated prodrug. The backbone of all hypoxic-activated prodrugs contains one of five different chemical moieties (nitro, quinine, aromatic and aliphatic N-oxides and transition metals), which are enzymatically reduced in tissues under hypoxic conditions. The hypoxic-activated prodrug is any prodrug with less or no activity relative to the corresponding drug and comprises the drug and one or more bioreducible groups. Such hypoxic-activated prodrugs include all prodrugs activated by various reducing agents and reductases, including but not limited to single-electron transferases (e.g., cytochrome P450 reductase) and two-electron transferases (or hydride transferring) enzymes. According to a preferred embodiment of the invention, the hypoxic-activated prodrug is TH-302. The method for synthesizing TH-302 is described in PCT applications WO 07 / 002931 and WO 08 / 083101. Preferred examples of this prodrug are selected from Group I: benzotriazine N-oxide, apaziquone (EO9), teirazamine (TPN), and SN30000; or from Group II: nitro compounds PR-104A, TH-302, TH-4000, and AQ4N.

[0146] In a preferred embodiment of the segregated targeted delivery system of the present invention, the bond between the iron-binding protein and the active ingredient contained in the complex is covalent and / or non-covalent; and / or the active ingredient contained in the complex is embedded / encapsulated by the iron-binding protein, preferably by ferritin or its multimers. In one embodiment, covalent and / or non-covalent coupling is carried out indirectly via linkers or spacers. If covalent bonding is desired, the active ingredient is directly or indirectly covalently coupled to one or more iron-binding proteins using the relevant thiol, amino, or carboxyl groups of the iron-binding protein. It is also conceivable that the complex contains different active ingredients. For example, one class of active ingredients may bind to iron-binding proteins (non-covalently), while another class is encapsulated. This method utilizes the different release rates of the active substance after delivery from the iron-binding protein to the target tissue and / or cells. For example, a drug derivative as the active ingredient can be reactively covalently linked to the 24-mer surface or lumen of the ferritin molecule using the relevant thiol, amino, or carboxyl groups. This type of useful reaction is well known in the art and can be employed by those skilled in the art to concretize active ingredients without any additional work. An example of such a reaction is described in Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies. MAbs. 2014 Jan-Feb; 6(1): 46-53.

[0147] In other respects, the present invention relates to a method for preparing the isolated targeted delivery system of the present invention, comprising the following steps:

[0148] a) Provide purified iron-binding protein as defined above;

[0149] b) Covalently or non-covalently linking the active ingredient to the iron-binding protein and / or encapsulating the active ingredient in the iron-binding protein;

[0150] c) Provide CD45 as defined above + White blood cells; and

[0151] d) Culturing CD45 in the presence of the complex of the iron-binding protein and the active ingredient generated in step b) + White blood cells, up to CD45 + The leukocytes are at least partially, preferably fully loaded, with the complex of the iron-binding protein and the active ingredient generated in step b).

[0152] The formation of adducts between proteins and drug moieties can be a non-covalent drug molecule that binds to a target protein, and can be as described below: In the case of ferritin, the drug can typically be encapsulated within a lumen (physical enclosure) by utilizing the association and dissociation properties of the ferritin macromolecule itself. The drug molecule is held in place by non-covalent interactions with amino acid residues on the lumen surface. Hemoglobin macromolecules also provide the possibility of non-covalent binding of selected drug molecules, which can be contained within the heme-binding pouch of the hemoglobin itself. Heme can be pouch-transferred and replaced by a drug having suitable hydrophobicity. On the other hand, all proteins considered in this invention can be covalently linked to drug molecules modified with a specifically active linker moiety that is reactive to the protein's own thiol or amino groups. Similarly, ferritin or hemoglobin can be linked to a cysteine ​​thiol-reactive drug with a peptide-based cleavable linker (e.g., a cathepsin-sensitive valine-citrulline sequence and a p-aminobenzylcarbamate spacer). As a prominent example, the antimitotic agent monomethylolpropamine E (MMAE) has been used. Peptide-based linkers stably bind proteins to cytotoxic compounds, thus preventing drug release from the protein under physiological conditions, helping to prevent toxicity to healthy cells and ensuring dose efficiency. The resulting protein-drug adduct can link to selected receptor types, namely CD163 for hemoglobin and TfR for ferritin or transferrin. Once bound, the protein-drug adduct is internalized via endocytosis, allowing for selective uptake by target cells. The drug-containing vesicles fuse with lysosomes and lysosomal cysteine ​​proteases, particularly cathepsin B, which initiates the breakdown of the valine-citrulline linker, preventing further antibody binding and allowing direct release into the tumor environment.

[0153] Alternatively, DM1-SMCC is a highly efficient mertansine derivative with a linker that specifically binds to lysine residues that form covalent complexes with ferritin, hemoglobin, or transferrin in reactions successfully described for antibodies. Specifically, hemoglobin, ferritin, or transferrin can react with DM1-SMCC to provide a covalent protein-drug adduct that can be cleaved intracellularly and release the active drug in a time-dependent manner. DM1 inhibits microtubule dynamics, inducing mitotic arrest and cell death.

[0154] In the context of this invention, the term "full load" refers to the maximum amount of iron-binding protein, preferably ferritin, complexed with the active ingredient, which can be bound by CD45. + Leukocyte uptake is preferred to be taken up by macrophages, and more preferably by activated macrophages.

[0155] In a third aspect, the present invention relates to the isolated targeted delivery system of the present invention, which is used as a drug.

[0156] In a fourth aspect, the present invention relates to pharmaceutical compositions comprising the isolated targeted delivery system of the present invention and a pharmaceutically acceptable carrier and / or suitable excipient. Because the isolated targeted delivery system contains living cells, carriers and excipients that preserve cell viability are preferred.

[0157] "Pharmaceutical acceptable" means approved by federal or state government regulatory agencies, listed in the United States Pharmacopeia, or listed in other generally recognized pharmacopoeias for use in animals, or more specifically for human use.

[0158] As used herein, the term "carrier" refers to a pharmacologically inactive substance, such as, but not limited to, diluents, excipients, surfactants, stabilizers, physiological buffers, or transporters, which is administered together with the therapeutically active ingredient. Such drug carriers can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids, such as water or salt solutions in oil, including but not limited to those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Salt solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Salt solutions are preferred carriers when the pharmaceutical composition is administered intravenously. Examples of suitable drug carriers are described in EW Martin's "Remington's Pharmaceutical Sciences".

[0159] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gum, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.

[0160] "Surfactants" include anionic surfactants, cationic surfactants and nonionic surfactants, such as, but not limited to, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100 and polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0161] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists.

[0162] "Physiological buffer solutions" include, but are not limited to, sodium chloride solutions, softened water, and suitable organic or inorganic buffer solutions, such as, but not limited to, phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4(2-hydroxyethyl)piperazinyl]ethanesulfonic acid), or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of the appropriate buffer usually depends on the desired molar concentration. For example, phosphate buffer is suitable for both injection and infusion solutions.

[0163] The term "adjuvant" refers to an agent that, at the cellular or humoral level, enhances, stimulates, activates, strengthens, or modulates the immune response to the active ingredient of a composition. For example, an immune adjuvant stimulates the immune system's response to an actual antigen but does not itself have an immunological effect. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g., Freund's complete and incomplete adjuvants), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virions, bacterial adjuvants (e.g., monophospholipid A or muramyl peptide), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine).

[0164] In a fifth aspect, the present invention relates to the isolated targeted delivery system of the present invention for the prevention / treatment of tumors, inflammatory diseases or ischemic areas in skin wounds, other wounds or after organ infarction (heart) or retinal ischemia, wherein the tumor is preferably a solid tumor, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer or tumors with hypoxic areas.

[0165] As used herein, the term “treatment” includes all types of preventive and / or therapeutic interventions permitted by medicine for various purposes, such as treatment, temporary relief, prevention, etc., including delaying or halting disease progression, causing lesions to regress or disappear, preventing attacks or suppressing recurrence.

[0166] The targeted delivery system according to the invention allows for the delivery of active ingredients to tumors that are normally inaccessible (e.g., due to solubility issues). It also enables the delivery of active ingredients to hypoxic tumors or hypoxic regions of tumors. The system further provides, for example, the delivery of active ingredients to any region of an organism undergoing hypoxic conditions during ischemic events or inflammatory processes.

[0167] As described above, the present invention also provides a method for delivering a targeted drug to a tumor mass. This method includes CD45. + Preparation of leukocytes, preferably activated macrophages, wherein the activated macrophages enable the macrophages to efficiently take up iron-binding proteins (ferritin, hemoglobin and / or transferrin), wherein the ferritin, hemoglobin and / or transferrin are loaded with an active ingredient (e.g., a drug / prodrug), tumor-targeting and the iron-binding proteins are transferred to cancer cells, and the active ingredient is released at the cancer cells.

[0168] This invention utilizes CD45 + Leukocytes, preferably activated macrophages loaded with iron-binding proteins linked to drugs / prodrugs, serve as tumor-targeting delivery systems. Unsatisfactory tumor responses to chemotherapy or difficulties in detecting them using imaging methods are primarily related to altered permeability of anticancer drugs to hypoxic areas due to poor vascular systems. However, even in avascular regions far from blood vessels, these avascular areas attract CD45. + Leukocytes, preferably activated macrophages, migrate. Therefore, they constitute a particulate delivery system to the tumor mass. Promising examples of such particles are iron-binding proteins. However, when used as a single agent, similar to other compounds, they do not reach hypoxic areas and accumulate in other organs.

[0169] The inventors used chemical methods to bind anticancer drugs, hypoxia-activated prodrugs (for therapeutic purposes), or isotopes to hemoglobin or transferrin, and loaded them onto CD45 by treating cells with an iron-binding protein solution as described in the examples. + In leukocytes (monocytes, macrophages, lymphocytes, and / or granulocytes), preferably loaded onto activated macrophages. The inventors observed that, upon administration to animals, the loaded CD45... + Leukocytes and preferably activated macrophages migrate to hypoxic tumor sites and release iron-binding proteins containing encapsulated active ingredients into cancer cells. This method allows for precise application of the active ingredient to the tumor site (especially hypoxic areas), preventing its accumulation in other organs. Example Section

[0170] Example 1 - Activation of macrophages

[0171] Macrophages used in this invention are obtained, differentiated, and activated as follows. To activate macrophages, they are first obtained from bone marrow precursors (see, for example, the paper: Weischenfeld and Porse, 2008, CSH Protoc, doi. 10.1101 / pdb.prot.5080) or blood mononuclear cells. Alternatively, they can be obtained from the peritoneum. Methods for the isolation, culture, differentiation, and polarization / activation of macrophages are well known to those skilled in the art. For example, they are described in detail by Murray et al. (Immunity, 2014, 41(1): 14-20).

[0172] In this practical implementation of the invention, bone marrow-derived macrophages are obtained from BALB / c or C57B1 / 6 mice. However, macrophages derived from canine blood monocytes or commercially available macrophage cell lines (monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human: THP-1, U937, or canine DH82 cell line) can also be used.

[0173] Shortly thereafter, the bone marrow-derived macrophages were seeded in 5 ml of medium (3 ml cells per plate) in plastic Petri dishes: DMEM: F12 + glutamine / glutamax + 10% FBS + penicillin / streptomycin and 20% L929 conditioned medium or M-CSF (50 ng / ml). Over the next five days, the medium was supplemented with one of the growth factors and activating compounds or a combination thereof as a mixture of cytokines.

[0174] Alternatively, the macrophages have been cultured in "M1 / M2 macrophage generating medium" (Promocell) or a commercially available equivalent or a homemade medium containing all the necessary cytokines and interleukins to be considered as activated.

[0175] To obtain macrophages from blood monocytes, centrifuge fresh blood (up to 12 hours) using a Histopaque 1077 system or equivalent, and seed with white blood cells (or white blood cells collected from the blood bank only) in an appropriate amount of preheated monocyte attachment medium (or equivalent, such as DMEM / RPMI supplemented with M-CSF), for example, 15 ml of medium per T-75 flask. For monocytes with a monocyte content ≥25%, the seeding density should be 1 million cells / cm³. 2 Up to 2 million / cm 2 For monocytes with a monocyte content of <25%, the seeding density should be 1.5 million cells / cm². 2 Up to 3 million / cm 2Then, the cells were cultured in an incubator for 1 to 1.5 hours at 5% CO2 and 37°C without any other intervention.

[0176] After cell attachment, wash them at least twice, then add an appropriate amount of complete "M1 macrophage or M2 macrophage generation medium DXF" to the cells (e.g., 20 ml per T-75 flask) and culture the cells at 37°C and 5% CO2 for 6 days without changing the medium. To activate macrophages, the entire medium should be replaced with medium supplemented with activating compounds.

[0177] The activating compounds used in this invention (for bone marrow-derived cells or for activating cells derived from monocyte-macrophage lines) are as follows: IL-4 (20 ng / ml), IFN-γ (at least 20 ng / ml), LPS (at least 10 ng / ml), IL-13 (at least 20 ng / ml), IL-10 (at least 20 ng / ml), dexamethasone (at least 20 µg / ml), oxLDL (at least 20 ng / ml), TNF-α (20 ng / ml), TGF-β (20 ng / ml), cortisol (150 ng / ml to 300 ng / ml), or combinations thereof as a mixture of cytokines. No activating compounds were added to obtain unactivated macrophages.

[0178] Reversal of macrophage polarization / activation (from classical activation to alternative activation) can be achieved, for example, by culturing macrophages in the appropriate cytokines listed above for at least 48 hours.

[0179] Example 2—Monocyte Isolation

[0180] To obtain monocytes in this practical implementation of the invention, bone marrow-derived macrophages or spleen-derived macrophages are obtained from BALB / c or C57B1 / 6 mice; however, canine blood monocytes or commercially available monocyte cell lines (monocyte-macrophage cell lines: mouse cells: RAW 264.7, J744; human cells: THP-1, U937 or canine DH82 cell line) are used.

[0181] To obtain blood monocytes, centrifuge fresh blood (within 12 hours) using the Histopaque 1077 system or an equivalent, and seed the white blood cells in an appropriate amount of preheated monocyte attachment medium (or an equivalent, such as DMEM / RPMI supplemented with 20 ng / ml M-CSF), for example, 15 ml of medium per T-75 flask. Alternatively, white blood cells collected from a blood bank (erythrocyte sedimentation rate, brown layer) can be used alone. For monocytes with a monocyte content ≥25%, the seeding density should be 1 million cells / cm³. 2Up to 2 million / cm 2 For monocytes with a monocyte content of <25%, the seeding density should be 1.5 million cells / cm². 2 Up to 3 million / cm 2 Then, the cells were cultured in an incubator for 1 to 1.5 hours at 5% CO2 and 37°C without any other intervention. After cell attachment, they were washed at least twice, and the attached cells were considered to be monocytes.

[0182] To obtain bone marrow-derived monocytes, in this practical implementation of the invention, bone marrow-derived macrophages were obtained from BALB / c or C57B1 / 6 mice. Shortly after, the bone marrow-derived precursor was seeded into 5 ml of medium (3 ml cells per plate) in plastic Petri dishes: DMEM: F12 + glutamine / glutamax + 10% FBS + penicillin / streptomycin and 20% L929 conditioned medium or 20 ng / ml M-CSF. Two days later, 5 ml of standard medium was added. Then, two days later, 0.5 ml / plate of L929 conditioned medium was added. Adhesive cells were considered monocytes.

[0183] To obtain spleen-derived monocytes, in this practical embodiment of the invention, the spleen is mechanically separated to obtain a single-cell suspension, which is then passed through a 70 μm cell filter. The cells are centrifuged and the supernatant is removed. After erythrocyte lysis, monocytes are isolated using a magnetic bead purification method, such as the EasySep mouse monocyte enrichment kit protocol and a suitable magnet.

[0184] To achieve better protein loading and migration before use, they can be pretreated with the following macrophage activating stimulants: IL-4 (20 ng / ml), IFN-γ (at least 20 ng / ml), LPS (at least 10 ng / ml), IL-13 (at least 20 ng / ml), IL-10 (at least 20 ng / ml), dexamethasone (at least 20 µg / ml), oxLDL (at least 20 ng / ml), TNF-α (20 ng / ml), TGF-β (20 ng / ml), cortisol (150 ng / ml to 300 ng / ml), or a combination thereof as a mixture of cytokines.

[0185] Example 3—Granocyte Isolation

[0186] To obtain granulocytes from blood, nine volumes of blood were diluted with one part ACD buffer (containing 0.17 M d-glucose, 0.10 M citrate, and 0.11 M trisodium citrate). The blood from this step was further diluted 1:1 with PBS and centrifuged. After removing the plasma and erythrocyte sedimentation rate (ESR) layer, the remaining cells were mixed with PBS to 80% of the initial volume of the first step (ACD-blood), and then diluted 4:12 with cold distilled water. Six parts of 2.7% NaCl solution were then added and centrifuged. After removing the supernatant, the cells were resuspended in RPMI-1640 medium. These cells were considered granulocytes.

[0187] Example 4 - Lymphocyte Isolation

[0188] To obtain spleen-derived lymphocytes, in this practical embodiment of the invention, the spleen is mechanically separated to obtain a single-cell suspension, which is then passed through a 70 μm cell filter. The cells are centrifuged and the supernatant is removed. After erythrocyte lysis, lymphocytes, such as EasySep mouse CD4 cells, are isolated using a magnetic bead purification method. + Enrichment kit protocol and appropriate magnets.

[0189] Example 5 - Preparation of Ferritin Complex

[0190] To bind ferritin to anticancer drugs (e.g., classic drugs such as cyclophosphamide, chlorambucil, melphalan, bendamustine, banoxantrone, or hypoxia-activated prodrugs such as TH-302), ferritin must be prepared prior to macrophage treatment. Soon after, it was obtained according to SEQ ID NO: 4 ( Figure 1Recombinant mouse protein was obtained. The expression vector pET-22b containing the gene encoding the synthetic ferritin (SEQ ID NO: 4) was transformed into *E. coli* BL21(DE3). *E. coli* cultures were grown at 37°C to an OD 600 of 0.6 in 1 L Luria-Bertani broth (LB) supplemented with ampicillin (100 mg / L). Protein expression was induced by the addition of 1 mM isopropyl thio-bD-galactopyranoside (IPTG), and the cultures were incubated overnight. Cells were collected by centrifugation (15000 g, 15 min) and resuspended in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg / mL DNase, and 10 mM MgCl2, followed by sonication. The lysate was centrifuged at 15000 g for 30 min, and the supernatant was centrifuged at 50°C for 10 min to remove denatured proteins, then centrifuged again at 70°C for 10 min. Add 30% (NH)₄SO₄ to the supernatant, stir at 4°C for 1 h, and centrifuge at 15000g for 30 min. Add 70% (NH)₄SO₄ to the supernatant, stir at 4°C for 1 h, and centrifuge at 15000g for 30 min. Resuspend the pellet in 20 mM Hepes (pH 7.5) and 150 mM NaCl, and dialyze overnight at 4°C to the same buffer. Load the protein onto a HILOAD 26 / 600 SUPERDEX 200 gel filter column (GE-Healthcare), then aseptically filter and store at 4°C. Figure 9 Using 21000M -1 m -1 The molar extinction coefficient was used to determine the protein concentration at 280 nm using spectrophotometry, and the absorbance at 595 nm was measured using the Bradford assay.

[0191] The ferritin includes recombinant mammalian ferritin H homopolymer and / or L homopolymer.

[0192] The ferritin obtained as described above was purified using standard methods to obtain a preclinical grade product free of endotoxins (see, for example: Ceci et al., 2011, Extremophiles 15(3):431-439; Vanucci et al., 2012, Int JNanomed 7:1489-1509). Shortly thereafter, the ferritin, sterilely stored in a stock solution containing 20 mM Hepes at pH 7.5, was diluted to a final concentration of 4 μM of the 24-mer in acidic solutions (final pH < 3.0) or in highly alkaline pH (pH > 9.5) (see, for example, Pontillo et al., 2016), thus allowing for the dissociation of the multimers. The drug was dissolved in a suitable solvent at a very high concentration, and then small volumes were added to the ferritin solution in excess of 200 moles. The pH was then neutralized by adding an appropriate amount of NaOH / HCl solution to allow for multimer remodeling. Current experimental methods demonstrate that three / four washes (concentration steps) with PBS in a 100 kDa molecular weight cutoff concentrator rapidly and completely remove the co-solvent and unencapsulated drug, fully restoring the drug-loaded ferritin nanocages. The resulting ferritin-drug complex is then rapidly freeze-dried in liquid nitrogen.

[0193] Depending on the choice of cosolvent and the inherent chemical properties of the drug molecules, it can be estimated that up to 150 to 180 drug molecules can be encapsulated / adsorbed in the 24-meric ferritin cage.

[0194] Furthermore, drugs can be covalently coupled to the amino acid side chains (lysine or cysteine) of ferritin by appropriately selecting drugs activated by phenylhydrazone, succinimide, or maleimide. Therefore, i) phenylhydrazone derivatives can decompose the ferritin surface and release the drug from it, ii) lysine-bound derivatives can become active after the protein is completely degraded into amino acids, or iii) cysteine-bound derivatives can be released intracellularly through the reductive hydrolysis of the maleimide thioether bond.

[0195] Example 6 - Preparation of hemoglobin-compound complex

[0196] As described by Rossi-Fanelli et al. (Archives of biochemistry and biophysics 77:478-492, 1958), human hemoglobin was prepared from fresh red blood cells. Shortly thereafter, heparinized blood obtained from healthy donors was centrifuged at 1600 rpm for 30 minutes (4°C) to precipitate RBCs. The erythrocyte sedimentation rate (ESR) layer was precisely removed by needle aspiration from the surface of the pellets. The plasma supernatant was discarded, and the RBC pellets were washed three times by resuspending the RBCs in isotonic 0.9% saline solution and centrifuging at 1600 rpm for 30 minutes at 4°C. After the final saline wash and centrifugation step, the RBC pellets were resuspended in distilled water buffered with 5 mM potassium phosphate buffer (PB, pH=7.2) at pH 7.2 and allowed to dissolve overnight with gentle stirring at 4°C. The dialyzed RBC lysates were then centrifuged at 13,000 rpm for 30 minutes at 4 °C, and the supernatant was loaded directly onto an ÄKTA Explorer system equipped with an XK 26 / 40 column packed with Q-sepharose XL resin (GE Healthcare) at room temperature. The column was equilibrated with buffer A (20 mM Tris-HCl, pH 8.2) at a flow rate of 12 mL / min and washed three times with the same buffer. Elution was performed by a linear gradient from 100% buffer A to 75% buffer B (20 mM Tris-Cl, plus 0.2 M NaCl, pH 8.20), followed by a discontinuous gradient of 100% buffer B. After elution, protein fractions were collected using a fraction collector. The obtained proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stored frozen at -80 °C.

[0197] Human hemoglobin (SEQ ID NO: 18 or SEQ ID NO: 22, see also) Figure 1 Hemoglobin can be readily covalently linked to suitable drug conjugates to accommodate hydrophobic drug molecules or even deliver cytotoxic small molecules bound to heme iron within the heme-binding pouch. Hb can be readily modified by selectively linking a suitable drug conjugate to a cysteine ​​residue at position 93 of the β-chain, the only titratable cysteine ​​residue on the protein surface. Maleimide-functionalized drugs, such as the tubulin inhibitor monomethylolpropamine (MMAE) or succinimide-functionalized mertansine analogs (DM1-SMCC), are prime examples of highly potent cytotoxic agents that can readily and specifically attach to the relevant cysteine ​​β93 residue (for maleimide-functionalized drugs) or to one or more lysine residues (for succinimide-functionalized drugs), respectively. These drugs can be conveniently bound to human hemoglobin according to the following steps:

[0198] Orestatin E analogue, namely maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl olrestatin E (vcMMAE), was obtained from MedChem Express (Princeton, New Jersey). The hemoglobin vcMMAE adduct was prepared as follows: Human hemoglobin solution was adjusted to a heme concentration of 120 μM with reaction buffer (50 mM phosphate buffer, pH 6.8, containing 0.1 mM EDTA) and incubated overnight at 4°C in the presence of 20% v / v acetonitrile solution with a 10-fold molar excess of vcMMAE. The maleimide group reacts effectively and specifically with free (reduced) thiol groups at pH 6.5 to 7.5 to form a stable thioether bond. The excess vcMMAE was purified and exchanged with D-PBS buffer using a PM100 ultrafiltration concentrator. The binding yield was approximately 80% of the total cysteine. The formation of the vcMMAE conjugate was confirmed by LC-MS analysis and titration of residual free thiol groups with p-chloromercuric benzoate. The concentration of the Hb-vcMMAE conjugate was determined by UV-Vis spectroscopy.

[0199] The following preparation of the lysine covalently linked and functionalized metansine analog DM1 SMCC (Alb Technology Ltd, Henderson, Nevada, USA) was performed. Human hemoglobin solution was adjusted to a heme concentration of 400 μM with reaction buffer (0.1 mM phosphate buffer, pH 7.4, containing 0.5 mM EDTA) and bound to a 20-fold molar excess of DM1-SMCC at 4 °C in the presence of 10% v / v DMSO solution for 16 h. Amine-reactive succinimide esters coupled with amines, thereby forming covalent adducts with lysine groups on the protein surface. Excess DM1-SMCC was removed, and the buffer was exchanged with D-PBS using a PM100 ultrafiltration concentrator. The binding yield was approximately 2.4 metansine molecules per hemoglobin tetramer. The formation of the DM1-SMCC conjugate was confirmed by LC-MS analysis. The concentration of the Hb-DM1-SMCC conjugate was determined by UV-Vis spectroscopy.

[0200] Example 7 - Preparation of transferrin-compound complex

[0201] Serum was obtained from healthy donors, and excess iron bicarbonate was added in the presence of citrate ions and bicarbonate as chelating agents to promote the binding of iron to transferrin. The reaction mixture contained 6.5 mg sodium bicarbonate and 153.16 g ferric citrate, pH 8, 4 °C, 1 h per 100 mL serum. Albumin was then precipitated by adding an alcohol solution to the serum sample at a ratio of 3.5 V / V at 4 °C for 2 h, pH 9.4, with rivanol (4%). The solution was then centrifuged at 3000 rpm for 20 min and finally filtered through a 0.8 mm syringe filter. Excess rivanol was then removed by gel filtration on a Sephadex G-25 column in 0.025 M ammonium sulfate. A first precipitation was then performed with 50% saturated ammonium sulfate at pH 6.5, followed by centrifugation at 3000 rpm for 10 min (to remove immunoglobulins). A second precipitation was then performed with 80% saturated ammonium sulfate to recover the transferrin precipitate. The solid precipitate was then dissolved in a 0.06 M Tris-HCl buffer containing 1 M NaCl at pH 8. The solution was dialyzed in the same buffer to completely remove ammonium sulfate. The protein solution was then concentrated to a maximum of 10 to 15 mg / mL using a Centiton PM50 centrifuge (estimated by the Bradford method) and loaded onto a Sephadex G-100 gel filter column (2, 4 × 80 cm) equilibrated in 1 M NaCl at a flow rate of 15 mL / h. The resulting transferrin was estimated to be 88% to 90% pure by SDS-polyacrylamide gel electrophoresis. Ion-exchange chromatography with DEAE Sephadex A-50 anion exchanger was then used as the final purification step. The transferrin sample was loaded onto a column equilibrated with 0.06 M Tris-HCl at pH 8 and eluted with a linear concentration gradient of 0.3 M Tris-HCl at pH 8. The protein purity was greater than 98%, with a yield of approximately 150 mg per 100 mL of serum.

[0202] Although it can only be modified with lysine due to the lack of a free, titratable cysteine ​​group, human total transferrin (SEQ ID NO: 28), which is similar to hemoglobin, Figure 1 Transferrin can be readily covalently linked to suitable drug conjugates. Therefore, succinimide-functionalized mertansine analogs (DM1-SMCC) have been used to covalently link to one or more lysine residues (succinimide-functionalized drugs). The drug has been conveniently bound to transferrin according to the following procedure:

[0203] The functionalized metansine analog DM1 SMCC for lysine covalent linkage was prepared as follows (Alb Technology Ltd, Henderson, Nevada, USA). Transferrin solution was adjusted to a concentration of 100 μM heme with reaction buffer (0.1 mM phosphate buffer, pH 7.4; EDTA was absent in this case due to possible iron chelation) and bound to a 20-fold molar excess of DM1-SMCC for 16 h at 4 °C in the presence of 8% v / v DMSO solution. Amine-reactive succinimide esters coupled with amines to form covalent adducts with lysine groups on the protein surface. Excess DM1-SMCC was removed and the buffer was exchanged with D-PBS using a PM100 ultrafiltration concentrator. The binding yield was approximately 1.5 metansine molecules per transferrin dimer. The formation of the DM1-SMCC conjugate was confirmed by LC-MS analysis. The concentration of the transferrin-DM1-SMCC conjugate was determined by UV-Vis spectroscopy.

[0204] Example 8 - Obtaining ferritin-loaded cells

[0205] The obtained cells are cultured in a ferritin solution at a concentration sufficient to ensure an appropriate ferritin / cell ratio for full loading and to ensure adequate drug concentration for therapeutic effect. The time and concentration can be varied depending on the number of molecules encapsulated / adsorbed into the ferritin cages, the state of cell activation, and the conditions and frequency of their intended application.

[0206] For example, to ensure proper ferritin loading, cells are cultured for 1 to 4 hours under standard culture conditions in a 0.2 mg / ml ferritin solution. The ferritin concentration can vary from at least 0.01 mg / ml to 4 mg / ml, and the culture time can also vary (5 minutes to 6 hours or longer). When adjusting the ferritin loading time and concentration onto cells, attention should be paid to the effects of ferritin and treatment conditions on cell viability. Cells obtained as described above are viable in a relatively short time (in minutes). Figure 8 Ferritin is readily absorbed by the cells. Once absorbed, they do not release it back into the culture medium. Figure 9 ).

[0207] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0208] Example 9 - Obtaining hemoglobin-loaded cells

[0209] The obtained cells are cultured in a hemoglobin solution at a concentration sufficient to ensure an appropriate hemoglobin / cell ratio for full cell loading and to ensure an appropriate drug concentration for therapeutic effect. The time and concentration can be varied depending on the number of molecules linked to the hemoglobin, the state of cell activation, and the conditions and frequency of their intended administration.

[0210] For example, to ensure proper hemoglobin loading, cells are cultured for 1 to 4 hours under standard culture conditions in a 0.1 mg / ml hemoglobin solution. The hemoglobin concentration can vary from at least 0.01 mg / ml to 0.2 mg / ml, and the culture time can vary (from 5 minutes to 4 hours or longer). When adjusting the time and concentration of hemoglobin loading onto cells, care should be taken to consider the effects of hemoglobin and treatment conditions on cell viability. Cells obtained as described above are viable in a relatively short time (in minutes). Figure 8 Hemoglobin is very easy to absorb from the body.

[0211] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0212] Example 10 - Obtaining cells loaded with transferrin

[0213] The obtained cells are cultured in a transferrin solution at a concentration sufficient to ensure an appropriate transferrin / cell ratio for full loading and to ensure adequate drug concentration for therapeutic effect. The time and concentration can be varied depending on the number of molecules linked to the transferrin molecule, the state of cell activation, and the conditions and frequency of their intended application.

[0214] For example, to ensure proper transferrin loading, cells are cultured under standard culture conditions in a 0.1 mg / ml transferrin solution for 1 to 4 hours. The transferrin concentration can vary from at least 0.01 mg / ml to 0.2 mg / ml, and the culture time can also vary (5 minutes to 4 hours or longer). When adjusting the time and concentration of transferrin loading onto cells, care should be taken to consider the effects of transferrin and treatment conditions on cell viability. Cells obtained as described above are viable in relatively short times (in minutes). Figure 8 Transferrin is very easy to obtain from the body.

[0215] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0216] Example 11 - Ferritin / hemoglobin / transferrin-macrophage complex as a useful tool for delivery to cancer cells

[0217] Macrophages derived from Example 1, as prepared in Examples 8, 9, and 10, readily transported ferritin, hemoglobin, and transferrin to cancer cells: mouse breast cancer, colon cancer, canine breast cancer, human breast cancer, pancreatic cancer, and bladder cancer. Figure 4 , Figure 10 Furthermore, this metastasis is more specific to cancer cells than to non-cancer cells. Figure 11 The ratio of macrophages to cancer cells is much higher. However, in the case of cancer cells, the ratio of the two cell types is crucial. The more macrophages there are, the better and faster the transport. The most efficient metastasis to cancer cells is observed when the ratio of macrophages to cancer cells is 1:1 or higher.

[0218] This transfer occurs not only when protein carriers bind to fluorescent labels (e.g., FITC or Alexa610), but also when they bind to / encapsulate other compounds, such as anticancer drugs. Figure 12 This demonstrates the transfer of ferritin encapsulated with the fluorescently hypoxia-activated prodrug, banoxantrone. This transfer of the compound, bound to the anticancer drug, induces apoptosis in cancer cells. Figure 6 , Figure 13 ).

[0219] Example 12 - Ferritin / hemoglobin / transferrin-monocyte complex as a useful tool for delivery to cancer cells

[0220] The mononuclear cells from Example 2, as prepared in Examples 8, 9, and 10, readily transported ferritin, hemoglobin, and transferrin to cancer cells. Figure 14 However, the ratio of the two cell types is crucial. The more monocytes there are, the better and faster the transport. Most efficient metastasis to cancer cells was observed when the ratio of monocytes to cancer cells was 1:1 or higher.

[0221] Example 13 - Ferritin / hemoglobin / transferrin-granulocyte complex as a useful tool for delivery to cancer cells

[0222] Granulocytes from Example 3, as prepared in Examples 8, 9, and 10, readily transported ferritin, hemoglobin, and transferrin to cancer cells. Figure 15 However, the ratio of the two cell types is crucial. The more granulocytes, the better and faster the transport. Most efficient metastasis to cancer cells was observed when the ratio of granulocytes to cancer cells was 1:1 or higher.

[0223] Example 14 - Ferritin / hemoglobin / transferrin-lymphocyte complex as a useful tool for delivery to cancer cells

[0224] Lymphocytes derived from Example 4, as prepared in Examples 8, 9, and 10, readily transport ferritin, hemoglobin, and transferrin to cancer cells. Figure 16 However, the ratio of the two cell types is crucial. The more lymphocytes, the better and faster the transport. Most efficient metastasis to cancer cells is observed when the ratio of lymphocytes to cancer cells is 1:1 or higher.

[0225] Example 15 - Leukocyte-protein carrier complex as a useful targeted drug delivery agent to hypoxic regions

[0226] The macrophages prepared as described above are injected into the tail vein of an animal with a tumor (the number of macrophages should be adjusted to suit the tumor size, stage of development, and presence of metastases). Figure 2 and Figure 17 As shown, they specifically reach the tumor (several hours later) and disperse throughout the animal's other organs. Figure 18 In addition, such as Figure 19 As shown, in the hypoxia model, they can also migrate to avascular and hypoxic sites and transfer carrier proteins to cancer cells. Figure 3 and Figure 20 ).

[0227] For imaging purposes, 1 million to 50 million macrophages were injected into the tail vein of animals with breast or colon cancer tumors. Prior to this, the macrophages were pre-labeled with a cell tracker and loaded with ferritin-FITC (as shown in Example 8). Eight hours after macrophage administration to the tumor mass, the presence of ferritin-FITC-loaded macrophages was detected using two-photon scanning. Figure 17 After pre-labeling macrophages with DIR cytoplasmic dye, whole-body imaging (IVIS) in animals was used to demonstrate their specific targeting of tumors and their migration to other organs. Figure 18 ).

[0228] One to ten million macrophages loaded with ferritin encapsulated with cyclophosphamide, melphalan, and ferritin encapsulated with chlorambucil were intravenously injected into mice with tumors (300,000 to 500,000 EMT6 cells were injected into the skin of the flank). Increased mouse survival was observed after three injections of macrophages every three days (on days 5, 8, and 11 after cancer cell injection, or on days 7, 10, and 13 after cancer cell injection) or five times daily. Figure 5 ).

[0229] Example 16 - Leukocyte-protein carrier complex or labeled leukocytes as a useful imaging tool

[0230] The targeted delivery system described in this invention allows ferritin to be conjugated to the contrast agent after targeting. For example... Figure 21 As shown, 1 ml to 50 ml of ferritin-loaded macrophages, as described in Example 8, are injected with a contrast agent (in this case, ferrihydrite, but with isotopes, for example...) 123 I obtained the same result) coupling or isotope labeling (in this case) 18 After F-FDG Figure 21 These can be easily detected by MRI, PET, or SPECT. In this embodiment ( Figure 7 Mice with breast tumors were imaged using MRI 3, 22, and 24 hours after intravenous injection of macrophages loaded with ferritin Fh. Mice were treated with macrophages (at time point 0). An increase in the diameter of blood vessels filled with injected macrophages (arrows) was then observed (with a significant decrease in T2 signal), and subsequently, the macrophages diffused into the tissue (dot pattern; arrows). These changes were observed in all mice examined (at the same time points).

[0231] Macrophages also use 18 F-FDG (5 mln to 50 mln) labeling was performed, and PET imaging was conducted 1 hour after intravenous administration to mice with tumors. These mice were inoculated with the 4T1 metastatic tumor cell line 3 weeks prior to the experiment, and metastases in the lungs, liver, and spleen were confirmed histopathologically. Figure 21 In the PET scan, it can be seen that macrophages migrate to areas with metastatic tumors, making them visible under PET.

[0232] While the foregoing description of the invention enables those skilled in the art to obtain and use what is considered the best present method, they will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples described herein. Therefore, the invention should not be limited to the foregoing embodiments, methods, and examples, but rather to all embodiments and methods within the scope and spirit of the invention.

[0233] The present invention also relates to the following aspects and preferred embodiments thereof. The definitions provided above similarly apply to the following aspects and embodiments.

[0234] 1. A targeted delivery system comprising activated macrophages loaded with ferritin carrying active ingredients.

[0235] 2. According to the targeted delivery system of Implementation Scheme 1, the active ingredient carried by ferritin is an anticancer drug.

[0236] 3. The targeted delivery system according to implementation scheme 2, wherein the anticancer drug is an apoptosis-inducing drug.

[0237] 4. The targeted delivery system according to implementation scheme 2, wherein the anticancer drug is selected from cyclophosphamide, chlorambucil, melphalan, bendamustine and banoxantrone.

[0238] 5. The targeted delivery system according to implementation scheme 1, wherein the active ingredient is a hypoxia-activated prodrug.

[0239] 6. The targeted delivery system according to embodiment 5, wherein the hypoxia-activated prodrug is TH-302.

[0240] 7. A method for preparing the targeted delivery system, the targeted delivery system comprising activated macrophages loaded with ferritin carrying an active ingredient, the method comprising the following steps:

[0241] a) Purify ferritin;

[0242] b) Obtaining ferritin carrying the active ingredient by linking ferritin to the active ingredient;

[0243] c) Activate isolated macrophages;

[0244] d) Culture macrophages for a period of time in the ferritin solution carrying the active ingredient obtained in step b) at a ferritin concentration sufficient to ensure that the ferritin carrying the active ingredient is fully loaded into the macrophages.

[0245] 8. The method according to embodiment 7, wherein the activated macrophages are bone marrow-derived macrophages.

[0246] 9. The method according to embodiment 7, wherein the activated macrophage is a blood-derived macrophage.

[0247] 10. The method according to implementation scheme 7, wherein the activated macrophages are derived from a macrophage cell line.

[0248] 11. The method according to any one of embodiments 7 to 10, wherein the activated macrophage is a macrophage polarized to M1 or M2.

[0249] 12. The method according to implementation scheme 11, wherein the activated macrophages have been polarized to M2.

[0250] 13. The method according to implementation scheme 11, wherein the iron metabolism of activated macrophages has been controlled.

[0251] 14. The method according to any one of embodiments 7 to 13, wherein the active ingredient carried by ferritin is an anticancer drug.

[0252] 15. The method according to embodiment 14, wherein the anticancer drug is an apoptosis / autophagy or necrosis inducing drug.

[0253] 16. The method according to embodiment 14, wherein the anticancer drug is selected from cyclophosphamide, chlorambucil, melphalan, bendamustine, and banoxantrone.

[0254] 17. The method according to any one of embodiments 7 to 13, wherein the active ingredient is a hypoxia-activated prodrug.

[0255] 18. The method according to embodiment 17, wherein the hypoxia-activated prodrug is TH-302.

[0256] 19. The targeted delivery system as defined in any one of Implementation Schemes 1 to 7, which is used as a targeted delivery system for anticancer drugs.

[0257] 20. A targeted delivery system as defined in any one of embodiments 1 to 7, used for the prevention / treatment of solid tumor growth.

[0258] 21. Use of a targeted delivery system as defined in any one of embodiments 1 to 7 in the treatment of inflammatory diseases.

[0259] 22. Use of the targeted delivery system as defined in any one of embodiments 1 to 7 in the treatment of ischemic areas or in imaging ischemic areas.

[0260] In a preferred embodiment, the present invention does not include the subject matter of items 1 to 22 above.

Claims

1. A segregated targeted delivery system comprising macrophages, the macrophages containing a complex of ferritin and an anticancer drug, wherein the ferritin and the anticancer drug are covalently coupled.

2. The isolated targeted delivery system according to claim 1, wherein the anticancer drug is an antimitotic agent.

3. The isolated targeted delivery system according to claim 2, wherein the antimitotic agent is a microtubule polymerization inhibitor.

4. The isolated targeted delivery system according to claim 3, wherein the microtubule polymerization inhibitor is olistatin.

5. The separated targeted delivery system according to claim 4, wherein the orestatin is monomethyl orestatin E (MMAE).

6. The isolated targeted delivery system according to claim 5, wherein the MMAE is linked to ferritin via a cysteine ​​side chain.

7. The isolated targeted delivery system according to claim 5 or 6, wherein the ferritin and the MMAE are covalently linked by a peptide-based cleavable linker.

8. The isolated targeted delivery system according to any one of claims 5 to 7, wherein the ferritin and the MMAE are covalently linked by a cathepsin-sensitive valine-citrulline sequence and a p-aminophenylcarbamate spacer.

9. The isolated targeted delivery system according to any one of claims 1 to 8, wherein the macrophages are activated macrophages, preferably CD11b. + Macrophages, preferably CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, CD11b + CD68 + Macrophages, preferably CD11b + CD86 + M1 macrophages, which preferably produce iNOS and / or secrete interleukin-12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + Major Histocompatibility Complex II + (MHCII + (Low or high expression) M2 macrophages, CD11b + CD200R + M2 macrophages, CD11b + CD163 + M2 macrophages, or activated macrophages that produce arginase and / or secrete interleukin-10 (IL-10).

10. The isolated targeted delivery system according to claim 9, wherein the activated macrophages: (i) Can be produced in vitro by culturing monocytes or macrophages with factors that can alter the expression of markers on macrophages, preferably (a) Produced by in vitro culture with at least one M1 inducer, (b) Produced by in vitro culture with at least one M2 inducer, (c) Or produced in vitro by culture of factors that can alter the ability of macrophages to secrete cytokines, preferably IL-10 and IL-12, chemokines and / or produce iNOS, arginase or other immunomodulatory enzymes. (ii) Characterized by the expression of at least one of the following antigens: high expression of CD64, CD86, CD16, CD32, MHCII, and / or the production of iNOS and / or IL-12; (iii) It can be produced in vitro by culturing monocytes or macrophages with factors that can induce macrophage phagocytosis; (iv) Characterized by the expression of at least one of the following antigens: CD204, CD206, CD200R, CCR2, transferrin receptor (TfR), CXC motif chemokine receptor 4 (CXCR4), CD163, and / or T cell immunoglobulin domain and mucin domain 2 (TIM-2), and / or low expression of MHCII. (v) Possesses the ability to devour; and / or (vi) It is capable of secreting cytokines or producing inducible nitric oxide synthase (iNOS) (or other pro-inflammatory compounds), arginase or other immunosuppressive / anti-inflammatory compounds, wherein the cytokines are preferably IL-12 or IL-10.

11. The targeted delivery system according to claim 10, wherein: (i) M1 inducers are selected from LPS, INF-γ, GM-CSF, and viral and bacterial infections; or (ii) M2 inducers are selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, thermally activated gamma globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor, adenosine, worm and fungal infections.

12. The isolated targeted delivery system according to any one of claims 1 to 11, wherein the ferritin is heavy (H) ferritin.

13. The isolated targeted delivery system of claim 12, wherein the H-ferritin is composed of a variant of human full-length H-ferritin, wherein the variant has at least 90% identity with human H-ferritin according to SEQ ID NO:6, and has human H-ferritin according to SEQ ID NO:6 being CD45 + At least 70% of the white blood cells' uptake capacity.

14. A method for preparing a separated targeted delivery system according to any one of claims 1 to 13, comprising the following steps: a) Provides purified ferritin; b) Covalently link olistatin to ferritin; c) Provide macrophages; and d) Culture macrophages in the presence of the ferritin produced in step b) until the macrophages are at least partially loaded with the ferritin produced in step b).

15. A pharmaceutical composition comprising a segregated targeted delivery system according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier and / or a suitable excipient.

16. Use of the isolated targeted delivery system according to any one of claims 1 to 14 in the preparation of a medicament for the prevention / treatment of tumors.

17. The use according to claim 16, wherein the tumor is a solid tumor, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, or colon cancer.

18. The use according to claim 16, wherein the tumor is a tumor having a hypoxic region.

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