Isolated targeted delivery system for the treatment of gliomas - Patent Application 20070122999

Abstract

The present invention provides CD45 antibodies containing intracellularly one or more iron-binding proteins and a complex of a pharmaceutically active agent, a label, or a pharmaceutically active agent and a label for use in methods of treating or diagnosing glioma. + It relates to an isolated targeted delivery system, including white blood cells.

Description

[Technical Field]

[0001] The present invention provides a CD45 iron-binding protein comprising a complex of a pharmaceutically active substance and / or label for use in a method for treating or diagnosing glioma. + It relates to an isolated targeted delivery system comprising white blood cells. [Background technology]

[0002] The prognosis for patients with glioma is poor. Regardless of treatment, most patients with glioblastoma (GBM) survive only a few months, with a median survival of 11–12 months. Only 3%–8% of patients survive longer than three years. Because these tumors exhibit diffuse, infiltrative growth, curative resection is usually impossible, and radiation therapy is ineffective. The blood-brain barrier prevents most compounds, including modern therapies (e.g., monoclonal antibodies, CAR-T), from penetrating the brain. Safe and efficient localized delivery of drugs into the brain is challenging. Furthermore, even when administered locally, active ingredients often fail to reach deep within the glioma or penetrate the entire tumor mass. Summary of the Invention

[0003] The present inventors have developed a CD45 complex containing an iron-binding protein and an active ingredient. + We have found that our isolated targeted delivery system containing white blood cells can specifically target glioma cells in the brain and exert a therapeutic effect. We have shown that administration of our isolated targeted delivery system containing an anticancer drug significantly extends survival in animal models of glioblastoma.

[0004] The targeted delivery system according to the present invention for use in methods for treating or diagnosing gliomas offers, inter alia, the following advantages over the prior art: (i) targeted delivery of active ingredients to gliomas that would normally be unable to reach the brain, (ii) targeted delivery of active ingredients to glioma cells, (iii) improved penetration of active ingredients deep into the glioma mass, (iv) improved distribution of active ingredients within the glioma mass, (v) protection of the active ingredient against inactivation in the blood circulation or clearance outside the body, (vi) delivery of active ingredients with poor pharmacokinetics into the brain, (vii) reduced toxicity of the active ingredient due to direct targeted delivery, (viii) higher therapeutic efficacy with lower doses of the active ingredient due to direct targeted delivery and local deposition in glioma cells, (ix) improved efficacy of systemic administration of the active ingredient, thereby preventing the risk of tissue damage during local delivery into the brain, and / or (x) improved therapeutic efficacy of alkylating agents, and (xi) improved therapeutic efficacy of temozolomide (TMZ)-resistant tumors.

[0005] The present invention provides CD45 antibodies containing intracellularly one or more iron-binding proteins and a complex of a pharmaceutically active agent, a label, or a pharmaceutically active agent and a label for use in methods of treating or diagnosing glioma. + It relates to an isolated targeted delivery system, including white blood cells. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 shows that intravenous or intratumoral injection of macrophages containing ferritin-drug conjugates in a mouse GL261 glioma model extends the survival time of tumor-bearing mice. [Figure 2] (A) A diagram showing a decrease in the total number of cancer cells after irradiation, and (B) A diagram showing an increase in the number of macrophages within the tumor after irradiation. [Figure 3] FIG. 1 shows that macrophage infiltration is increased in glioma-containing brains compared to healthy brains. [Figure 4]FIG. 1 shows that administration of ferritin-containing macrophages increases ferritin levels in gliomas compared to administration of ferritin alone. [Figure 5] Figure 1 shows that cell-to-cell contact is required for ferritin transfer from macrophages to cancer cells. Ferritin transfer between cells isolated by Transwell inserts was analyzed. HFt-AF488-loaded THP-1 macrophages (HFt(+)) were seeded onto Transwell inserts or directly with MDA-MB-231 cancer cells growing on 24-well plates and cultured for 24 hours. Ferritin-free macrophages (HFt(-)) were used as a control. Flow cytometry analysis of AF488 fluorescence of cancer cells after 24 hours of co-culture with macrophages. Bar graphs represent the average geometric mean fluorescence of AF488-positive cancer cells. AF488-positive cancer cells were detected when directly co-cultured with macrophages but not when isolated from macrophages by Transwell inserts, indicating that cell-to-cell contact is required for delivery of ferritin-active agent (label) conjugates to cancer cells. [Figure 6]Figure 1 shows the role of secretion and cell-cell contact in the transfer of ferritin from macrophages to cancer cells. EMT6 cancer cells were directly co-cultured with HFt-AF488-loaded RAW264.7 macrophages or cultured with culture supernatant. Culture supernatant was collected from RAW264.7-HFt-AF488 macrophages cultured for 24 hours in normal growth medium, RAW264.7-HFt-AF488 macrophages cultured for 24 hours in medium produced by cancer cells, or co-cultures of RAW264.7-HFt-AF488 macrophages with cancer cells. Ferritin-free macrophages were used as a control. (a) Representative density plots show the distribution of AF488 fluorescence in EMT6 cells. For all conditions, the density plot on the left is the no-ferritin control. Bar graphs represent the average (b) geometric mean fluorescence and (c) percentage of AF488-positive cancer cells from three independent experiments. For all conditions, the left bar represents the control without ferritin. After 4 hours, efficient uptake of HFt-AF488 by cancer cells was observed only in direct co-culture, but not in culture with culture medium. After 24 hours, uptake of HFt-AF488 by cancer cells was higher and more frequent in direct co-culture than in culture with culture medium. The data indicate that cell-to-cell contact is required for delivery of the ferritin-active ingredient (labeled) conjugate to cancer cells, and that secretion of the ferritin-active ingredient (labeled) conjugate into the medium by macrophages and subsequent uptake by cancer cells is insufficient to ensure efficient delivery of the ferritin-active ingredient (labeled) conjugate to cancer cells. [Figure 7] FIG. 1 shows that intratumoral injection of macrophages containing ferritin-drug conjugates in a ZH-161 glioma model extends the survival time of tumor-bearing mice. [Figure 8] FIG. 1 shows the systemic distribution of macrophages containing ferritin-drug conjugates after intratumoral administration. DETAILED DESCRIPTION OF THE INVENTION

[0007] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0008] The terms "peptide" or "polypeptide" are used indistinguishably in the context of the present invention to refer to a chain of at least two amino acids linked by peptide bonds. Thus, the term "polypeptide" in the context of the present invention is also used to refer to an amino acid chain comprising more than 50, more than 100 or more than 150 amino acids.

[0009] The term "amino acid" encompasses naturally occurring amino acids as well as amino acid derivatives. In the context of this specification, amino acids are identified using single-letter codes (Hausman RE, Cooper GM (2004). The cell: a molecular approach. Washington, DC: ASM Press. p. 51. ISBN 978-0-87893-214-6). Amino acids identified with the letter X correspond to any amino acid. Amino acids identified with the letter B correspond to either D (asparagine) or N (aspartic acid). Amino acids identified with the letter Z correspond to either E (glutamine) or Q (glutamic acid).

[0010] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to polymeric or oligomeric macromolecules made from nucleotide monomers. A nucleotide monomer is composed of a nucleobase, a pentose sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the nucleic acid molecules referred to include, but are not limited to, ribonucleic acid (RNA) and its various forms (e.g., ssRNA, LNA, etc.), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be synthesized chemically, for example, by the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). An "aptamer" is a nucleic acid that binds to a polypeptide with high affinity. Aptamers can be isolated from large pools of diverse single-stranded RNA molecules by selection methods such as SELEmir146-a (see, e.g., Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; U.S. Pat. No. 5,582,981). Aptamers can also be synthesized and selected in their mirror-image form, e.g., as L-ribonucleotides (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms isolated in this way have the advantage of being less susceptible to degradation by naturally occurring ribonucleases and therefore more stable.

[0011] The term "identity" is used throughout this specification in reference to polypeptide and nucleotide sequence comparisons. When comparing two sequences and calculating the percentage sequence identity, if no reference sequence is specified, the sequence identity is calculated with reference to the longer of the two sequences being compared, unless otherwise specifically indicated. When a reference sequence is specified, the sequence identity is determined based on the full length of the reference sequence, as indicated by the SEQ ID NO:, unless otherwise specifically indicated. For example, compared to a reference 300-amino acid-long polypeptide sequence, a polypeptide sequence consisting of 200 amino acids may exhibit a maximum percentage of sequence identity of 66.6% (200 / 300), while a sequence having a length of 150 amino acids may exhibit a maximum percentage of sequence identity of 50% (150 / 300). If 15 of the 150 amino acids are different from each amino acid in the 300-amino acid-long reference sequence, the level of sequence identity drops to 45%. The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined through sequence alignment. Such alignments can be performed using several art-known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), available 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 used are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The degree of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). BLAST protein searches are performed using the BLASTP program, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis can be supplemented by established homology mapping methods such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. Structure-based alignment of multiple protein sequences and / or structures using information from sequence database searches, 3D structures, and available homologs with user-defined constraints can also be used (Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of distantly related proteins. Bioinformatics 2007, 23:802-808; 3DCoffee@igs: a web server 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 referred to in this application, these percentages are calculated with respect to the full length of the longer sequence unless specifically indicated otherwise.

[0012] The term "antibody" as used in the context of the present invention refers to a glycoprotein that belongs to the immunoglobulin superfamily, and the terms antibody and immunoglobulin are often used interchangeably. Antibodies refer to protein molecules produced by plasma cells and are used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Antibodies recognize a unique portion of a foreign target, their antigen.

[0013] As used herein, the term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antibody fragment" include fragment antigen-binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAbs), single chain variable fragments (scFv), variable fragments (Fv), V, and the like. H Domain, V L These include domains, single domain antibodies, nanobodies, IgNARs (immunoglobulin new antigen receptors), dis-scFvs, bispecific T cell engagers (BITEs), dual affinity retargeting (DART) molecules, triple bodies, diabodies, single chain diabodies, alternative scaffold proteins, and fusion proteins thereof.

[0014] The term "antigen" is used to refer to a substance, preferably an immunogenic peptide, that comprises at least one epitope, preferably an epitope that elicits a B-cell or T-cell response or a B-cell and a T-cell response.

[0015] An "epitope," also known as an antigenic determinant, is a portion of a substance, e.g., an immunogenic polypeptide, that is recognized by the immune system. Preferably, this recognition is mediated by the binding of an antibody, B cell, or T cell to the epitope of interest. In this context, the term "binding" preferably relates to specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The term "epitope" includes both conformational and nonconformational epitopes. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0016] The immunogenic polypeptide according to the present invention is preferably derived from a pathogen selected from the group consisting of a virus, a bacterium and a protozoan. However, in an alternative embodiment of the invention, the immunogenic polypeptide is a tumor antigen, i.e., a polypeptide or a fragment of a polypeptide that is specifically expressed by cancer.

[0017] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0018] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA technology are used, as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0019] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, any permutations and combinations of all elements described in this application should be considered to be disclosed by the specification of this application.

[0021] targeted delivery system The present invention provides CD45 antibodies containing intracellularly one or more iron-binding proteins and a complex of a pharmaceutically active agent, a label, or a pharmaceutically active agent and a label for use in methods of treating or diagnosing glioma. + It relates to an isolated targeted delivery system comprising white blood cells.

[0022] The term "treatment" as used herein includes any type of medically approved preventative and / or therapeutic intervention for various purposes, including slowing or stopping the progression of a disease, causing a lesion to regress or disappear, preventing the onset of a disease, or preventing recurrence, for the purpose of cure, palliative care, prevention, etc. When the isolated targeted delivery system is provided for use in a method for treating glioma, the isolated targeted delivery system comprises a pharmaceutically active substance. When the isolated targeted delivery system is provided for use in a method for diagnosing glioma, the isolated targeted delivery system comprises a label. The method for diagnosis is an in vivo diagnostic method. In a preferred embodiment, the isolated targeted delivery system is provided for use in a method for treating glioma.

[0023] The term "glioma" refers to a cancer of the glial cells that surround nerve endings in the brain. In a preferred embodiment, the glioma is a glioblastoma. A glioma or glioblastoma can be a primary tumor or a secondary tumor.

[0024] In some embodiments, the glioma is resistant to chemotherapy. In some embodiments, the glioma is resistant to alkylating agent chemotherapy. In some embodiments, the glioma is a temozolomide (TMZ)-resistant glioma. In some embodiments, the glioma expresses O-6-methylguanine-DNA methyltransferase (MGMT).

[0025] The therapeutic benefit of TMZ depends on its ability to alkylate / methylate DNA, which most often occurs at the N-7 or O-6 positions of guanine residues. However, some tumor cells can repair this type of DNA damage by synthesizing a protein encoded by the O-6-methylguanine-DNA methyltransferase (MGMT) gene, which can reduce the therapeutic effectiveness of TMZ.

[0026] Surprisingly, the present inventors have demonstrated that treatment with the isolated targeted delivery system of the present invention, particularly a ferritin-drug conjugate, more particularly a ferritin-MMAE conjugate, and even more particularly an isolated targeted delivery system comprising HFt-vcMMAE, significantly extends survival in an in vivo model of TMZ-resistant glioma.

[0027] In some embodiments, the glioma is a stage III glioma or a stage IV glioma (glioblastoma). In some embodiments, the glioma is a malignant glioma, such as glioblastoma multiforme or anaplastic astrocytoma.

[0028] In some embodiments, the isolated targeted delivery system is used in a method for treating glioma in a human individual experiencing progressive disease or relapse during or after treatment with an alkylating agent, particularly TMZ.

[0029] In the present context, the term "active ingredient" is used to refer to at least one pharmaceutically active substance and / or at least one label. Preferably, the active ingredient is a pharmaceutically active substance.

[0030] The term "targeted delivery" refers to the direct delivery of a therapeutic or diagnostic agent (collectively referred to herein as "active ingredient") to a subject, e.g., a patient, particularly to a cell, more particularly to cells within the patient's body. Targeted delivery results in an increased concentration of the active ingredient in a specific region of the body compared to administration of the active ingredient alone, a complex of the active ingredient with an iron-binding protein, or another delivery system. In particular, targeted delivery results in an increased concentration of the active ingredient within tumor tissue, particularly within gliomas, more particularly within glioma cells, compared to administration of the active ingredient alone, a complex of the active ingredient with an iron-binding protein, or another delivery system. Targeted delivery also includes "targeted therapeutic-diagnostic delivery," meaning that both a therapeutic agent and a diagnostic agent are delivered simultaneously, preferably to the affected area, thereby enabling simultaneous treatment and diagnosis and / or treatment monitoring.

[0031] In a preferred embodiment, the active ingredient is preferably CD45 + Direct delivery to glioma cells is via direct transfer from leukocytes to glioma cells (direct cell-to-cell transfer), preferably via mechanisms involving cell-to-cell contact and / or fusion of cell membranes.

[0032] In some embodiments, the targeted delivery system is administered via intratumoral injection. In some embodiments, the targeted delivery system is administered via intravenous injection.

[0033] In this application, the term "targeted pharmaceutically active agent delivery system" is used to refer to a system capable of delivering a pharmaceutically active agent to a targeted area, i.e., capable of targeted delivery within the patient's body, preferably to an affected area.

[0034] In this application, the term "targeted label delivery system" is used to refer to a system that is capable of delivering a label to a targeted area, i.e., targeted delivery within the patient's body, preferably to an affected area.

[0035] In the present application, the term "targeted therapeutic diagnostic delivery system" is used to refer to a system that can simultaneously deliver a pharmaceutically active substance and a conjugate with a label to a targeted area, i.e., can deliver targeted delivery to a patient's body, preferably to an affected area, thereby allowing for simultaneous treatment and diagnosis and / or treatment monitoring.

[0036] The term "targeted delivery system" is used generally to refer to "targeted pharmaceutically active agent delivery systems," "targeted labeled delivery systems," and "targeted therapeutic diagnostic delivery systems." Targeted delivery systems are described in WO 2016 / 207257, WO 2016 / 207256, and WO 2017 / 222398, which are incorporated herein by reference.

[0037] Conjugates and Linkers Within the isolated targeted delivery system of the present invention, the active ingredient may be covalently or non-covalently bound to the iron-binding protein or encapsulated by the iron-binding protein or its multimer. The term "complex" also encompasses the inclusion of the active ingredient within an iron-binding protein or its multimer, particularly within ferritin multimers that form the "ferritin cage." When the active ingredient is encapsulated within an iron-binding protein or its multimer, the encapsulation may occur with or without covalent or non-covalent bonds. In some embodiments, the active ingredient can be encapsulated (physically trapped) within the internal cavity of a ferritin oligomer by exploiting the association / dissociation properties of the ferritin macromolecule itself. In such embodiments, the active ingredient is held in place by non-covalent interactions with amino acid residues within the internal surface of the cavity. Hemoglobin macromolecules also offer the possibility of non-covalent binding of selected pharmaceutically active substances and / or labeling molecules, which may be accommodated within the heme-binding pocket of hemoglobin itself. The heme in the pocket can be displaced and replaced by a pharmaceutically active agent and / or label with an appropriate hydrophobicity profile.

[0038] CD45 + When leukocytes internalize iron-binding proteins, the formation of a complex binds the active ingredient to CD45 + The active ingredient can be transported into leukocytes. Therefore, it is preferable that the active ingredient is bound to an iron-binding protein so as not to interfere with the transport mechanism. This can be easily tested by those skilled in the art using uptake assays known in the art and described in WO 2016 / 207257, WO 2016 / 207256, and WO 2017 / 222398. The complex containing the active ingredient binds to CD45. + When taken up by leukocytes and transported to target cells in the body, the complex is preferably stable enough to survive from intracellular transport to the target area in the body. Therefore, it is preferable to deliver the complex, rather than the active ingredient alone, into target cells in the target area. This characteristic allows the CD45 receptor that delivers the active ingredient to be used. +Possible harmful effects of the active ingredient on white blood cells or other cells of the body that are not target cells, such as cytotoxicity, are also reduced.

[0039] In a preferred embodiment, the active ingredient and the iron-binding protein are linked by a covalent and / or non-covalent bond, preferably by a covalent bond. When the active ingredient is linked to the iron-binding protein by a covalent bond, such coupling is preferably via amino acid residues known to be located in surface regions that are not involved in binding of the iron-binding protein to receptors involved in endocytosis.

[0040] When the iron-binding protein and the active ingredient are linked by a covalent bond, they may be linked directly or indirectly via a linker. In a preferred embodiment, the iron-binding protein and the active ingredient are linked by a covalent bond via a linker.

[0041] Polyalanine, Polyglycine, Carbohydrate, (CH2) n Linkers such as groups or polypeptide linkers are known to those skilled in the art. The linker may be biodegradable or non-biodegradable, preferably biodegradable. In a preferred embodiment, the linker is cleavable. The linker may be a peptide linker, a disulfide linker, a hydrazone linker, or a carbohydrate-containing linker. Preferably, the linker is a peptide linker. In some embodiments, the peptide linker is cleavable by a protease. A carbohydrate-containing linker may be cleavable by β-glucuronidase. A hydrazone linker can be cleaved by acid hydrolysis. A disulfide linker can be cleaved by cytosolic reductive cleavage. In a preferred embodiment, the linker, preferably a peptide linker, is cleavable by a lysosomal protease, more preferably a lysosomal cysteine ​​protease, and even more preferably a cathepsin.

[0042] In some embodiments, the linker comprises a reactive group that, upon activation by a defined stimulus, causes cleavage of a covalent bond within the linker. Suitable reactive groups include, for example, a photoactivatable group (i.e., a group that can be activated by ultraviolet light, such as 3-amino-3-(-2-nitro)phenylpropionic acid or its photoactivatable structural equivalent), a dithionite-activatable group (e.g., an azobenzene moiety), or a periodate-activatable group (e.g., a 1,2-dihydroxy moiety, a 1-amino-2-hydroxy moiety, or a 4-amino-4-deoxy-L-threonic acid). In other embodiments, the linker is cleavable via a pH shift.

[0043] By appropriate selection of a phenylhydrazone-, succinimide-, or maleimide-activated drug, pharmaceutically active agents or labels can also be covalently attached to the amino acid side chains (lysine or cysteine) of iron-binding proteins. Phenylhydrazone derivatives can degrade the iron-binding protein and release the drug therefrom, lysine-linked derivatives can become active after complete degradation of the protein to amino acids, or cysteine-linked derivatives can be released intracellularly by reductive hydrolysis of the maleimide thioether linkage.

[0044] In some embodiments, the linker is a dipeptide linker, particularly Val-Cit, Val-Ala, or Ala-Ala, or a tripeptide linker and a tetrapeptide linker.

[0045] In some embodiments, the linker is a heterobifunctional crosslinker containing an N-hydroxysuccinimide (NHS) ester group and a maleimide group, which allows for covalent conjugation of amine- and sulfhydryl-containing molecules. In some embodiments, the linker is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or sulfo-SMCC.

[0046] Preferably, the cleavable linker is cleaved in the lysosomal compartment.

[0047] In a most preferred embodiment, the linker is a maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl (mc-vc-PAB) linker.

[0048] In some embodiments, the iron-binding protein is conjugated to an active ingredient or a linker via a cysteine ​​or lysine residue, preferably a cysteine ​​residue. To form a covalent bond, a relevant thiol, amino, or carboxyl group on the iron-binding protein is used to directly or indirectly covalently couple an active ingredient that is reactive to thiol or amino groups to the iron-binding protein. The active ingredient may be modified with a specific active moiety, i.e., a linker.

[0049] Thus, ferritin or hemoglobin may be linked to cysteine ​​thiol-reactive pharmaceutically active substances and / or labels with peptide-based cleavable linkers (e.g., cathepsin-sensitive valine-citrulline sequences and para-aminobenzyl carbamate spacers). A notable example is the use of the antimitotic drug monomethyl auristatin E (MMAE). The peptide-based linker stably couples the protein to the cytotoxic compound, preventing the drug from being readily released from the protein under physiological conditions, helping to prevent toxicity to healthy cells and ensuring drug efficacy. The resulting adducts of iron-binding proteins and pharmaceutically active substances and / or labels can bind to selected receptor types, such as CD163 for hemoglobin and TfR for transferrin, respectively. Upon binding, the adducts of iron-binding proteins and pharmaceutically active substances and / or labels are internalized by endocytosis and thus selectively taken up by cells. The vesicles containing the active ingredient fuse with lysosomes, where lysosomal cysteine ​​proteases, particularly cathepsin B, begin to degrade the cleavable peptide linkers, particularly the valine-citrulline linker, so that the active ingredient, particularly MMAE, is no longer bound to iron-binding proteins and is released directly into the tumor environment.

[0050] Alternatively, DM1-SMCC is an efficient mertansine derivative with a linker that specifically binds to lysine residues, forming covalent complexes with ferritin, hemoglobin, or transferrin in a reaction well described for antibodies. In particular, hemoglobin, ferritin, or transferrin can react with DM1-SMCC and be cleaved intracellularly, resulting in a covalent protein-drug adduct that releases the active drug in a time-dependent manner. Inhibition of microtubule dynamics by DM1 induces mitotic arrest and cell death.

[0051] Methods for preparing complexes of iron-binding proteins and active ingredients are described in WO 2016 / 207257, WO 2016 / 207256, and WO 2017 / 222398.

[0052] The term "fully loaded" in the context of the present invention means that the maximum amount of iron-binding protein, preferably ferritin, complexed with the pharmaceutically active agent, label, or pharmaceutically active agent and label is bound to CD45 + It is used to indicate that it can be taken up by white blood cells, preferably macrophages, more preferably activated macrophages.

[0053] It is also contemplated that isolated targeted delivery systems may contain different active ingredients. For example, one type of active ingredient may be covalently bound to a ferritin polypeptide, while another type is encapsulated in a complex. This approach takes advantage of the different release rates of the active ingredients from the complex upon delivery to the targeted tissue and / or cell. For example, by exploiting the reactivity of the associated thiol, amino, or carboxyl groups, the active ingredient can be covalently attached to the ferritin molecule either on the surface or within the internal cavity of the 24-mer. Such useful reaction types are known in the art, and those skilled in the art can employ them for specific active ingredients without any additional work. Examples of such reactions are described in Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies. MAbs. 2014 Jan-Feb;6(1):46-53.

[0054] For therapeutic diagnostic applications, i.e., applications in which the complex contains both a label and a pharmaceutically active agent, it is preferred that the label be covalently attached to the iron-binding protein and the pharmaceutically active agent be non-covalently bound to the iron-binding protein and / or be trapped in an internal cavity formed upon assembly of the ferritin polypeptide multimers.

[0055] iron-binding proteins In some embodiments, the iron-binding protein is selected from the group consisting of ferritin, preferably heavy chain (H) ferritin, light chain (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, hemopexin, transferrin, and lactoferrin.

[0056] Human transferrin and ferritin proteins have been considered effective carriers for delivering small molecules or toxin conjugates that specifically target cancer cells. However, despite considerable efforts to date, conjugates of transferrin or ferritin with drugs have not achieved clinical success (Luck AN et al. 2013, Adv Drug Deliv Rev 65(8):1012-9).

[0057] Ferritin is a hollow, globular protein complex composed of 24 ferritin monomer subunits assembled into a cage-like structure. Ferritin is the major intracellular iron storage protein. It is produced by almost all living organisms and is present in all cell types. The ferritin gene is highly conserved among species. In vertebrates, two ferritin monomers exist: light (L) and heavy (H) chains, with molecular weights of 19 kDa and 21 kDa, respectively. The vertebrate ferritin 24-mer can be a homo-oligomer consisting of either the L or H chain, or a hetero-oligomer consisting of both the L and H chains (Theil EC, 1987, Annual Review of Biochemistry. 56 (1): 289-315). Typically, ferritin complexes have inner and outer diameters of approximately 8 nm and 12 nm, respectively. Ferritin has been shown to be internalized by endocytosis upon binding to CD71. The interaction of ferritin with CD71 is mediated through the ferritin heavy chain (Li L et al, Proc. Natl. Acad. Sci. USA 107 (8) (2010) 3505-3510). Ferritin is not abundant in plasma, but it can be easily produced in high yields as a recombinant protein in common protein expression systems such as Escherichia coli cells.

[0058] Purified transferrin can be efficiently conjugated to a variety of molecules, including anticancer drugs, 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 lysine groups on the protein surface are readily available for covalent attachment.

[0059] Hemoglobin has been considered in the past as a possible drug carrier due to its versatility in chemical conjugation with drugs, its abundance in blood, and its relative stability (Somatogen, 1993, WO 1993 / 008842). However, due to the lack of receptor targeting properties, biomedical applications other than as a blood substitute or anti-sickling agent have not been cultivated. In fact, Hb can only be recognized by the CD163 (haptoglobin / hemoglobin receptor) epitope from leukocytes, particularly monocytes-macrophages. The CD45 receptor described in this application + Leukocyte-, particularly macrophage-, based protein delivery has placed hemoglobin at the center as a target-specific carrier of pharmaceutically active substances and / or labels. Hemoglobin can be easily covalently linked to appropriate pharmaceutically active substances and / or labels, can accommodate hydrophobic pharmaceutically active substances and / or labels within its heme-binding pocket, or can even transport small molecules, such as cytotoxic molecules, linked to the heme iron. Hb can be easily modified by selectively attaching appropriate drug conjugates to the β93 cysteine ​​residue, the only titratable cysteine ​​on the protein surface. Maleimide-functionalized drugs, such as the tubulin inhibitor monomethyl auristatin (MMAE) or the DNA crosslinker pyrrolobenzodiazepine dimer (PBD), are notable examples of highly potent cytotoxic agents that can be easily and specifically linked to the relevant cysβ93 residue.

[0060] Alternatively, the lysine residues on the Hb surface (at least 10 titratable lysine residues per Hb tetramer) may be readily amenable to drug conjugation via a cleavable succinimide linker. Hemoglobin also possesses the unique ability to release noncovalently bound heme groups at acidic pH values. The resulting apo-hemoglobin can accommodate several hydrophobic molecules in the empty heme pocket, as shown in the case of paclitaxel (Meng Z et al. 2015 J Pharm Sci 104(3):1045-55) or fluorescent labels (e.g., chlorin e6, hypericin, and phthalocyanine derivatives) (Dong J et al. J Photochem Photobiol B 2014, 140:163-172).

[0061] In a preferred embodiment of the targeted delivery system for use in the present invention, the iron-binding protein is selected from the group consisting of ferritin, preferably heavy chain (H) ferritin, light chain (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, hemopexin, 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, hemopexin, transferrin, and lactoferrin encompass structural variants of naturally occurring proteins and thus relate to proteins having at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and more preferably at least 100% of the iron ion(s) binding capacity of the respective wild-type proteins. The iron-binding proteins used in the context of the present invention are preferably of mammalian origin, more preferably of mouse, rat, dog, ape, particularly chimpanzee, or human origin, and most preferably of human origin. Consensus sequences of preferred iron-binding proteins and preferred structural variants are disclosed in WO 2016 / 207257.

[0062] In a preferred embodiment, the iron-binding protein is ferritin, preferably mammalian ferritin. The mammalian ferritin may be mouse, rat, dog, ape, particularly chimpanzee, or human ferritin. In a preferred embodiment, the mammalian ferritin is mouse, rabbit, rat, or human ferritin, preferably human ferritin. In an even more preferred embodiment, the human ferritin is human heavy chain ferritin.

[0063] Amino acid substitutions within the iron-binding protein are preferably selected so that they do not excessively alter the conformation of the polypeptide. As an example, a "small amino acid" should be replaced with another small amino acid. A "small amino acid" in the context of the present invention is preferably an amino acid having a molecular weight of less than 125 daltons. Preferably, a small amino acid in the context of the present invention is selected from the group consisting of the amino acids glycine, alanine, serine, cysteine, threonine, and valine, or derivatives thereof. As another example, an amino acid with a hydrophobic side chain should be replaced with another amino acid with a hydrophobic side chain.

[0064] Any ferritin used in an isolated targeted delivery system for use according to the present invention should retain the properties of wild-type ferritin with respect to complex formation (a cage-like structure consisting of 24 ferritin monomer subunits) and iron uptake.

[0065] In some embodiments, the ferritin comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 1-5, and has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the ability of wild-type ferritin, particularly human wild-type ferritin, to bind to iron ion(s) and / or form a ferritin 24-mer. SEQ ID NO: 3 is a mammalian consensus sequence. In SEQ ID NO: 3, 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 can be present or absent and, if present, represents any amino acid, preferably He; 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 He; X at position 105 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 any naturally occurring amino acid, preferably Asn, X at position 181 is absent or any naturally occurring amino acid, preferably Glu, and X at position 182 is absent or any naturally occurring amino acid, preferably Ser. SEQ ID NO: 5 is a mammalian consensus sequence.In SEQ ID NO: 5, 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 can be present or absent and, if present, represents any amino acid, preferably He; X at position 22 can represent any amino acid, preferably Asn; and 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; and X at position 110 can be any naturally occurring amino acid, preferably X at position 164 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ser; X at position 166 can be any naturally occurring amino acid, preferably Met or Leu, preferably Leu; X at position 178 can be any naturally occurring amino acid, preferably Asp or His, more preferably Asp; X at position 181 can be absent or any naturally occurring amino acid, preferably Asn; X at position 182 can be absent or any naturally occurring amino acid, preferably Glu; and X at position 183 can be absent or any naturally occurring amino acid, preferably Ser.In some embodiments, the ferritin comprises an amino acid sequence according to SEQ ID NO: 4, optionally including 1 to 5, 1 to 10, 1 to 15, 1 to 20, or 1 to 25 amino acid mutations outside of positions 54, 72, 87, and / or 144, particularly outside of position 54, and 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% of the ability of wild-type ferritin, particularly human wild-type ferritin, to bind to iron ion(s) and / or form a ferritin 24-mer, particularly the ability to form a ferritin 24-mer.

[0066] Drugs and Labels The terms "drug" or "pharmaceutically active substance" are used synonymously in the context of the present invention and refer to any compound that modulates or regulates cellular activity, or any compound that can be activated to modify or regulate cellular activity, preferably in the body of a patient, i.e., a prodrug. Examples of such active ingredients include so-called "small molecules" and peptides. The term "small molecule" is used in the context of the present invention to refer to a hydrocarbon with a molecular weight of less than 1,500 g / mol or a pharmaceutically active radioisotope.

[0067] In a preferred embodiment, the pharmaceutically active agent is an anti-cancer agent selected from the group consisting of proteins, peptides, nucleic acids, non-protein, non-nucleic acid compounds having a molecular weight of less than 1.5 kD, photosensitizing compounds, viruses, and pharmaceutically active radioisotopes.

[0068] Preferred pharmaceutically active radioisotopes are alpha- or beta-emitting radioisotopes that also emit cell-damaging amounts of gamma rays selected from the group consisting of lutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, cesium-131, palladium-103, radium-233, iodine-125, and boron-10, or preferably radioisotopes that emit cell-damaging amounts of alpha rays selected from the group consisting of actinium-225, bismuth-213, lead-212, and polonium-212. Also preferred are nanoparticles (e.g., gold, silver, graphene) or complexes of the above-mentioned compounds and isotopes linked to these nanoparticles.

[0069] When the pharmaceutically active agent is a virus, the pharmaceutically active agent is preferably an oncolytic virus.

[0070] When the drug is a nucleic acid, it is preferable that the drug is a nucleic acid encoding miRNA, siRNA, chemically modified RNA, LNA, ssRNA, DNAzyme, or a pharmaceutically active protein, such as an antibody, an antibody mimetic, a cytokine, a prodrug-converting enzyme, an immunogenic peptide, etc.

[0071] In a preferred embodiment, the anti-cancer drug is a cytostatic agent, a cytotoxic agent or a prodrug thereof.

[0072] The preferred anticancer drug is selected from apoptosis / autophagy or necrosis inducers. The apoptosis / autophagy or necrosis inducer can be any drug that can effectively induce apoptosis / autophagy or necrosis even in cells with abnormal cell proliferation. These drugs are preferably used in complex with one or more ferritins.

[0073] In preferred embodiments, the anti-cancer drug is selected from the group consisting of apoptosis inducers, alkylating agents, antimetabolites, antibiotics, antimitotics, DNA modifying agents, DNA minor groove interstrand crosslinking drugs, inhibitors of DNA synthesis, inhibitors of RNA synthesis, epothilones, nuclear receptor agonists and nuclear receptor antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, inhibitors of cell cycle dependent protein kinases (CDKs), inhibitors of cyclooxygenase and / or lipoxygenase, biogenic fatty acids, biogenic fatty acid derivatives including prostanoids and leukotrienes, inhibitors of protein kinases, inhibitors of protein phosphatases, inhibitors of lipid kinases, platinum coordination complexes, ethylene imines, methylmelamines, triazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkylsulfonates, folic acid analogs, anthracenediones, substituted ureas, and methylhydrazine derivatives, enediyne antibiotics, maytansinoids, auristatin derivatives, immune checkpoint inhibitors, and inhibitors of tumor-specific proteins or tumor-specific markers, preferably Rho-GDP dissociation inhibitors, more preferably Grp94 inhibitors or AXL inhibitors, tubulin inhibitors, or topoisomerase inhibitors.

[0074] In a preferred embodiment, the anticancer drug is acediasulfone, aclarubicin, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, vanoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, capecitabine, carmustine, celecoxib, calicheamicin, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, deruxtecan, dibromopropamidine, diethylstilbestrol, docetaxel, doxorubicin, dolastatin 10, dolastatin 15, dynemicin A, enediyne, epirubicin steroids, epothilone B, epothilone D, estramustine phosphate, estrogen, ethinyl estradiol, etoposide, exatecan derivatives, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fosfestrol, furazolidone, gemcitabine, gonadotropin-releasing hormone analogs, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon alpha, irinotecan, leuprolide, lomustine, lutecan, mafenide sulfateolamide), maytansine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mepacrine, mercaptopurine, mertansine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, neocarzinostatin, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, nimorazole, Trofurantoin, nitrogen mustard, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, prosine, procarbazine, pyrimethamine, pyrrolobenzodiazepines, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride chloride), semustine, streptozocin, sn-38, sulfacarbamide, sulfacetamide, sulfachloropyridazine, sulfadiazine, sulfadiclamide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanol, sulfamethizole, sulfamethoxazole, cotrimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfapyridine, sulfa Preferably, the anticancer drug is selected from the group consisting of phenazole, sulfathiazole, sulfisomidine, staurosporine, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triaziconazole, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinorelbine, and zorubicin. More preferably, the anticancer drug is selected from the group consisting of auristatins, banoxantrone, bendamustine, chlorambucil, calicheamicin, dynemicin A, maytansine, melphalan, mertansine, neocarzinostatin, and pyrrolobenzodiazepines.

[0075] Even more preferably, the anticancer drug is an auristatin, in particular monomethyl auristatin E or monomethyl auristatin F, or deruxtecan.

[0076] In some embodiments, the anticancer drug is an immunomodulatory agent that activates or inhibits the activity of immune cells, and preferably the immunomodulatory agent is a ligand or antagonist of a pattern recognition receptor, particularly a Toll-like receptor, a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a stimulator of interferon genes (STING) protein. Physiologically, these receptors recognize signals of the types known as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

[0077] In a preferred embodiment, the anticancer drug is a growth-inhibitory protein or peptide, preferably a cell cycle inhibitor, or an antibody or antibody-like binding protein that specifically binds to a growth-promoting protein, or a nucleic acid, preferably a nucleic acid encoding a growth-inhibitory protein or an antibody or antibody-like binding protein that specifically binds to a growth-promoting protein, or an siRNA, oligonucleotide, LNA, or DNAzyme.

[0078] The term "prodrug" as used in the context of the present invention refers to any active ingredient that, after administration, is metabolized or otherwise converted into a component (or drug) that is biologically active or more active in at least one property. Compared to a drug, a prodrug is chemically modified to be less active or inactive than the drug, but the chemical modification is such that the corresponding drug is produced by metabolic or other biological processes after the prodrug is administered to a patient. A prodrug may, for example, have altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor compared to an active drug (see, e.g., Nogrady, 1985, Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.

[0079] In some embodiments, the pharmaceutically active agent is a hypoxia-activated prodrug, preferably a hypoxia-activated prodrug selected from the group consisting of benzotriazine N-oxides, apaziquone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH-302, TH-4000, and AQ4N. The use of an active ingredient that is activated under hypoxic conditions can provide additional targeting specificity and / or further reduce adverse effects of the active ingredient. Thus, in particularly preferred embodiments, the active ingredient is a hypoxia-activated prodrug. The backbone of all hypoxia-activated prodrugs is the presence of one of five different chemical moieties (nitro groups, quinines, aromatic and aliphatic N-oxides, and transition metals) that are enzymatically reduced under hypoxic conditions in tissues. A hypoxia-activated prodrug is any prodrug that is less active or inactive than the corresponding drug and contains a drug and one or more bioreducible groups. Such hypoxia-activated prodrugs include all prodrugs activated by various reducing agents and reductases, including, but not limited to, single-electron transferases (such as cytochrome P450 reductase) and two-electron transfer (or hydride transfer) enzymes. According to a preferred embodiment of the present invention, the hypoxia-activated prodrug is TH-302. Methods for synthesizing TH-302 are described in PCT Publication Nos. WO 07 / 002931 and WO 08 / 083101.

[0080] In some embodiments, the pharmaceutically active agent is an antigen or a nucleic acid encoding an antigen.

[0081] The terms "label" or "diagnostic agent" are used interchangeably herein and refer to any type of compound suitable for diagnostic purposes. In preferred embodiments, the label is selected from the group consisting of a fluorescent dye, a radioisotope / fluorescent-emitting isotope, a detectable polypeptide or a nucleic acid encoding a detectable polypeptide, and an imaging agent, or the label comprises a chelator that complexes with a divalent or trivalent metal cation. More preferably, the label is selected from a fluorescent dye, a radioisotope, and an imaging agent. An imaging agent is a dye or other substance that helps to indicate abnormal areas in the body.

[0082] Preferred fluorescent dyes are selected from the group consisting of the following classes of fluorescent dyes: xanthenes (e.g., fluorescein), acridines (e.g., acridine yellow), oxazines (e.g., oxazine 1), cyanines (e.g., Cy7 / Cy3), styryl dyes (e.g., Dye-28), coumarins (e.g., Alexa Fluor 350), porphines (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™ 700DX), and conjugates and combinations of these classes of dyes.

[0083] Preferred radioactive / fluorescent emitting isotopes are alpha emitting isotopes, gamma emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, e.g. 65 Tb, fluorescent emitting isotopes, e.g. 18 F, 51 Cr, 67 Ga, 68 Ga, 89 Zr, 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, 67 Cu, 188 Re, 186 Re, 198 Au, and 199 Ags, as well as conjugates and combinations of the above with proteins, peptides, small molecule inhibitors, antibodies, or other compounds (e.g., 18 F-FDG, 89 Zr oxide, or 64 Cu-porphyrin).

[0084] Preferred detectable polypeptides are autofluorescent proteins, preferably green fluorescent proteins, or adsorption (absorption: a b and any structural variants thereof with altered sorption and / or emission spectra.

[0085] Preferred contrast agents are selected from paramagnetic agents such as Gd, Eu, W, and Mn, preferably complexed with a chelating agent. Further options are superparamagnetic iron (Fe) complexes and particles, compounds containing atoms with high atomic numbers, i.e., iodine for computed tomography (CT), microbubbles, and carriers such as liposomes containing these contrast agents. In a preferred embodiment, the label comprises a chelating agent that complexes with divalent or trivalent metal cations.

[0086] Preferred chelating agents are selected from the group consisting of 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-hydrazinopyridine-3-carboxylic acid (HYNIC).

[0087] white blood cells The ability of a given cell or population of cells to internalize ferritin depends on the expression of receptors involved in this internalization process. Receptors that mediate ferritin internalization include, for example, TfR, CXCR4, scavenger receptors, CD163, and TIM-2. Those skilled in the art are well aware of methods for measuring the amount of ferritin uptake, and preferred methods for measuring uptake are described in the Examples section below.

[0088] The term "leukocyte" (or "white blood cell") is used in the context of this invention to refer to cells of the immune system involved in protecting the body from both infectious diseases and foreign invaders. All white blood cells are produced and derived from pluripotent cells in the bone marrow known as hematopoietic stem cells. White blood cells are found throughout the body, including the blood and lymphatic systems. All white blood cells have a nucleus, distinguishing them from other blood cells, anucleated red blood cells (RBCs) and platelets. White blood cell types can be classified in standard ways. Two sets of broadest categories classify them by structure (granulocytes or agranulocytes) or by cell division lineage (myeloid or lymphoid cells). These broadest categories can be further divided into five major types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils exhibit phagocytosis. Further subtypes can be classified, for example, lymphocytes include B cells, T cells, and NK cells. Granulocytes are distinguished from agranulocytes by their nuclear shape (lobulated vs. round, i.e., polymorphonuclear vs. mononuclear) and their cytoplasmic granules (presence or absence, or more precisely, visible or not visible by light microscopy). Another dichotomy is by lineage. Myeloid cells (neutrophils, monocytes, eosinophils, and basophils) are distinguished from lymphoid cells (lymphocytes) by their hematopoietic lineage (cell differentiation lineage).

[0089] CD45 expression is a characteristic of subgroups of leukocyte cells, namely monocytes, monocyte-macrophages, lymphocytes, granulocytes, and NK cells, and in particular, CD45 + White blood cells are suitable for use in connection with the targeted delivery system of the present invention because they are attracted to specific tissues and cells within the body and can deliver one or more iron-binding proteins and one or more pharmaceutically active agents, labels, or complexes of pharmaceutically active agents and labels to or into the cells. This subgroup of white blood cells is hereinafter referred to as "CD45 + White blood cells" or "CD45 +Preferably, the monocytes are not dendritic cells, the differentiation of which is controlled by one or more of the following transcription factors: IFN regulatory factor 8 (IRF8), nuclear factor interleukin (IL) 3-regulatory protein (NFIL3), basic leucine zipper transcription factor ATF-like 3 (BATF3) or transcription factor RelB (NF-KB subunit)-RELB, Spi-1 proto-oncogene (PU / 1), recombining binding protein suppressor of hairless (RBPJ), IFN regulatory factor 4 (IRF4), or transcription factor E2-2 (also known as TCF4).

[0090] CD45 as defined above unless of clonal origin + It will be understood by those skilled in the art that white blood cells are a mixed population of various white blood cells that share the common property of expressing the CD45 surface antigen. + Subpopulations of cells within the diverse group of white blood cells are characterized throughout this specification by additional functional and / or structural characteristics. + The term "expresses the CD45 surface antigen" indicates that the majority or essentially all of the cells in a population of cells express the CD45 surface antigen.

[0091] "Expression" in this context means that the majority or essentially all of the cells in a population of cells express the marker (also referred to herein as a surface antigen). In this context, as with other cell surface antigens, the term "express" indicates that the surface antigen is produced within the cell and detectably exposed on the surface of the cell. The level of expression, and therefore the number of surface antigens detectably exposed on the surface of a cell, can vary greatly from cell to cell. Generally, a cell is considered positive for a cell surface antigen if at least 5, and preferably at least 10, copies of the surface antigen are detectably exposed on the surface of the cell, i.e., " +". Those skilled in the art are well aware of methods for detecting, quantifying and selecting cells that are positive (or negative) for a given cell surface antigen. A preferred method involves fluorescence activated cell sorting (FACS). In this technique, fluorescently labeled antibodies are used to bind to cell surface antigens on a population of cells, which are then separated into single cells that are characterized as positive or negative for the given cell surface antigen based on the fluorescence intensity measured on the single cells. In some embodiments of the invention, high or low expression of a given protein is indicated. This means that the protein is detectably expressed in both cases, albeit at different levels, i.e., " + " means that the expression level is high. High expression and low expression each refer to different absolute numbers of proteins per cell for different proteins. Thus, a given protein can be considered to be expressed at a high level when there are more than 500 detectable copies of that protein per cell, and at a low level when there are 1-50 detectable copies of that protein per cell. However, another protein can be considered to be expressed at a high level when there are more than 5000 detectable copies per cell, and at a low level when there are 1-500 detectable copies per cell. Methods are known in the art for quantifying the number of proteins expressed or produced in cells using flow cytometry and Becton Dickinson Quantibrite™ bead technology (see, e.g., Pannu, KK, 2001, Cytometry. 2001 Dec 1;45(4):250-8) or mass spectrometry (see, e.g., Milo, R., 2013, Bioessays, 35(12): 1050-1055).

[0092] For the purposes of the present invention, the term "high expression" of a given protein refers to expression of a protein that is highly expressed in healthy cells, particularly CD45 +The term "low expression" of a given protein refers to detectable expression of that protein that is at least 70% of the highest expression level found in a population of leukocytes, i.e., the number of copies per cell. + The highest expression level seen in a population of leukocytes, i.e., detectable expression of the protein that is 30% or less of the copy number of the protein per cell, is referred to. Preferably, the "highest expression level" refers to the highest expression level seen in a population of leukocytes, i.e., detectable expression of the protein that is 30% or less of the copy number of the protein per cell. Preferably, the "highest expression level" refers to the highest expression level seen in healthy cells of various subjects, particularly CD45 + The expression level is determined as the average of the highest expression levels found in white blood cells. In some embodiments, a preferred subpopulation of cells is characterized as "producing" a given protein. This is understood to mean that the protein may not necessarily be detectable on the surface of the cell, but may only be present intracellularly. Those skilled in the art are well aware of how to detect and / or quantify the production of a protein within a cell and / or how to select cells that produce such a protein. Alternatively, a cell population can be defined by the expression of a specific transcription factor. Methods for determining the expression of a given protein or its encoding mRNA in a population of cells or even in a single cell are known in the art, for example by in vivo labeling with antibodies, FISH assays, in vivo single molecule fluorescence microscopy (Crawford, R. et al. Biophys J. (2013) 105(11): 2439) alone or in combination with fluorescence activated cell sorting (FACS), or PrimeFlow technology (e Bioscience), (Adam S. Venable et. al., (2015) Methods in Molecular Biology).

[0093] The term "differentiated monocytes" is used in the context of the present invention to refer to monocytes differentiated from committed precursors called macrophage-DC precursors (MDPs), which reside primarily in the bone marrow (but can also be present in the spleen) and differentiate into either dendritic cells or macrophages. In mice, they are divided into two main subpopulations: (i) high expression of CX3CR1, CCR2, and Ly6C - CD11b with low expression + cells, and (ii) low expression of CX3CR1, CCR2, and Ly6C + High expression of CD11b + After leaving the bone marrow, the cells + Monocytes express Ly6C in circulation. - Similarly, in human monocyte differentiation, CD14 ++ Classical monocytes leave the bone marrow and express CD14 in the peripheral blood circulation. ++ CD16 + intermediate monocytes, sequentially CD14 + CD16 ++ Differentiation into non-classical monocytes has been observed (Yang et al. 2014; Biomark Res 2(1) doi. 10.1186 / 2050-7771-2-1). Preferably, the differentiated monocytes are not dendritic cells, the differentiation of which is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IIRF4 (IRF4), and / or TCF4, and more preferably are not dendritic cells.

[0094] Macrophages are tissue-resident professional phagocytes and antigen-presenting cells (APCs) that differentiate from circulating peripheral blood monocytes (PBMs). The term "activated macrophages" is used in the context of the present invention to refer to any polarized macrophage. Macrophage activation is generally achieved by incubation 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, classically activated macrophages (CAMs), and M2-macrophages and alternatively activated macrophages (AAMs). Classically activated M1-macrophages include immune effector cells with an acute inflammatory phenotype. They are highly aggressive against bacteria and produce large amounts of lymphokines (Murray and Wynn, 2011, J LeukocBiol, 89(4):557-63). Alternatively activated, anti-inflammatory M2-macrophages can be divided into at least three subgroups. These subtypes have a variety of distinct functions, including immune regulation, maintenance of tolerance, and tissue repair / wound healing. The term "M1 inducer" is used in the context of the present invention to refer to compounds that direct the differentiation of PBMs into M1-type macrophages. The term "M2 inducer" is used in the context of the present invention to refer to compounds that direct the differentiation of PBMs into M2-type macrophages. Those skilled in the art are aware of numerous methods for promoting differentiation into either M1 or M2 macrophages. The term "macrophage phagocytosis" refers to the process by which macrophages engulf solid particles to form internal vesicles known as phagosomes. The phrase "viral / bacterial / fungal / helminthic protein or product" refers to molecules produced by or derived from viruses, bacteria, fungi, or helminths during viral / bacterial / fungal / helminthic infections.

[0095] CD45 included in targeted delivery systems for use according to the present invention + White blood cells express CD34 + It can be produced from hematopoietic progenitor cells.

[0096] In a preferred embodiment, CD45 + The leukocytes are selected from the group consisting of monocytes, differentiated monocytes, preferably macrophages, lymphocytes, and granulocytes. + Preferably, the leukocytes are macrophages, preferably activated macrophages.

[0097] Regarding monocytes, monocytes express CD11b + Monocytes, preferably CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CD14 + CD16 + Monocytes, CD11b + CD14 + HLA-DR monocytes, CD11b + CD14 + CD115 + Monocytes, CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CCR1 + Monocytes, CD11b + CCR2 + Monocytes, CD11b + CX3CR + Monocytes, CD11b + CXR4 + Monocytes, CD11b + CXR6 + Monocytes and CD11b + CD14 + CD33 + Preferably, they are selected from the group consisting of monocytes.

[0098] For differentiated monocytes, macrophages, activated macrophages, preferably CD11b + Macrophages, more preferably CD11b + CD16 + Macrophage, CD11b + CD32 +Macrophage, CD11b + CD64 + Macrophage, CD11b + CD68 + Macrophages, preferably CD11b + CD86 + M1 macrophages, preferably those that produce iNOS and / or secrete interleukin 12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + HLA-DR + M2 macrophages, CD11b + CD200R + M2 macrophages, CD11b + CD163 + M2 macrophages or activated macrophages that produce arginase and / or secrete interleukin 10 (IL-10), and dendritic cells (DCs), preferably CD11b + CD11c + DC, CD11b + CD80 + DC, CD11c + CD80 + DC, CD11c + CD86 + DC, CD11c + HLA-DR + DC or CD11c + CD123 + Preferably, the differentiated monocyte-macrophage is selected from the group consisting of Lox1 + Foam cells, CXCR7 + Foam cells and NRF2 + Not foam cells.

[0099] In some embodiments, the macrophages are undifferentiated macrophages. In some embodiments, the macrophages are naive macrophages. In some embodiments, the macrophages are M0 macrophages. In preferred embodiments, the macrophages are mildly polarized toward M2. In some embodiments, the macrophages are M2 macrophages. Those skilled in the art are aware of surface markers expressed by M2 macrophages or macrophages mildly polarized toward M2.

[0100] Preferably, the differentiated monocytes express at least one chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CXCR4, and CXCR6, or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony-stimulating factor receptor (CD115), granulocyte colony-stimulating factor receptor (CD114), and granulocyte-macrophage colony-stimulating factor receptor (CD116 and CD131).

[0101] In a preferred embodiment, the monocytes or differentiated monocytes are (i) CD34 + It can be produced from hematopoietic progenitor cells, (ii) producible by incubating monocytes in vitro with at least one inducer, preferably an M1 inducer or an M2 inducer, more preferably at least one M2 inducer; (iii) characterized by the expression of at least one of the following antigens: TfR, CD163, CD14, CD16, CD33, CXCR4, 25f9, HLA-DR, and / or CD115, and optionally CD172a and / or CXCR4, in particular at least one of TfR, CD163, CD14, CD16, CD33, 25f9, CD172a, and / or CD115, or at least one of TfR, CD163, CD14, CD16, CXCR4, 25f9, and / or CXCR1; and / or (iv) have the ability to phagocytose;

[0102] Preferably, (i) the M1-inducing factor is selected from the group consisting of LPS, GM-CSF, INF-γ, a viral or bacterial protein or product; (ii) The M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, an immune complex of an antigen and an antibody, IgG, heat-activated gamma globulin, glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, a PPARγ agonist, leukocyte inhibitory factor, cancer culture supernatant, cancer cells, adenosine, and a protein or product of a helminth or a fungus.

[0103] In a preferred embodiment, the activated macrophages are (i) a factor capable of changing an expression marker on macrophages, preferably (a) at least one M1-inducing factor; (b) at least one M2 inducer, or (c) factors capable of altering the ability of macrophages to secrete cytokines, preferably IL-10 and IL-12, chemokines, and / or to produce iNOS, arginase, or other immunomodulatory enzymes; can be produced by incubating monocytes or macrophages in vitro with (ii) characterized by expression of at least one of the following antigens: CD64, CD86, CD16, CD32, HLA-DR, and / or production of iNOS and / or IL-12; (iii) can be produced by incubating monocytes or macrophages in vitro with a factor capable of inducing the ability of macrophages to phagocytose; (iv) characterized by expression of at least one of the following antigens: CD204, CD206, CD200R, CCR2, transferrin receptor (TfR), C-X-C motif chemokine receptor 4 (CXCR4), CD163, and / or exhibiting low expression of HLA-DR; (v) having the ability to phagocytose; and / or (vi) capable of secreting cytokines, preferably IL-12 or IL-10, or producing inducible nitric oxide synthase (iNOS), a pro-inflammatory compound, an arginase immunosuppressant compound, or an anti-inflammatory compound.

[0104] Preferably, (i) the M1 inducer is selected from the group consisting of LPS, INF-γ, GM-CSF, and a viral or bacterial protein or product; or (ii) The M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, an immune complex of an antigen and an antibody, IgG, heat-activated gamma globulin, glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, a PPARγ agonist, leukocyte inhibitory factor, adenosine, a helminth or fungal protein or product.

[0105] Preferably, the differentiated monocytes, preferably macrophages, are characterized by expressing at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven, or all of TfR, CD163, CD14, CD16, CD33, 25f9, CD172a, and / or CD115, or at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven, or all of TfR, CD163, CD14, CD16, CXCR4, 25f9, and / or CXCR1.

[0106] For lymphocytes, CD3 + and CD4 + or CD8 + T lymphocytes, or CD19 + , CD20 + , CD21 + , CD19 + CD20 + , CD19 + CD21+ , CD20 + CD21 + , or CD19 + CD20 + CD21 + Preferably, the antigen-binding protein is selected from the group consisting of B lymphocytes, and natural killer (NK) cells.

[0107] In a preferred embodiment of the targeted delivery system of the present invention, the lymphocytes (i) CD34, which can be obtained from blood, spleen, or bone marrow, or as known to those skilled in the art and also described, for example, in Lefort 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. + can be produced from precursor cells, (ii) are immunocompetent lymphocytes; (iii) expresses an antigen-specific T cell receptor, and / or (iv) characterized by 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 antigens, and preferably capable of producing immunoglobulins.

[0108] In a particularly preferred embodiment, CD45 + The lymphocytes are NK cells, (i) is obtainable from blood, spleen, or bone marrow, or is CD34 + producible from progenitor cells; and / or (ii) lack of CD3 expression and less than CD56 + and / or CD94 + , CD158a + CD158f +CD314 + CD335 + The gene is characterized by the expression of at least one of the following:

[0109] For granulocytes, neutrophils, preferably CD66b + Neutrophils, eosinophils, and basophils, preferably CD193 + Preferably, they are selected from the group consisting of eosinophils.

[0110] In a preferred embodiment of the targeted delivery system of the present invention, the granulocytes are (i) CD34, which can be obtained from blood, spleen, or bone marrow, or as described, for example, in Kuhs et al. 2015, CurrProtocImmunol 111:7.23-1-7.23.16; Coquery et al. 2012, Cytometry A 81(9): 806-814; Swemydas and Lionakis 2013, J Vis Exp 77: 50586. + can be produced from precursor cells, (ii) characterized by expression of at least one of CD66b and / or CD193: (iii) polymorphonuclear leukocytes characterized by the presence of granules in their cytoplasm; and / or (iii)(iV) characterized by expression of at least one of the following: TfR, CD163, TIM-2, and / or CXCR4.

[0111] A targeted delivery system for use in the present invention will still provide the outlined advantages even if not all cells in a population of cells have a particular characteristic, as long as a majority of the cells in the population have that characteristic. Accordingly, the following describes certain preferred cellular characteristics of a targeted delivery system for use in the present invention.

[0112] In a preferred embodiment, the CD45 is included in the targeted delivery system. + The white blood cells, preferably macrophages, are derived from isolated peripheral blood mononuclear cells (PBMCs). Preferably, the targeted delivery system comprises CD45+ The white blood cells, preferably macrophages, are primary cells, i.e., cells isolated directly from human tissue, particularly peripheral blood. + The white blood cells, preferably macrophages, are preferably not cells of an immortalized cell lineage.

[0113] In some embodiments, the CD45 included in the targeted delivery system + The white blood cells are derived from the patient to be treated. In such cases, the cells loaded with the complexes will be autologous to the patient. It is also envisioned that the patient will be HLA-typed before treatment with the targeted delivery system of the present invention, so that the cell type used for a given patient is HLA-matched to that patient. In these two preferred embodiments, the cells are primary cells or are derived from primary cells by a few differentiation steps. Alternatively, the cells may be derived from an immortalized, but preferably untransformed, cell line.

[0114] CD45 + The blood used for the separation of white blood cells is preferably obtained from the patient to be treated or from a healthy donor. Alternatively, the blood can be obtained from a blood bank. The use of umbilical cord blood is also contemplated herein.

[0115] CD45 + Methods for loading white blood cells with a complex of an iron-binding protein and an active ingredient are described in WO 2016 / 207257, WO 2016 / 207256, and WO 2017 / 222398.

[0116] Mechanism The present invention provides a drug / prodrug-linked iron-binding protein-loaded CD45 antibody as a delivery system for targeting glioma. +The CD45 receptor of the targeted delivery system of the present invention for use in the treatment of gliomas is utilized. The poor response of gliomas to chemotherapy or the difficulty of detecting gliomas using imaging methods is mainly related to the reduced penetration of anti-cancer drugs into tumors due to the blood-brain barrier. However, the CD45 receptor of the targeted delivery system of the present invention for use in the treatment of gliomas is utilized. + Leukocytes, preferably activated macrophages, can cross the blood-brain barrier and migrate to the area of ​​glioma. In the brain, increased interstitial fluid pressure within the tumor and the blood-tumor barrier prevent the penetration of therapeutic agents into glioma. The CD45 targeting delivery system of the present invention for use in the treatment of glioma + Leukocytes, preferably activated macrophages, can reach deeper into tumors. When administered locally, the targeted delivery system according to the present invention ensures better drug distribution within tumor tissue compared to local administration of drugs or drugs in complex with iron-binding proteins. In summary, the targeted delivery system according to the present invention constitutes an effective delivery system for active ingredients throughout the glioma tumor mass.

[0117] The inventors have demonstrated that the isolated targeted delivery system for use according to the present invention, when administered intravenously or intratumorally to animals, induces the release of loaded CD45 + We observed that leukocytes, preferably activated macrophages, migrate to the glioma site and release complexes of iron-binding proteins and active ingredient(s) into cancer cells (Figures 3, 4, and 8). The present inventors have demonstrated that cell-to-cell contact is necessary for efficient transfer of complexes, particularly conjugates, comprising ferritin and an active agent from macrophages to cancer cells, and that secretion of complexes, particularly conjugates, comprising ferritin and an active agent by macrophages and subsequent uptake by cancer cells is not sufficient to ensure efficient delivery (Figures 5 and 6). This is because the transfer of the targeted delivery system of the present invention is a direct cell-to-cell transfer, and therefore CD45 +It has been shown that cell-to-cell contact or at least very close proximity between leukocytes (macrophages) and cancer cells is required. This direct delivery has the advantage that the active agent is specifically delivered to the target cells without increasing the extracellular concentration in the brain outside the tumor cells, thereby increasing efficacy and reducing side effects. Therefore, the method of the present invention advantageously allows for precise administration of the active ingredient(s) to the glioma site, particularly to glioma cells.

[0118] Combination therapy In some embodiments of the isolated targeted delivery system for use according to the present invention, treating or diagnosing a glioma comprises irradiating the glioma followed by administering the isolated targeted delivery system.

[0119] The present inventors found that a combination of treatments involving irradiation of gliomas followed by administration of a targeted delivery system further extended the survival time of mice in an aggressive glioma model (Example 3, Tables 5 and 6). Injection of macrophages containing ferritin-drug conjugates after irradiation significantly extended survival time and was much more effective than injection of ferritin-drug conjugates alone (Example 3, Table 6).

[0120] The beneficial effects of combining irradiation with subsequent administration of a targeted delivery system are not limited to gliomas, but also apply to other cancers. Thus, in a further aspect, the present invention provides a CD45 antibody containing intracellularly one or more iron-binding proteins and a complex of a pharmaceutically active substance, a label, or a combination of a pharmaceutically active substance and a label for use in a method for treating or diagnosing cancer. + The present invention provides an isolated targeted delivery system comprising white blood cells, wherein the treatment or diagnosis of glioma comprises administering the isolated targeted delivery system after irradiating the glioma. + All embodiments relating to white blood cells, complex formation, iron-binding proteins, pharmaceutically active agents and labels also apply to aspects relating to the treatment or diagnosis of cancer.

[0121] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition for the treatment or diagnosis of glioma, comprising an isolated targeted delivery system as described in relation to the first aspect of the invention and a pharmaceutically acceptable carrier and / or suitable excipient(s). All embodiments described in relation to the first aspect of the invention also apply to the pharmaceutical composition.

[0122] Because the isolated targeted delivery system comprises living cells, carriers and excipients are preferably selected to keep the cells viable.

[0123] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans.

[0124] The term "carrier," as used herein, refers to a pharmacologically inert substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer, or vehicle, administered with a pharmaceutically active substance. Such pharmaceutical carriers can be liquid or solid. Liquid carriers include, but are not limited to, sterile liquids such as saline solution and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0125] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0126] "Surfactants" include, but are not limited to, anionic, cationic, and nonionic surfactants such as sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, and polysorbates, e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

[0127] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists.

[0128] "Physiological buffers" include, but are not limited to, sodium chloride solution, demineralized water, and suitable organic or inorganic buffers, such as, but not limited to, phosphate buffer, citrate buffer, Tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazino]ethanesulfonic acid) or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of the respective buffer generally depends on the desired buffer molarity. Phosphate buffers are suitable, for example, for injection and infusion solutions.

[0129] Treatment method In another aspect, the present invention provides a method for treating or diagnosing glioma, comprising administering to a patient in need thereof an effective amount of an isolated targeted delivery system as described in relation to the first aspect of the invention. All embodiments described in relation to the first aspect of the invention also apply to the method of treatment or diagnosis. [Example]

[0130] Example 1 - BMDM-Ft-vcMMAE Treatment of Murine Glioma GL261 Tumors Intratumoral and intravenous injection of bone marrow-derived macrophages (BMDMs) containing ferritin-drug conjugates (BMDM-Ft-vcMMAE) was performed in a murine GL261 glioma model.

[0131] The ferritin-drug conjugate Ft-vcMMAE was generated by covalently linking the anticancer drug monomethylauristatin E (MMAE) to ferritin using a maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl (mc-vc-PAB) linker. Maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl-monomethylauristatin E (vcMMAE) was obtained from MedChem Express (Princeton, NJ). The conjugate was prepared as follows: A human ferritin solution was adjusted to a concentration of 120 μM using reaction buffer (50 mM phosphate buffer (pH 6.8) containing 0.1 mM EDTA) and conjugated with a 10-fold molar excess of vcMMAE in the presence of 20% (vol / vol) acetonitrile solution at 4°C overnight. The maleimide group reacts efficiently and specifically with free (reduced) sulfhydryls at pH 6.5-7.5 to form stable thioether bonds. Excess vcMMAE was purified using a PM 100 ultrafiltration concentrator and buffer-exchanged with D-PBS. The conjugation yield was approximately 80% of the total cysteine. The formation of the Ft-vcMMAE conjugate was confirmed by LC-MS analysis and titration of residual free thiol groups with p-chloromercuribenzoate. The concentration of the Ft-vcMMAE conjugate was determined by UV-vis spectroscopy.

[0132] Ft-vcMMAE was loaded into macrophages by incubating them in Ft-vcMMAE solutions with concentrations of 0.5 mg / ml to 0.75 mg / ml for 1 to 4 hours under standard culture conditions.

[0133] PBS, macrophages without ferritin-drug conjugates, and ferritin-drug conjugates without macrophages were used as controls for the injections. The intravenous treatment group received four doses of 5 million macrophages containing ferritin-drug conjugates. The intratumoral treatment group received only two doses of 2 million macrophages loaded with ferritin-drug conjugates. As seen in Tables 1 and 2 below and Figures 1A and 1B, injection of macrophages containing ferritin-drug conjugates significantly extended survival after tumor implantation and was much more effective than injection of ferritin-drug conjugates or macrophages alone.

[0134] Table 1 - Intratumoral treatment of GL-261 glioma-bearing mice with BMDM-HFt-vcMMAE [Table 1-1]

[0135] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 1-2]

[0136] Table 2 - Intravenous treatment of GL-261 glioma-bearing mice with BMDM-HFt-vcMMAE [Table 2-1]

[0137] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 2-2]

[0138] Example 2 - BMDM-Ft-vcMMAE Treatment of Murine Glioma CT-2A Tumors Intratumoral and intravenous injection of macrophages containing ferritin-drug conjugates (BMDM-Ft-vcMMAE) in a mouse CT-2A glioma model was performed as described in Example 1. As seen in Tables 3 and 4 below, injection of macrophages containing ferritin-drug conjugates significantly extended survival time after tumor implantation and was much more effective than injection of ferritin-drug conjugates alone or macrophages alone.

[0139] Table 3 - Intratumoral treatment of CT-2A glioma-bearing mice with BMDM-HFt-vcMMAE [Table 3-1]

[0140] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 3-2]

[0141] Table 4 - Intravenous treatment of CT-2A glioma-bearing mice with BMDM-HFt-vcMMAE [Table 4-1]

[0142] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 4-2]

[0143] Example 3 - Radiation promotes tumor infiltration by BMDM-Ft-vcMMAE In a mouse GL261 glioma model, low-dose irradiation (2 Gy of gamma rays applied on three consecutive days before the first of two administrations of the targeted delivery system: 2 Gy three times) or higher-dose irradiation (4 Gy of gamma rays applied on the day before each of two administrations of the targeted delivery system: 4 Gy two times) was performed (IR treatment). Macrophages were then intravenously administered. Macrophages were differentiated from the bone marrow of a donor mouse strain carrying the CD45.1 variant of the CD45 marker. In experiments involving in vivo imaging, macrophages were stained with the membrane dye CellBrite™ NIR790, a far-red fluorescent marker.

[0144] After IR treatment combined with macrophage injection, brains were harvested, enzymatically dissociated, stained, and subjected to flow cytometry analysis. The injected macrophages can be distinguished from endogenous CD45 cells using anti-CD45.1 antibodies.

[0145] We show that after radiation, the number of CD45.1 cells (intravenously administered macrophages) increases within the tumor mass (Fig. 2B), while IR treatment leads to a reduction in tumor cells (Fig. 2A).

[0146] Example 4 - Low-dose radiation promotes tumor invasion and improves the efficacy of BMDM-Ft-vcMMAE intravenous treatment against GL261 glioma In a mouse GL261 glioma model, low-dose radiation (2 Gy of gamma radiation administered on three consecutive days prior to the first of two doses of the targeted delivery system: three doses of 2 Gy) was administered, followed by intravenous administration of macrophages containing a ferritin-drug conjugate (BMDM-Ft-vcMMAE).

[0147] As can be seen in Tables 5 and 6 below, treatment with macrophages containing ferritin-drug conjugates after radiation further extends survival. Without wishing to be bound by theory, the inventors speculate that radiation increases inflammation within the tumor, which attracts macrophages containing ferritin-drug conjugates (BMDM-Ft-vcMMAE), which infiltrate the tumor more than in the absence of radiation.

[0148] Furthermore, in the irradiated group (Table 6), injection of macrophages containing ferritin-drug conjugates is much more effective than injection of ferritin-drug conjugates alone or macrophages alone.

[0149] Table 5 - Intravenous treatment of GL-261 glioma-bearing mice with BMDM-Ft-vcMMAE [Table 5-1]

[0150] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 5-2]

[0151] Table 6 - Intravenous treatment of GL-261 glioma-bearing mice with BMDM-Ft-vcMMAE in combination with low-dose radiation (radiotherapy) [Table 6-1]

[0152] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 6-2]

[0153] Example 5 - Relative infiltration of BMDM into diseased brains Healthy and glioma-bearing mice were intravenously injected with macrophages and stained with the membrane dye CellBrite™ NIR790, a far-red fluorescent marker. Brain infiltration was examined by determining in vivo far-red fluorescence in the brain compared to the rest of the body. Brain infiltration was increased in glioma-bearing mice (Figure 3).

[0154] Example 6 - Penetration of BMDM-Ft into glioma Intratumoral injection of ferritin-Alexa Fluor 488 conjugate (Ft-AF488) or macrophages containing the conjugate (BMDM-Ft-AF488) was performed in a mouse glioma model. Macrophages were differentiated from the bone marrow of donor mice bearing a CD45.1 mutant. 24 hours later, mice were sacrificed, and brain sections were analyzed. CD45.1 immunostaining was performed to visualize macrophages. Injection of BMDM-Ft-AF488 resulted in the localization of macrophage-ferritin conjugates in tumor tissue, but not in healthy brain (Figure 4). In contrast, injection of Ft-AF488 did not result in the localization of ferritin conjugates in tumor tissue. Some ferritin conjugates were also detectable in healthy brain.

[0155] Example 7 - HMDM-Ft-vcMMAE treatment of human glioma ZH-161 tumors In the ZH-161 glioma model, human ZH-161 tumor cells are injected into mice. The ZH-161 glioma model is known for its unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter, MGMT expression, and resistance to temozolomide (TMZ), as described in Le Rhun et al. (Int J Cancer. 2019 Jul 1;145(1):242-253.doi: 10.1002 / ijc.32069) and Silginer et al. (Cell Death Dis. 2017 Apr 20;8(4):e2753.doi: 10.1038 / cddis.2017.171). In the ZH-161 glioma model, macrophages containing a ferritin-drug conjugate (MDM-Ft-vcMMAE: B Intratumoral injection of ferritin-drug conjugates (MDM-Ft-vcMMAE) was performed as described in Example 1. As can be seen in Table 7 below and Figure 7, injection of macrophages containing ferritin-drug conjugates significantly extended survival time after tumor implantation and was much more effective than injection of ferritin-drug conjugates alone or macrophages alone.

[0156] [Table 7-1]

[0157] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg correction of p-values): [Table 7-2]

[0158] Example 8 - Biodistribution In a biodistribution study, we assessed the systemic distribution of macrophages containing ferritin-drug conjugates after intratumoral administration. Remarkably, these results demonstrated that the therapeutic agent was restricted to brain tissue (Figure 8). The administered dose remained largely localized in the brain, with only minor amounts, if any, detected in other major organs and tissues. Furthermore, the amount of drug detected in brain tissue remained fairly constant up to 72 hours after injection, highlighting the potential for sustained drug presence and prolonged therapeutic efficacy. This unique characteristic not only highlights the efficacy of targeted treatment of gliomas with macrophages containing ferritin-drug conjugates, but also suggests reduced potential for off-target side effects and systemic toxicity.

[0159] array SEQ ID NO:1 - Human ferritin heavy chain MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0160] SEQ ID NO:2 - Mouse ferritin heavy chain MTTASPSQVRQNYHQDAEAAINRQINLELYASYVYLSMSCYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDRDDWESGLNAMECALHLEKSVNQSLLELHKLATDKNDPHLCDFIETYYLSEQVKSIKELGDHVTNLRKMGAPEAGMAEYLFDKHTLGHGDES

[0161] SEQ ID NO: 3 - Mammalian ferritin consensus MTTASXSQVRQNYXQXSEAAXXRQINLELXASYVYLSMSXYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIXLXDIKKPDXDDWESGLNAMECALXLEKXVNQSLLELHKLATDKNDPHLCDFIETXYLXEQVKXIKELGDHVTNLRKMGAPEXGXAEYLFDKHTLGXSDXXX

[0162] Sequence number 4-FT_2 MTTASTSQVRENYHEDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAEYFLHQSHEEREHAEKLMELQNQRGGRIFLQDIQKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVEAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0163] SEQ ID NO: 5 - Alternative mammalian ferritin consensus MTTASXSQVRQNYXQXSEAAXXRQINLELXASYVYLSMSXYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIXLXDIKKPDXDDWESGLNAMECALXLEKXVNQSLLELHKLATDKNDPHLCDFIETXYLXEQVKXIKELGDHVTNLRKMGAPEXGXAEYLFDKHTLGXSDXXX [Explanation of symbols]

[0164] Drawing translation Figure 1 Survival [%] Survival rate [%] Days (post tumor implantation) Treatment Macrophages Ferritin-drug conjugate Macrophages comprising ferritin-drug conjugate

[0165] Figure 2 Total number of cancer cells Total number of CD45.1+ CD45.1 + Total number of Control

[0166] Figure 3 [% of total fluorescence] Healthy Glioma

[0167] Figure 4 Macrophage-HFt conjugates Free Ft anti-CD45.1 anti-CD45.1 Healthy brain Tumor

[0168] Figure 5 geo-MFI Geometric MFI Co-culture Co-culture

[0169] Figure 6 Plain medium Regular medium Conditioned medium of co-culture Culture supernatant of co-culture Conditioned medium of cancer cells Culture supernatant of cancer cells Co-culture Co-culture 4 hours 4 hours 24 hours 24 hours Alexa Fluor 488 fluorescence Alexa Fluor 488 fluorescence geo-MFI Geometric MFI AF488 positive [%] AF488 positive [%] Control

[0170] Figure 7 Survival probability (%) Time post glioma implantation [days] Treatment Macrophages Ferritin-drug conjugate Macrophages comprising ferritin-drug conjugate

[0171] Figure 8 MMAE [pg / mg wet tissue] MMAE [pg per mg wet tissue] Time post injection [h] Brain Blood blood Heart Liver Pancreas Lung Kidney Ovary Bone marrow Spleen

Claims

1. CD45 containing intracellularly one or more iron-binding proteins and a complex of a pharmaceutically active substance, a label, or a pharmaceutically active substance and a label for use in a method of treating or diagnosing glioma + An isolated targeted delivery system comprising white blood cells.

2. (i) the iron-binding protein and the pharmaceutically active substance or the label are linked by a covalent and / or non-covalent bond, and / or (ii) The isolated targeted delivery system for use according to claim 1, wherein the pharmaceutically active substance or the label is encapsulated by the iron-binding protein or a multimer thereof.

3. 2. The isolated targeted delivery system for use according to claim 1, wherein the iron-binding protein and the pharmaceutically active substance or the label are covalently linked.

4. 4. The isolated targeted delivery system for use according to claim 3, wherein the iron-binding protein and the pharmaceutically active substance or the label are covalently linked via a cleavable linker, preferably the cleavable linker is a peptide-based linker cleavable by a lysosomal protease, preferably a lysosomal cysteine ​​protease, more preferably cathepsin B, and most preferably the linker is a maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl (mc-vc-PAB) linker.

5. 5. The isolated targeted delivery system for use according to any one of claims 1 to 4, wherein the iron-binding protein is conjugated to the pharmaceutically active agent, the label or the linker via a cysteine ​​or lysine residue, preferably a cysteine ​​residue.

6. CD45 + The isolated targeted delivery system according to any one of claims 1 to 5, wherein the leukocytes are selected from the group consisting of monocytes, differentiated monocytes, preferably macrophages, lymphocytes and granulocytes.

7. (i) the monocytes are CD11b + Monocytes, preferably CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CD14 + CD16 + Monocytes, CD11b + CD14 + HLA-DR monocytes, CD11b + CD14 + CD115 + Monocytes, CD11b + CD14 + Monocytes, CD11b + CD16 + Monocytes, CD11b + CCR1 + Monocytes, CD11b + CCR2 + Monocytes, CD11b + CX3CR + Monocytes, CD11b + CXR4 + Monocytes, CD11b + CXR6 + Monocytes and CD11b + CD14 + CD33 + monocytes; (ii) the differentiated monocytes are macrophages, activated macrophages, preferably CD11b + Macrophages, more preferably CD11b + CD16 + Macrophage, CD11b + CD32 + Macrophage, CD11b + CD64 + Macrophage, CD11b + CD68 + Macrophages, preferably CD11b + CD86 + M1 macrophages, preferably those that produce iNOS and / or secrete interleukin 12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + HLA-DR + M2 macrophages, CD11b + CD200R + M2 macrophages, CD11b + CD163 + M2 macrophages or activated macrophages that produce arginase and / or secrete interleukin 10 (IL-10), and dendritic cells (DCs), preferably CD11b + CD11c + DC, CD11b + CD80 + DC, CD11c + CD80 + DC, CD11c + CD86 + DC, CD11c + HLA-DR + DC or CD11c + CD123 + DCs, and preferably, the differentiated monocyte-macrophages are selected from the group consisting of Lox1 + Foam cells, CXCR7 + Foam cells and NRF2 + Not foam cells (iii) the lymphocytes are CD3 + and CD4 + or CD8 + T lymphocytes, or CD19 + , CD20 + , CD21 + , CD19 + CD20 + , CD19 + CD21 + , CD20 + CD21 + , or CD19 + CD20 + CD21 + or selected from the group consisting of B lymphocytes, and natural killer (NK) cells; (iv) the granulocyte is a neutrophil, preferably a CD66b + Neutrophils, eosinophils, and basophils, preferably CD193 + 7. The isolated targeted delivery system for use according to claim 6, wherein the isolated targeted delivery system is selected from the group consisting of eosinophils.

8. The monocytes or the differentiated monocytes are (i) CD34 + It can be produced from hematopoietic progenitor cells, (ii) producible by incubating monocytes in vitro with at least one inducer, preferably an M1 inducer or an M2 inducer, more preferably at least one M2 inducer; (iii) characterized by expression of at least one of the following antigens: TfR, CD163, CD14, CD16, CD33, CXCR4, 25f9, HLA-DR, and / or CD115; and / or (iv) having the ability to phagocytose; Here, preferably, (i) the M1-inducing factor is selected from the group consisting of LPS, GM-CSF, INF-γ, a viral or bacterial protein or product; (ii) The isolated targeted delivery system for use according to any one of claims 6 to 9(7), wherein the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, an immune complex of an antigen and an antibody, IgG, heat-activated gamma globulin, glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, a PPARγ agonist, leukocyte inhibitory factor, cancer culture supernatant, cancer cells, adenosine, and a protein or product of a helminth or a fungus.

9. The activated macrophages are (i) a factor capable of changing an expression marker on macrophages, preferably (a) at least one M1-inducing factor; (b) at least one M2 inducer; or (c) factors capable of altering the ability of said macrophages to secrete cytokines, preferably IL-10 and IL-12, chemokines and / or to produce iNOS, arginase, or other immunomodulatory enzymes; can be produced by incubating monocytes or macrophages in vitro with (ii) characterized by expression of at least one of the following antigens: CD64, CD86, CD16, CD32, and HLA-DR, and / or production of iNOS and / or IL-12; (iii) can be produced by incubating monocytes or macrophages in vitro with a factor capable of inducing the ability of macrophages to phagocytose; (iv) characterized by expression of at least one of the following antigens: CD204, CD206, CD200R, CCR2, transferrin receptor (TfR), C-X-C motif chemokine receptor 4 (CXCR4), CD163, and / or exhibiting low expression of HLA-DR; (v) having the ability to phagocytose; and / or (vi) capable of secreting cytokines, preferably IL-12 or IL-10, or producing inducible nitric oxide synthase (iNOS), a pro-inflammatory compound, an arginase immunosuppressant compound, or an anti-inflammatory compound; Here, preferably, (i) the M1-inducing factor is selected from the group consisting of LPS, INF-γ, GM-CSF, and a viral or bacterial protein or product; or 8. The isolated targeted delivery system for use according to claim 7, wherein (ii) the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, an immune complex of an antigen and an antibody, IgG, heat-activated gamma globulin, glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, a PPARγ agonist, leukocyte inhibitory factor, adenosine, a helminth or fungal protein or product.

10. 10. The isolated targeted delivery system for use according to any one of claims 1 to 9, wherein the iron-binding protein is selected from the group consisting of ferritin, preferably heavy chain (H) ferritin, light chain (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, hemopexin, transferrin, and lactoferrin.

11. 11. The isolated targeted delivery system for use according to any one of claims 1 to 10, wherein the pharmaceutically active agent is an anti-cancer drug selected from the group consisting of proteins, peptides, nucleic acids, non-protein, non-nucleic acid compounds with a molecular weight of less than 1.5 kD, photosensitizing compounds, viruses, and pharmaceutically active radioisotopes.

12. The anticancer drug is Apoptosis inducers, alkylating agents, antimetabolites, antibiotics, antimitotics, DNA modifying agents, DNA minor groove interstrand crosslinking agents, inhibitors of DNA synthesis, inhibitors of RNA synthesis, epothilones, nuclear receptor agonists and nuclear receptor antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, inhibitors of cell cycle-dependent protein kinase (CDK), inhibitors of cyclooxygenase and / or lipoxygenase, biogenic fatty acids, biogenic fatty acid derivatives including prostanoids and leukotrienes, inhibitors of protein kinases, inhibitors of protein phosphatases antitumor agents, inhibitors of lipid kinases, platinum coordination complexes, ethyleneimines, methylmelamines, triazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkylsulfonates, folic acid analogs, anthracenediones, substituted ureas, and methylhydrazine derivatives, enediyne antibiotics, maytansinoids, auristatin derivatives, immune checkpoint inhibitors, and inhibitors of tumor-specific proteins or tumor-specific markers, preferably Rho-GDP dissociation inhibitors, more preferably Grp94 inhibitors or AXL inhibitors, tubulin inhibitors, or topoisomerase inhibitors; Acediasulfone, aclarubicin, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, vanoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, capecitabine, carmustine, celecoxib, calicheamicin, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, deruxtecan, dibromopropamidine, diethylstilbestrol, docetaxel, docetaxel Xorubicin, dolastatin 10, dolastatin 15, dynemicin A, enediyne, epirubicin, epothilone B, epothilone D, estramustine phosphate, estrogen, ethinyl estradiol, etoposide, exatecan derivatives, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fosfestrol, furazolidone, gemcitabine, gonadotropin-releasing hormone analogues, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxypropane Roguetone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon alpha, irinotecan, leuprolide, lomustine, lutecan, mafenide sulfate olamide, maytansine, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mepacrine, mercaptopurine, mertansine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, neocarzinostatin, niflatoxin ru, nifuroxazide, nifuralazine, nifurtimox, nimustine, nimorazole, nitrofurantoin, nitrogen mustard, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, promethazine, procarbazine, pyrimethamine, pyrrolobenzodiazepine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustine, streptozocin, sn-38,Sulfacarbamide, sulfacetamide, sulfachloropyridazine, sulfadiazine, sulfadiclamide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanol, sulfamethizole, sulfamethoxazole, cotrimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperine, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporine, tamoxifen, taxol, teniposide, tartiposide, te structone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triazicon, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinorelbine, and zorubicin, and preferably selected from the group consisting of auristatins, banoxantrone, bendamustine, chlorambucil, calicheamicin, dynemicin A, maytansine, melphalan, mertansine, neocarzinostatin, and pyrrolobenzodiazepines; an immunomodulatory agent that activates or inhibits the activity of immune cells, preferably said immunomodulatory agent being a ligand or antagonist of a pattern recognition receptor, in particular a Toll-like receptor, a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a stimulator of interferon genes (STING) protein; and / or 12. The isolated targeted delivery system for use according to claim 11, which is a growth-inhibitory protein or peptide, preferably a cell cycle inhibitor, or an antibody or antibody-like binding protein that specifically binds to a growth-promoting protein, or a nucleic acid, preferably a nucleic acid encoding a growth-inhibitory protein or an antibody or antibody-like binding protein that specifically binds to a growth-promoting protein, or an siRNA, oligonucleotide, LNA, or DNAzyme.

13. 12. The isolated targeted delivery system for use according to claim 11, wherein the anticancer drug is an auristatin, in particular monomethyl auristatin E or monomethyl auristatin F, or deruxtecan.

14. The pharmaceutically active agent is a hypoxia-activated prodrug, preferably selected from the group consisting of benzotriazine N-oxides, apaziquone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH-302, TH-4000, and AQ4N; or an antigen or a nucleic acid encoding the antigen; 11. The isolated targeted delivery system for use according to any one of claims 1 to 10, wherein

15. The label is selected from the group consisting of a fluorescent dye, a fluorescent-emitting isotope, a radioisotope, a detectable polypeptide or a nucleic acid encoding a detectable polypeptide, and an imaging agent, or the label comprises a chelator that complexes with a divalent or trivalent metal cation, wherein preferably the chelating agent is selected from the group consisting of 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-hydrazinopyridine-3-carboxylic acid (HYNIC); the contrast agent preferably comprises a paramagnetic agent selected from Gd, Eu, W, and Mn, or ferrihydrite; The radioactive isotope / the fluorescent emitting isotope may be an alpha ray emitting isotope, a gamma ray emitting isotope, an Auger electron emitting isotope, an X-ray emitting isotope, a fluorescent isotope, e.g. 65 Tb, fluorescent emitting isotopes, e.g. 18 F, 51 Cr, 67 Ga, 68 Ga, 89 Zr, 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, 67 Cu, 188 Re, 186 Re, 198 Au, and 199 and conjugates and combinations of the above with proteins, peptides, small molecule inhibitors, antibodies, or other compounds; the fluorescent dye is selected from the group consisting of the following classes of fluorescent dyes: xanthenes, acridines, oxazines, cyanines, styryl dyes, coumarins, porphines, metal-ligand complexes, fluorescent proteins, nanocrystals, perylenes, and phthalocyanines, as well as conjugates and combinations of these classes of dyes; and / or 15. The isolated targeted delivery system for use according to any one of claims 1 to 14, wherein said detectable polypeptide is an autofluorescent protein, preferably green fluorescent protein, or any structural variant thereof with altered adsorption and / or emission spectra.

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