Cell targeting label delivery system

Abstract

The present invention relates to an isolated cell-targeted delivery system comprising CD45+ leukocytes, in said cells a complex comprising one or more iron-binding proteins and / or markers, to a method for preparing such an isolated cell-targeted delivery system, and to the use of the system for therapeutic diagnosis, in particular for the diagnosis of cancer, in particular metastatic cancer, 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 201680047506.5 and invention title "Cell-Targeted Marker Delivery System". Technical Field

[0002] This invention relates to a segregated cell-targeted delivery system containing CD45. + Leukocytes, containing complexes of one or more iron-binding proteins and / or markers, the present invention also relates to methods for preparing such isolated cell-targeted delivery systems, and the use of such systems for diagnosis, particularly for the diagnosis of cancer, especially metastatic cancer. Background Technology

[0003] Current imaging tools are capable of detecting large metastatic tumors (greater than 0.5 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 delivering contrast agents via the bloodstream to label and image these metastatic tumors is not feasible. 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 characteristic 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, micrometastases are often refractory to treatment. Although the solid tumors from which micrometastases originate usually respond well to conventional therapy, they often regrow at the site of the primary tumor or at the site of the metastasis. This constitutes 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 one or more markers that are complexed in vitro with or without one or more iron-binding proteins, and deliver these complexes in vivo to cells or cells, preferably to tumor cells or 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 provides one or more of the following advantages in particular: (i) specific delivery of one or more markers to cells that attract the aforementioned CD45. +(ii) delivery of markers to diseased cells, preferably to tissues containing leukocytes; (iii) protection of markers from inactivation in the bloodstream or clearance from the body; (iv) delivery of markers preferably to cells in poorly vascularized or non-vascularized areas of the disease, such as metastases, large tumors, rheumatic lesions, avascular wounds, or hypoxic areas of the skin; (v) reduced marker toxicity; (v) delivery of markers with poor pharmacokinetics; (vi) reduced marker side effects due to targeted delivery; (vii) lower doses of markers with higher diagnostic efficacy due to targeted delivery; and (viii) administration of markers linked to iron-binding proteins loaded on CD45. + The risk of local tissue damage at the marker application site is lower in white blood cells; and / or (ix) the possibility of detecting small metastases with high hypoxia (Pérez-Herrero E, Fernández-Medarde A. 2015, Eur J PharmBiopharm 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 preferably have the ability to internalize iron-binding proteins, and the cells contain a complex of one or more iron-binding proteins and one or more markers.

[0008] In a second aspect, the present invention relates to a segregated targeted delivery marker system comprising CD45 + White blood cells, CD45 + Leukocytes are preferably capable of being labeled, and contain one or more markers, preferably radioactive markers or combinations thereof.

[0009] In a third 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:

[0010] a) Provide an iron-binding protein, preferably purified;

[0011] b) Covalently or non-covalently linking the marker to iron-binding proteins and / or encapsulating the marker in iron-binding proteins;

[0012] c) Provide CD45 + White blood cells; and

[0013] d1) Culturing CD45 in the presence of the complex of the iron-binding protein and the marker generated in step b) + White blood cells, up to CD45 +Leukocytes are at least partially loaded with the complex of iron-binding proteins and markers produced in step b), and / or

[0014] d2) Culturing CD45 in the presence of markers + White blood cells up to CD45 + White blood cells were at least partially marked with markers.

[0015] In a fourth aspect, the present invention relates to a segregated targeted delivery system according to the first aspect of the invention or a segregated targeted delivery system that can be prepared by the method according to the third aspect of the invention, which is used as a diagnostic reagent.

[0016] In a fifth aspect, the present invention relates to diagnostic compositions comprising a segregated targeted delivery system of the first aspect of the invention or a segregated targeted delivery system that can be prepared by the method of the third aspect of the invention, and a pharmaceutically acceptable carrier and / or suitable excipient.

[0017] In a sixth aspect, the present invention relates to a segregated targeted delivery system according to the first aspect of the invention or a segregated targeted delivery system that can be prepared according to the method of the third aspect of the invention, for the diagnosis of tumors, inflammatory diseases or ischemic areas, wherein the tumors are preferably solid tumors and / or their metastases, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer or their metastases, or tumors with hypoxic areas, wherein the ischemic areas are particularly in skin wounds or ischemic areas following organ infarction (heart) or retinal ischemia. Attached Figure Description

[0018] 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 a 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 a 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 (SEQ ID NO: 23 and SEQ ID NO: 24) of the common amino acid sequence in mammalian transferrins 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 (SEQ ID NO: 26 and SEQ ID NO: 27) of human transferrin 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.

[0019] Figure 2 : Macrophages within mouse tumor masses (TRITC-stained before injection, loaded with FITC-modified ferritin).

[0020] 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.

[0021] Figure 4 : MRI images of mouse mammary tumors. Mice were treated with macrophages loaded with ferritin Fh (intravenously) (at time point 0 h). An increase in the diameter of blood vessels (arrows) filled with injected macrophages (significantly reduced T2-signal) and subsequent macrophage diffusion into the tissue (dot pattern; arrows) was then observed. These changes were observed in all mice examined (at the same time points).

[0022] Figure 5 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.

[0023] Figure 6 This indicates the stability of ferritin stored in macrophages.

[0024] Figure 7 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).

[0025] Figure 8 : Whole-body imaging of mice that received intravenous labeled macrophages, showing the accumulation of macrophages at tumor sites and their distribution in other organs.

[0026] Figure 9 : Shows macrophage migration to hypoxic tissue, from a cross-section of a mouse tumor, the mice having been administered pre-labeled macrophages intravenously, and the hypoxic region of the tumor visualized using the hypoxia marker pimonidazolone.

[0027] Figure 10 : This shows the signal recorded by PET in a whole-body analysis of mice with metastatic 4T1 cancer. The 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

[0028] 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.

[0029] 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.

[0030] Several references are cited throughout this specification. Each reference cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether mentioned above or below, 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.

[0031] definition

[0032] 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, J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

[0033] Throughout this specification and the claims, unless the context requires, the word "comprising" and its variations shall be understood to mean including 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.

[0034] The terms "targeted marker delivery" or "targeted contrast agent delivery" refer to the delivery of markers, particularly contrast agents, to a patient or person to be diagnosed, resulting in an increased concentration of the markers, particularly the contrast agents, 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 1 to 24 hours after application of the targeted delivery system of the invention, the concentration of the markers, particularly the contrast agents, 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 markers, particularly the contrast agents, in the same number of cells or volume of biopsy tissue from a non-diseased area. More preferably, after application of the targeted delivery system of the present invention, preferably 2 to 24 hours later, the concentration of the marker, particularly the contrast agent, 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 marker, particularly the contrast agent, in the undiseased area of ​​the body. When assessed based on systemic distribution, it is preferable to deliver at least 5%, preferably at least 10%, more preferably at least 15% of the marker, particularly the contrast agent, applied to the patient or person to be diagnosed to the diseased area of ​​the body. Targeted delivery of the marker, particularly the contrast agent, limits the potential harmful effects of the marker, particularly the contrast agent, on the diseased area of ​​the body.

[0035] The terms “targeted marker delivery system” or “targeted delivery system” are used synonymously in this application and refer to a system that preferably delivers markers, particularly contrast agents, to a target area within the patient’s body, i.e., a system capable of targeted delivery.

[0036] In this invention, the term "marker" refers to any kind of compound suitable for diagnostic purposes. Preferred compounds are selected from fluorescent dyes, radioactive isotopes, and contrast agents. Contrast agents are dyes or other substances that help visualize abnormal areas in the body. In one embodiment, the term marker refers to a compound containing a chelating agent that forms a complex with a divalent or trivalent metal cation. Preferred radioactive isotopes / fluorescent emission isotopes are selected from alpha emission isotopes, gamma emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, and fluorescent isotopes, for example... 65 Tb fluorescent isotopes, for example 18 F, 51 Cr 67 Ga、 68 Ga、 111 In、99m Tc, 140 La、 175 Yb、 153 Sm、 166 Ho、 88 Y、 89 Zr、 90 Y、 149 Pm, 177 Lu、 47 Sc、 142 Pr、 159 Gd, 212 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101m15 Rh、 119 Sb, 128 Ba、 123 I, 124 I, 131 I, 197 Hg, 211 At、 169 Eu、 203 Pb, 212 Pb, 64 Cu、 67 Cu、 188 Re、 186 Re、 198 Au and 199 Ag, and combinations thereof with proteins, peptides, small molecule inhibitors, antibodies or other compounds, such as... 18 F-fluorodeoxyglucose (F-fluorodeoxyglucose) 18 F-FDG) or 64 Cu-porphyrin. Preferred fluorescent dyes are selected from the following types: oxanthracenes (e.g., fluorescein), acridines (e.g., acridine yellow), oxazines (e.g., oxazine 1), cyanides (e.g., Cy7 / Cy3), styryl dyes (e.g., Dye-28), coumarins (e.g., Alexa Fluor 350), porphyrins (e.g., chlorophyll B), metal ligand complexes (e.g., PtOEPK), fluorescent proteins (e.g., APC, R-phycoerythrin), nanocrystals (e.g., QuantumDot 705), perylenes (e.g., Lumogen Red F300), and phthalocyanines (e.g., IRDYE). TM 700DX) and combinations and combinations of these types of dyes or radiofluorescent dyes 65Tb. Preferred contrast agents are selected from paramagnetic agents, such as Gd, Eu, W, and Mn, preferably in combination with chelating agents. Other options are superparamagnetic iron (Fe) complexes and particles, compounds containing high atomic number atoms such as iodine for computed tomography (CT), microbubbles, and carriers, such as liposomes containing these contrast agents.

[0037] 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, the term "peptide" in the context of this invention also refers to an amino acid chain having more than 50, more than 100, or more than 150 amino acids.

[0038] 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 comparison purposes, Gapped BLAST, as described by Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, is used. When using the BLAST and Gapped BLAST procedures, the default parameters for each procedure are used. Sequence matching analysis can 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.

[0039] 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 agranulocytes) 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. Further subtypes can be classified; for example, among lymphocytes there are B cells, T cells, and NK cells. 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).

[0040] 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 markers, particularly contrast agents, to or into 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 and quantity of surface antigens detectably exposed on the cell surface can therefore 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, and is thus 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 the different absolute amounts 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 a given protein is expressed at a high level; if there are 1 to 50 detectable copies per cell, it can be considered that a 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 another protein 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 was found in white blood cell populations, specifically in healthy CD45. + At least 70% of the copy number of each cell in the white blood cell population. The term "low expression" of a given protein means that the detectable expression of that protein is at a level found in healthy CD45 cells. + The highest expression level found in white blood cell populations, specifically in healthy CD45 +The protein copy number in each cell of the leukocyte population is 30% or less. Preferably, the "highest expression level" is determined to be 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 may only be present inside the cell. Those skilled in the art are well aware of how to detect and / or quantify proteins produced within cells and / or select cells that produce such proteins.

[0041] 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).

[0042] 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, tolerance maintenance, 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.

[0043] The term "phagocytosis by macrophages" refers to the process by which macrophages engulf solid particles to form internal vesicles called phagosomes.

[0044] 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.

[0045] In a first aspect, the present invention relates to a segregated targeted delivery marker system comprising CD45 + Leukocytes, preferably capable of internalizing iron-binding proteins, contain a complex of one or more iron-binding proteins and one or more markers, particularly contrast agents.

[0046] The applicant unexpectedly discovered that CD45 + Leukocytes, preferably monocytes or activated monocytes and macrophages, preferably activated macrophages, preferably M1 macrophages, more preferably M2 macrophages, capture and deliver markers in a quantity sufficient to be detected using an imaging system so that they can be used to track their location in vivo. 18In the case of F-FDG, this is particularly preferred in the presence of monocytes and activated monocytes, macrophages, activated macrophages, preferably M1 macrophages, and most preferably M2 macrophages. Administration of the marker-loaded substance ( 18 F-FDG), preferably radiolabeled cells, can be used to visualize organs with accumulated labeled cells via positron emission tomography (PET) imaging. Therefore, in a second aspect, the present invention relates to a segregated targeted delivery system of labeled cells containing CD45. + Leukocytes are preferably tagged with one or more markers, preferably radioactive markers, or combinations thereof. A preferred embodiment of the second aspect of the invention involves direct cell-to-cell marker attachment or labeling with a marker. The marker may be located within the cell or on its surface, preferably on the cell surface.

[0047] The following preferred embodiments further illustrate the first and second aspects of the invention in each case.

[0048] 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 exhibit a tendency to internalize one iron-binding protein relative to another, thus resulting in higher complex concentrations and / or less leakage of the complex from the cell. This CD45 is described in more detail below. + Leukocyte subsets.

[0049] In the context of this invention, the phrase "a complex of one or more iron-binding proteins and markers" refers to a composition in which one or more marker 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 markers can be direct or indirect. In the latter case, the marker 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). nGroups or linking polypeptides. 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 marker 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 marker to be transported into the cell. Therefore, it is preferred that the marker 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 absorption assays known in the art, and is described in the Examples section below. Preferably, the complex is stable enough to be retained during transport within cells of the target region in vivo. Therefore, it is preferred to deliver the complex, rather than the marker alone, to cells or cells in the target region. This property also reduces the marker's effect on CD45. + Potentially harmful effects on leukocytes, such as cytotoxicity. If the marker 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 the 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 markers.

[0050] CD45 + The leukocytes are derived from the patient to be treated, in which case the cells loading the complex are homologous to the patient's own. 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 cells produced from primary cells through a small number of differentiation steps. Alternatively, CD45... + White blood cells can be from immortalized cells, but unconverted CD45 cells are preferred. + Leukocyte cell lines. Therefore, used for CD45. + The blood from which white blood cells, preferably macrophages, are separated is preferably 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.

[0051] The inventors noted that it can be made by CD34 + CD45 produced by hematopoietic progenitor cells + Leukocyte subsets are particularly well-suited for targeted, specific delivery of markers. 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.

[0052] 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 has 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 have 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 have the functional and / or structural properties summarized herein, but the pharmaceutical composition can still be used to treat diseases if the majority of cells have the corresponding functional and / or structural properties.

[0053] 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 complexes intracellularly (i.e., avoiding marker leakage and targeted release), 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...

[0054] (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;

[0055] (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 producing inducible nitric oxide synthase (iNOS) and / or secreting 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, CD11cMHCII, 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 absorb 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.

[0056] (iii) Monocytes or activated monocytes expressing at least one chemokine receptor or at least one growth factor receptor, wherein the chemokine is preferably selected from CCR1, CCR2, CXR4 and CXR6, and the growth factor receptor is 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;

[0057] (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

[0058] (v) Granulocytes are selected from neutrophils, eosinophils, and basophils, with the neutrophils preferably being CD66b. + Neutrophils, preferably CD193 eosinophils.+ Eosinophils.

[0059] In a preferred embodiment of the targeted delivery system of the present invention, activated macrophages:

[0060] (i) Can be produced in vitro by culturing monocytes or macrophages or their precursors with factors capable of altering the expression of markers on macrophages, preferably

[0061] (a) Produced by in vitro culture with at least one M1 inducer,

[0062] (b) Produced by in vitro culture with at least one M2 inducer,

[0063] (c) Or produced in vitro by culture 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. In addition to leukocyte inhibitors, leukopenic follicle-inhibiting factor (LIF), adenosine, worm and fungal infections, lipopolysaccharide (LPS), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and viral and bacterial infections, it was observed that activation of monocytes with M1 inducers, especially LPS, induced the expression of iNOS, while activation of monocytes with M1 inducers, especially LPS, induced the non-expression of arginase-1. Activation of monocytes with M2 inducers, especially IL-4, induced the expression of arginase-1, while activation of monocytes with M2 inducers, especially IL-4, induced the non-expression of iNOS.

[0064] (ii) Characterized by the expression of at least one of the antigens CD64, CD86, CD16, and CD32, high expression of MHCII, and / or the production of iNOS and / or IL-12;

[0065] (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;

[0066] (iv) Characterized by expression of at least one of the 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; activated macrophages with these properties are particularly well-suited to contain complexes containing ferritin as an iron-binding protein;

[0067] (v) Possesses the ability to devour; and / or

[0068] (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.

[0069] 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.

[0070] 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 the administered M1 macrophages are observed to concentrate at the tumor site, while approximately 35% of the M2 macrophages show tumor-specific targeting. However, this general advantage of M2 macrophages is offset when using complexes containing hemoglobin and markers, as a considerable amount of this 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 tissue.

[0071] In a preferred embodiment of the targeted delivery system of the present invention, monocytes:

[0072] (i) Can be generated by CD34 + Production of hematopoietic precursor cells;

[0073] (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;

[0074] (iii) Characterized by the expression of at least one of the following antigens: TfR, CD163, TIM-2, CD14, CD16, CD33 and / or CD115;

[0075] (iv) Characterized by the expression of at least one of the following antigens: TfR, CD163, TIM-2, CXCR4, CD14 and / or CD16; and / or

[0076] (v) It has the ability to devour.

[0077] 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.

[0078] The inventors unexpectedly discovered that monocytes are suitable for targeted delivery of activators to hypoxic tissues, preferably tumors or their metastases, and that monocytes treated with M2 activator are even more suitable for targeted delivery of activators to hypoxic tissues, preferably tumors or their metastases. This specificity makes monocytes treated with M2 activator more suitable for targeting tumors and hypoxic sites.

[0079] In a preferred embodiment of the targeted delivery system of the present invention, lymphocytes:

[0080] (i) Can be obtained from blood, spleen or bone marrow, or can be obtained from CD34 + Precursor cell generation, as is known to those skilled in the art, 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;

[0081] (ii) are immune-active lymphocytes;

[0082] (iii) Expressing antigen-specific T cell receptors; and / or

[0083] (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.

[0084] In a preferred embodiment of the targeted delivery system of the present invention, granulocytes:

[0085] (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;

[0086] (ii) Characterized by the expression of at least one of CD66b and / or CD193;

[0087] (iii) is a segmented leukocyte, characterized by the presence of granules in its cytoplasm; and / or

[0088] (iii) Characterized by the expression of at least one of the following substances: TfR, CD163, TIM-2 and / or CXCR4.

[0089] 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 comprise 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 ability of the corresponding wild-type protein to bind iron ions. 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.

[0090] 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).

[0091] Human transferrin and ferritin have been considered as effective carriers for delivering small molecules to cancer cells specifically targeted. To date, despite considerable efforts, no transferrin or ferritin complex has been successfully used in clinical practice (Luck AN et al., 2013, Adv Drug Deliv Rev 65(8): 1012-9).

[0092] Ferritin possesses a cage-like structure and the ability to bind iron, making it suitable for encapsulating markers within its lumen. Ferritin is scarce 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 the specific delivery of encapsulated drugs to TfR-expressing CD45 cells. + White blood cells. In addition, H-ferritin targets the complexed markers to the cell nucleus (thus directly delivering DNA-binding proteins into the cell nucleus).

[0093] Purified transferrin can effectively bind to a variety of molecules via covalent linkers that are appropriately released intracellularly (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.

[0094] Due to its multifunctionality in binding to various molecular chemistry, its abundant presence in blood, and its relative stability, hemoglobin has historically been considered a carrier (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... +Leukocyte-based protein delivery, particularly macrophage-based protein delivery, allows the central portion of hemoglobin to move as a carrier of target-specific markers. Hemoglobin can readily covalently bind suitable markers, accommodating hydrophobic marker molecules within the heme-binding pouch, by exchanging heme-free fluorescent porphyrins or porphyrins conjugated to radiolabeled markers, or even transporting marker-bound small molecules linked to heme iron. Hb can be readily modified by selectively linking suitable marker-binding compounds to β93-cysteine ​​residues, which are the only titratable cysteine ​​residues on the protein surface. Maleimide-functionalized markers are examples of markers that can readily and specifically attach to the associated β93-cysteine ​​residue. Alternatively, lysine residues on the Hb surface (at least 10 titratable lysine residues per Hb tetramer) can be readily labeled via cleavable succinimide linkers. Hemoglobin also provides the unique ability to release non-covalently bound heme groups at acidic pH. The resulting Apo-hemoglobin can contain several hydrophobic molecules with significant fluorescent properties (e.g., chloro-6, hyperycin, phthalocyanine derivatives) within an empty heme bag (Dong J et al., J Photochem Photobiol B2014, 140: 163-172).

[0095] Regardless of the conjugation / adsorption / binding method, this invention demonstrates that hemoglobin (Hb), transferrin (Tf), and ferritin are specialized marker carriers once loaded into suitable cellular systems with tumor-targeting properties, such as activated macrophages. The simple, rapid, inexpensive, and safe purification steps for these proteins also provide significant added value.

[0096] 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 absorbed 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 and 450 amino acids, preferably between 150 and 400 amino acids, more preferably between 200 and 350 amino acids, and even more preferably between 250 and 320 amino acids, then transferrin contains an N-terminal domain and a C-terminal domain, which are important for binding iron and being absorbed by CD45. + Leukocyte uptake is essential. The N-terminal domain contains amino acids 1 to 82 of full-length common 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 full-length common transferrin (SEQ ID NO: 25) or full-length human transferrin (SEQ ID NO: 28). In each case, X shown in the common 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.

[0097] 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 and CD45 +White blood cells preferably have at least 70% of the uptake capacity of M2 white blood cells. 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.

[0098] 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.

[0099] 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.

[0100] In a preferred embodiment, H-ferritin comprises or is composed of a mammalian common sequence obtained by comparing the full-length H-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, H-ferritin having the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 7, preferably SEQ ID NO: 2 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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, it comprises L-ferritin composed of the amino acid sequence according to SEQ ID NO: 12, CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0106] 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 uptake capacity of leukocytes preferably M2 macrophages is at least 70%. 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.

[0107] 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.

[0108] 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.

[0109] In a preferred embodiment, the α-hemoglobin comprises or is composed of the minimum mammalian common sequence obtained by comparing the full-length α-hemoglobin according to SEQ ID NO: 15. Preferably, it comprises or is composed of 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 the α-hemoglobin CD45 composed of the amino acid sequence according to SEQ ID NO: 15. + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0110] 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.

[0111] In a preferred embodiment, the α-hemoglobin comprises or is constituted by 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, it comprises α-hemoglobin consisting of the amino acid sequence according to SEQ ID NO: 16, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M2 macrophages.

[0112] 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.

[0113] In a preferred embodiment, the α-hemoglobin comprises a minimal mammalian common sequence obtained by comparing the full-length β-hemoglobin according to SEQ ID NO: 19 and its variants, or a minimal mammalian common sequence obtained by comparing the full-length β-hemoglobin according to SEQ ID NO: 19 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 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.

[0114] In a preferred embodiment, the α-hemoglobin comprises or is composed of the minimum human amino acid sequence obtained according to SEQ ID NO: 21 for human β-hemoglobin. 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.

[0115] In a preferred embodiment, the β-hemoglobin comprises or is composed of 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 composed 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.

[0116] 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.

[0117] 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 preferred transferrin comprises or is constituted by the amino acid sequence shown in SEQ ID NO: 25 and its variants, wherein the 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 constituted by the amino acid sequence according to SEQ ID NO: 25, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0118] 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.

[0119] 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.

[0120] 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, which is CD45 + White blood cells preferably have at least 70% of the uptake capacity of M1 macrophages.

[0121] Regardless of the given amino acid sequence, preferred ferritins also include proteins whose 24-mer subunit assemblies conform to a four-helix bundle protein module fall into a given sequence alignment of a distant relative protein defined by 3D structure-based alignment.

[0122] The inventors unexpectedly observed that monocytes, macrophages, and preferably M2 macrophages were able to take up sufficient amounts of the markers to make them visible using imaging methods at sites of accumulation (e.g., in tumors or their metastases, in hypoxic sites). Unexpectedly, the inventors found that lymphocytes and M2 macrophages were better at taking up complexes containing one or more ferritins and one or more markers, M1 macrophages were better at taking up complexes containing one or more hemoglobins and one or more markers, and macrophages were better at taking up complexes containing one or more transferrins and one or more markers. 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 markers 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 markers is used to load M1 macrophages.

[0123] In a preferred embodiment of the targeted delivery system of the present invention, the marker is selected from fluorescent dyes, fluorescent isotopes, radioactive isotopes, detectable peptides or nucleic acids encoding such detectable peptides and contrast agents.

[0124] In a preferred embodiment of the targeted delivery system of the present invention, the marker includes a chelating agent that forms a complex with a divalent or trivalent metal cation.

[0125] In a preferred embodiment of the targeted delivery system of the present invention, the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), and 6-hydrazylpyridine-3-carboxylic acid (HYNIC).

[0126] In a preferred embodiment of the targeted delivery system of the present invention, the contrast agent includes a paramagnetic agent, preferably selected from Gd, Eu, W and Mn or ferrihydride.

[0127] In a preferred embodiment of the targeted delivery system of the present invention, the radioactive isotope / fluorescent emission isotope is selected from alpha radiation radioactive isotopes, gamma radiation radioactive isotopes, Auger electron radioactive isotopes, X-ray radioactive isotopes, and fluorescent emission isotopes, for example... 18 F, 51 Cr, fluorescence 65 Tb, 67 Ga、 68 Ga、 111 In、 99m Tc, 140 La、 175 Yb、 153 Sm、 166 Ho、 88 Y、 90 Y、 149 Pm, 177 Lu、 47 Sc、 142 Pr、 159 Gd, 212 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101m15 Rh、 119 Sb, 128 Ba、 123 I, 124 I, 131 I, 197 Hg, 211 At、 169 Eu、 203 Pb, 212 Pb,64 Cu、 89 Zr、 67 Cu、 188 Re、 186 Re、 198 Au and 199 Ag, and combinations of the above isotopes with proteins, peptides, small molecule inhibitors, antibodies or other compounds (e.g., 18 F-FDG or 64 Cu-porphyrin).

[0128] In a preferred embodiment of the targeted delivery system of the present invention, the fluorescent dye is selected from the following categories of fluorescent dyes: oxane, acridine, oxazine, cyanine, styryl dyes, coumarin, porphyrin, metal ligand complexes, fluorescent proteins, nanocrystals, perylene and phthalocyanine, and combinations and mixtures of these categories of dyes.

[0129] In a preferred embodiment of the targeted delivery system of the present invention, the detectable peptide is an autofluorescent protein, preferably a green fluorescent protein, or any structural variant thereof having altered absorption and / or emission spectra.

[0130] As outlined above, the targeted delivery system of the present invention is particularly suitable for delivering markers to hypoxic regions.

[0131] In a preferred embodiment of the isolated targeted delivery system of the present invention, the bond between the iron-binding protein and the marker contained in the complex is covalent and / or non-covalent; and / or the marker contained in the complex is embedded / encapsulated by the iron-binding protein, preferably by ferritin or its polymers. In one embodiment, the covalent and / or non-covalent coupling is indirect via linkers or spacers. If covalent bonding is desired, the marker 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 markers. For example, one class of markers may bind to the iron-binding protein (non-covalently), while another class of markers is encapsulated. This method utilizes the different release rates of the markers after delivery from the iron-binding protein to the target tissue and / or cells.

[0132] In a third aspect, the present invention relates to a method for preparing the separated targeted delivery system of the present invention, comprising the following steps:

[0133] a) Provide purified iron-binding protein as defined above;

[0134] b) Covalently or non-covalently linking the marker to iron-binding proteins and / or encapsulating the marker in iron-binding proteins;

[0135] c) Provide CD45+ white blood cells as defined above; and

[0136] d1) Culture CD45+ leukocytes in the presence of the iron-binding protein and marker complex generated in step b) until the CD45+ leukocytes are at least partially, preferably fully loaded with the iron-binding protein and marker complex generated in step b), and / or

[0137] d2) Culturing CD45 in the presence of markers + White blood cells up to CD45 + White blood cells were at least partially marked with markers.

[0138] In another aspect, the present invention relates to a separated targeted delivery system, which can be produced by a method according to a second aspect of the invention.

[0139] The formation of adducts between proteins and markers can be a non-covalent marker binding to the target protein, and can be described as follows: In the case of ferritin, the marker can typically be encapsulated within a lumen (physical enclosure) by utilizing the association and dissociation properties of the ferritin macromolecule itself. The marker molecule is appropriately 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 marker molecules, whereby the drug molecule can be contained within a heme-binding pouch of the hemoglobin itself. The heme can be replaced by the pouch and by a marker having appropriate hydrophobicity. On the other hand, all proteins considered in this invention can be covalently linked to markers modified with a specifically active linker moiety that is reactive to the thiol or amino groups of the protein itself. Similarly, ferritin or hemoglobin can be linked to a cysteine ​​thiol-reactive marker with a peptide-based cleavable linker (e.g., a cathepsin-sensitive valine-citrulline sequence and a p-aminobenzylcarbamate spacer). Peptide-based linkers stably bind proteins to markers, preventing the markers from being released from the proteins under physiological conditions. This helps prevent toxicity and exudation to healthy cells and ensures the specificity of the markers. The resulting protein-marker adducts can bind to selected receptor types: CD163 for hemoglobin and TfR for ferritin or transferrin. Once bound, the protein-marker adduct is internalized via endocytosis and selectively taken up by target cells. The marker-containing vesicles fuse with lysosomes and lysosomal cysteine ​​proteases, particularly cathepsin B, which initiates the breakdown of the valine-citrulline linker. The marker is no longer bound to the antibody and is released directly into the tumor environment.

[0140] Alternatively, a marker with a linker that specifically binds to lysine residues can be used, which in the reaction form a covalent complex with ferritin, hemoglobin, or transferrin.

[0141] In the context of this invention, the term "fully loaded" refers to the maximum amount of iron-binding protein, preferably ferritin, complexed with the marker, which can be taken up by CD45+ leukocytes, preferably macrophages, more preferably activated macrophages.

[0142] In a fourth aspect, the present invention relates to a segregated targeted delivery system according to the first aspect of the invention or a segregated targeted delivery system that can be produced by the method according to the second aspect of the invention, which is used as a diagnostic reagent.

[0143] In a fifth aspect, the present invention relates to diagnostic compositions comprising a segregated targeted delivery system according to the first aspect of the invention or a segregated targeted delivery system that can be produced by the method according to the second aspect of the invention, and a pharmaceutically acceptable carrier and / or suitable excipients. Because the segregated targeted delivery system contains living cells, carriers and excipients that maintain cell viability are preferred.

[0144] "Pharmaceutical acceptable" means approved by federal or state government regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly for human use.

[0145] 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 loads, which is administered together with a labeled substance. 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".

[0146] 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.

[0147] "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.

[0148] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists.

[0149] "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.

[0150] In a sixth aspect, the present invention relates to a segregated targeted delivery system according to the first aspect of the invention or a segregated targeted delivery system that can be produced by the method according to the second aspect of the invention, for the diagnosis of tumors, inflammatory diseases or ischemic areas, wherein the tumors are preferably solid tumors and / or metastases thereof, preferably breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, or metastases thereof, or tumors having hypoxic areas, wherein the ischemic areas are particularly ischemic areas following skin wounds, other wounds or organ infarction (heart) or retinal ischemia.

[0151] The targeted delivery system according to the invention enables the delivery of markers, preferably contrast agents, to tumors that are typically inaccessible (e.g., due to solubility issues or lack of a vascular system). It also enables the delivery of markers, preferably contrast agents, to hypoxic tumors or hypoxic regions of tumors. The system further provides, for example, the delivery of contrast agents to any region of an organism undergoing hypoxic conditions during ischemic events or inflammatory processes.

[0152] As described above, the present invention also provides a method for targeted delivery of markers to tumor masses. This method includes preparing CD45... + Leukocytes enable macrophages to efficiently take up markers or iron-binding proteins (ferritin, hemoglobin, and / or transferrin), wherein the ferritin, hemoglobin, and / or transferrin transport the markers preferably as contrast agents.

[0153] And it targets tumors.

[0154] Furthermore, this invention allows radiolabeled markers to be targeted into tumor masses. These methods include CD45+ leukocytes, preferably activated macrophages, loaded with ferritin encapsulating a contrast agent or modified with a contrast agent used for MRI imaging (e.g., ferrohydride) or PET / SPECT imaging (e.g., alpha or beta radiation radioisotopes that also emit cellular-damaging amounts of gamma radiation, 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 cellular-damaging amounts of alpha radiation, preferably selected from actinium-225, bismuth-213, lead-212, and polonium-212).

[0155] This invention utilizes CD45 loaded with iron-binding protein linked to a marker, preferably a contrast agent. + Leukocytes, preferably activated macrophages, can be used as a tumor-targeting delivery system. Alternatively, CD45 labeled with a marker, preferably a contrast agent, can be used. + Leukocytes, preferably activated macrophages, form a marker delivery system to target tumors and visualize them using imaging methods (e.g., PET). The difficulty in detecting them using imaging methods is mainly related to altered contrast agent penetration into hypoxic areas, which are mostly observed in solid tumors, especially their micrometastases, due to poor vascular systems. However, these avascular areas attract CD45. + Leukocytes, preferably activated macrophages, even migrate in areas far from blood vessels. Thus, they constitute a particulate delivery system to the tumor mass. A promising example of such particles is 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.

[0156] The inventors used chemical methods to bind markers 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. + Among leukocytes (monocytes, macrophages, lymphocytes, and / or granulocytes), preferably 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 with encapsulating markers into cancer cells. This method allows for precise application of markers to tumor sites (especially hypoxic areas), preventing their accumulation in other organs. Example Section

[0157] Example 1 - Activation of macrophages

[0158] 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).

[0159] 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 mouse cells: RAW 264.7, J744, human cells: THP-1, U937, or canine cells DH82) can also be used.

[0160] 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 mixture thereof as a cytokine mixture.

[0161] 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.

[0162] To obtain macrophages from blood monocytes, fresh blood (within 12 hours) is separated using the Histopaque 1077 system or an equivalent. White blood cells (or white blood cells collected directly from the blood bank) are seeded in an appropriate amount of pre-warmed monocyte attachment medium (or an 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] Example 2—Monocyte Isolation

[0167] 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 lineage mouse cells: RAW 264.7, J744, human: THP-1, U937, or canine DH82 cell line) are used.

[0168] To obtain blood monocytes, fresh blood (within 12 hours) is separated using the Histopaque 1077 system or an equivalent, and white blood cells are seeded 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, without any other intervention, the cells were cultured in an incubator at 5% CO2 and 37°C for 1 to 1.5 hours. After cell attachment, they were washed at least twice, and the attached cells were considered to be monocytes.

[0169] 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. Attached cells were considered monocytes.

[0170] 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.

[0171] To achieve better protein loading and migration before use, they can be pretreated with 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.

[0172] Example 3—Granocyte Isolation

[0173] 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.

[0174] Example 4 - Lymphocyte Isolation

[0175] 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.

[0176] Example 5 - Preparation of Ferritin Complex

[0177] 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) or to imaging contrast agents (e.g., ferric hydrides or isotopes), ferritin must be prepared prior to macrophage treatment. Soon after, it was obtained according to SEQ ID NO: 4 ( Figure 1Recombinant mouse protein. The expression vector pET-22b containing the gene encoding ferritin synthesis (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.

[0178] The ferritin includes recombinant mammalian ferritin H homopolymer and / or L homopolymer.

[0179] 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 an acidic solution (final pH < 3.0) or in a highly alkaline pH (pH > 9.5) (see, for example, Pontillo et al., 2016), thereby dissociating the multimers. The drug was dissolved in a suitable solvent at a very high concentration, and then a small volume was 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 reconstruction. 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, and fully recover the drug-loaded ferritin nanocages. The resulting ferritin-drug complex is then rapidly freeze-dried in liquid nitrogen.

[0180] Based 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.

[0181] The marker can also be covalently coupled to the amino acid side chains (lysine or cysteine) of ferritin by appropriately selecting phenylhydrazone, succinimide, or maleimide-activated markers. Therefore, i) the phenylhydrazone derivative can be degraded and the marker released from the ferritin surface; ii) the lysine-bound derivative can become active after the protein is completely degraded into amino acids; or iii) the cysteine-bound derivative can be released intracellularly via the reductive hydrolysis of the maleimide thioether bond.

[0182] Example 6 - Preparation of hemoglobin-compound complex

[0183] Human hemoglobin was prepared from fresh red blood cells as described by Rossi-Fanelli et al. (Archives of biochemistry and biophysics 77: 478-492, 1958). Shortly after, 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 an isotonic 0.9% saline solution and centrifuging at 1600 rpm for 30 minutes at 4°C. After the final saline wash and centrifugation steps, 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 lyse overnight with gentle stirring at 4°C. The dialyzed RBC lysates were then centrifuged at 13,000 rpm for 30 min 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 from 100% buffer A to 75% buffer B (20 mM Tris-Cl, plus 0.2 M NaCl, pH 8.20). 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.

[0184] Human hemoglobin (SEQ ID NO: 18 or SEQ ID NO: 22, see also) Figure 1 Heme iron can be readily covalently linked to suitable drug conjugates, accommodating hydrophobic drug molecules or even delivering cytotoxic small molecules linked 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. During remodeling, the apo-protein solution must be kept in an ice bath. A 1.5-fold molar excess of CuT-CPP (Cu-TCPP 4,4',4'',4'''-(porphyrin-5,10,15,20-tetramethyl)tetra(benzoic acid)-Cu in 0.1M NaOH is used. 67 The protein solution must be added dropwise to the apo-globulin solution in 0.2M KPi buffer at pH 7.0, rapidly vortexed at room temperature, and then placed back in an ice bath for 30 minutes. The protein solution must then be filtered through a syringe filter before use.

[0185] Example 7 - Preparation of transferrin-compound complex

[0186] Serum was obtained from healthy donors, and excess iron was added in the presence of citrate ions and bicarbonate as chelating agents; bicarbonate promoted 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 with rivanol (4%) by adding an alcohol solution to the serum sample at a ratio of 3.5 V / V for 2 h at 4°C, pH 9.4. 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 transferrin from the 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.

[0187] Similar to hemoglobin, human all-transferrin (SEQ ID NO: 28, Figure 1 It can be readily covalently linked to suitable marker conjugates. During reconstitution, the apo-protein solution must be kept in an ice bath. A 1.5-fold molar excess of CuT-CPP (Cu-TCPP 4,4',4'',4'''-(porphyrin-5,10,15,20-tetramethyl)tetra(benzoic acid)-Cu in 0.1M NaOH is used. 67The protein solution must be added dropwise to the apo-globulin solution in 0.2M KPi buffer at pH 7.0, rapidly vortexed at room temperature, and then placed back in an ice bath for 30 minutes. The protein solution must then be filtered through a syringe filter before use.

[0188] Example 8 - Obtaining ferritin-loaded cells

[0189] The obtained cells are cultured in a ferritin solution at a concentration sufficient to ensure an appropriate ferritin / cell ratio for full loading and an adequate contrast agent concentration for proper imaging. 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.

[0190] 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 time and concentration of ferritin loading onto cells, care should be taken to consider the effects of ferritin and treatment conditions on cell viability. Cells obtained as described above are readily loaded in a relatively short time (in minutes). Figure 5 They ingest ferritin. Once they ingest ferritin, they do not release it into the culture medium. Figure 6 ).

[0191] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0192] Example 9 - Obtaining hemoglobin-loaded cells

[0193] The obtained cells are cultured in a hemoglobin solution at a concentration sufficient to ensure an appropriate hemoglobin / cell ratio for full loading and to ensure an appropriate contrast agent content for proper imaging. The time and concentration can be varied depending on the number of molecules linked to hemoglobin, the state of cell activation, and the conditions and frequency of their intended application.

[0194] 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 also 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 readily cultured in relatively short times (in minutes). Figure 5 Ingesting hemoglobin.

[0195] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0196] Example 10 - Obtaining cells loaded with transferrin

[0197] 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 contrast agent content for proper imaging. 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.

[0198] 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 (from 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 readily loaded in a relatively short time (in minutes). Figure 5 Transferrin is obtained from the body.

[0199] However, for the laboratory's own purposes, those skilled in the art can readjust the above conditions and optimize the scheme.

[0200] Example 11 - Labeling cells with radioactive isotopes

[0201] In this invention, using 18 Cells were labeled with F-FDG for imaging on PET. Cells had been isolated from the plate and labeled with sufficient amounts to ensure optimal cell uptake. 18 The concentration of F-FDG is used 18 Cells were cultured in F-FDG solution to allow for radioactive detection at sites of cell accumulation. In this practical invention, cells were pretreated with a mixture of different cytokines, growth factors, and other factors affecting their activation state to enhance their uptake of the marker, and then heated in a warm solution containing 20 to 60 MBq. 18 Cells are cultured in F-FDG solution. After labeling, the cells should be centrifuged, and the supernatant should be removed. This step should be repeated until no radioactivity is detected in the supernatant.

[0202] Example 12 - Ferritin / hemoglobin / transferrin / marker-leukocyte complex as a useful tool for tumor delivery

[0203] Macrophages from Example 1 prepared as described in Examples 8, 9, 10, and 11; monocytes from Example 2 prepared as described in Examples 8, 9, 10, and 11; granulocytes from Example 3 prepared as described in Examples 8, 9, 10, and 11; and lymphocytes from Example 4 prepared as described in Examples 8, 9, 10, and 11 readily transport ferritin, hemoglobin, transferrin, and markers to the tumor in amounts sufficient for detection using an imaging system. Figure 2 , Figure 3 , Figure 4 , Figure 7 and Figure 8 ).

[0204] Example 13 - Leukocyte-protein carrier complex as a useful targeted drug delivery agent to hypoxic regions

[0205] 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 , Figure 3 , Figure 4 , Figure 7 , Figure 8 and Figure 10 As shown, (several hours later) they specifically reach the tumor and also disperse throughout other organs of the animal. Furthermore, as... Figure 9 As shown, in the hypoxia model, they can also migrate to avascular and hypoxic sites.

[0206] 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 cell tracers and loaded with ferritin-FITC (as shown in Example 8). Eight hours after macrophage administration, the presence of ferritin-FITC-loaded macrophages was detected using two-photon imaging of the tumor block. Figure 7 Macrophages were pre-labeled with XenoLight DiR lipophilic DIR cytoplasmic dye, and whole-animal imaging (IVIS) was used to demonstrate their specific targeting of tumors and their migration to other organs. Figure 8 ).

[0207] Example 14 - Leukocyte-protein carrier complex or labeled leukocytes as a useful imaging tool

[0208] The targeted delivery system described in this invention constitutes a very useful imaging tool. For example... Figure 4 and Figure 10As shown, after injecting 1 ml to 50 ml of ferritin-loaded macrophages, the ferritin is mixed with a contrast agent (in this example, ferrihydrite, however, with an isotope, for example) as described in Example 8. 123 I obtained the same result) coupling or isotope labeling (in this case, ). 18 F-FDG)( Figure 10 These can be easily detected by MRI, PET, or SPECT. In this embodiment ( Figure 4 Mice with breast tumors were imaged using MRI 3, 22, and 24 hours after intravenous injection of macrophages loaded with ferritin Fh. Figure 4 As shown, the ferritin / macrophage complex is a very useful imaging tool. Mice were treated with macrophages (at time point 0 h). An increase in the diameter of blood vessels (arrows) filled with injected macrophages 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).

[0209] Macrophages also use 18 F-FDG (1 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 10 In the PET scan, it can be seen that macrophages migrate to areas with metastatic tumors, making them visible under PET.

[0210] 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.

[0211] 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.

[0212] 1. A targeted delivery system comprising activated macrophages loaded with contrast agent-laden ferritin.

[0213] 2. The targeted delivery system according to implementation scheme 1, wherein the contrast agent is an iron hydride or an isotope.

[0214] 3. A method for preparing a targeted delivery system, wherein the targeted delivery system comprises activated macrophages loaded with ferritin carrying a contrast agent, the method comprising the following steps:

[0215] a) Purify ferritin;

[0216] b) Obtain contrast-loaded ferritin by linking ferritin and the contrast agent;

[0217] c) Activate isolated macrophages;

[0218] d) In the contrast agent-loaded ferritin solution obtained in step b), culture macrophages for a period of time at a ferritin concentration sufficient to ensure that the contrast agent-loaded ferritin is fully loaded into the macrophages.

[0219] 4. The method according to implementation scheme 3, wherein the activated macrophages are bone marrow-derived macrophages.

[0220] 5. The method according to implementation scheme 3, wherein the activated macrophages are blood-derived macrophages.

[0221] 6. The method according to implementation scheme 3, wherein the activated macrophages are derived from a macrophage cell line.

[0222] 7. The method according to any one of embodiments 3 to 6, wherein the activated macrophage is a macrophage polarized to M1 or M2.

[0223] 8. According to the method of implementation scheme 7, wherein the activated macrophages have been polarized to M2.

[0224] 9. According to the method of implementation scheme 7, the iron metabolism of activated macrophages has been controlled.

[0225] 10. The method according to any one of embodiments 3 to 9, wherein the contrast agent is an iron hydride or an isotope.

[0226] 11. A targeted delivery system as defined in either Implementation Scheme 1 or 2, which is used as an imaging tool.

[0227] In a preferred embodiment, the present invention does not include the subject matter of items 1 to 11 above.

Claims

1. Use of a segregated targeted delivery system in the preparation of diagnostic compositions for diagnosing tumors, said segregated targeted delivery system comprising CD45. + White blood cells, in the CD45 + White blood cells contain a complex of one or more iron-binding proteins and markers. The CD45 mentioned above + White blood cells are macrophages The marker is a radioactive isotope. The iron-binding protein mentioned above is ferritin. The radioactive isotope is encapsulated by polymers of the ferritin, and The isolated targeted delivery system delivers the marker into tumor cells.

2. The use according to claim 1, wherein the macrophage is an activated macrophage.

3. The use according to claim 2, wherein the activated macrophages produce arginase and / or secrete interleukin-10 (IL-10).

4. The use according to claim 1, wherein the macrophages are CD11b. + Macrophages.

5. The use according to claim 4, wherein CD11b + Macrophages are CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, or CD11b + CD68 + Macrophages.

6. The use according to claim 4, wherein CD11b + Macrophages are CD11b + CD86 + M1 macrophages.

7. The use according to claim 6, wherein CD11b + CD86 + M1 macrophages are macrophages that produce iNOS and / or secrete interleukin-12 (IL-12).

8. The use according to claim 4, wherein CD11b + Macrophages are CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + Major Histocompatibility Complex II + (MHCII + M2 macrophages, CD11b + CD200R + M2 macrophages, or CD11b + CD163 + M2 macrophages.

9. The use according to claim 2, wherein the activated macrophages: (i) It can be produced in vitro by culturing monocytes or macrophages with factors that can alter the expression of markers on macrophages; (ii) Characterized by the expression of at least one of the antigens CD64, CD86, CD16, and CD32, high expression of 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 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 can secrete cytokines or produce inducible nitric oxide synthase (iNOS), arginase or other immunosuppressive / anti-inflammatory compounds.

10. The use according to claim 9, wherein the in vitro culture of macrophages and factors capable of altering the expression markers on macrophages is (a) In vitro culture with at least one M1 inducer, (b) In vitro culture with at least one M2 inducer, (c) Or in vitro culture with factors that can alter the ability of macrophages to secrete cytokines, chemokines and / or produce iNOS, arginase or other immunomodulatory enzymes.

11. The use according to claim 10, wherein the cytokines in (c) are IL-10 and IL-12.

12. The use according to claim 9, wherein the cytokine in (vi) is IL-12 or IL-10.

13. The use 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.

14. The use according to claim 1, wherein the ferritin is heavy (H) ferritin, light (L) ferritin and / or mitochondrial ferritin.

15. The use according to claim 1, wherein the radioactive isotope is selected from alpha radiation emission isotopes, gamma radiation emission isotopes, Auger electron emission isotopes, X-ray emission isotopes, and combinations of the above isotopes with proteins, peptides, small molecule inhibitors, antibodies or other compounds.

16. The use according to claim 15, wherein the radioactive isotope is 64 Cu.

17. The use according to claim 1, wherein the marker is encapsulated by ferritin polymers in the absence of covalent and / or non-covalent bonds.

18. The use according to claim 1, wherein the tumor is a solid tumor, its metastases, or a tumor with hypoxic regions.

19. The use according to claim 1, wherein the tumor is breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, or a metastasis thereof.

20. A method for preparing a separated targeted delivery system comprising macrophages, wherein the macrophages contain a complex of ferritin and a radioactive isotope, comprising the following steps: a) Provides purified ferritin; b) Encapsulating radioactive isotopes in ferritin polymers; c) Provide macrophages; and d) Culture macrophages in the presence of the complex of ferritin and radioisotope produced in step b) until the macrophages are at least partially loaded with the complex of ferritin and radioisotope produced in step b).

21. A diagnostic composition for diagnosing tumors, comprising a segregated targeted delivery system as defined in claim 1 and a pharmaceutically acceptable carrier and / or suitable excipient, wherein the segregated targeted delivery system delivers the marker to tumor cells.

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