Isolated targeted delivery system for treatment of glioma

Abstract

The present invention relates to an isolated targeted delivery system comprising CD45 + leukocytes comprising one or more than one iron binding protein and a pharmaceutically active substance, a marker or a complex of a pharmaceutically active substance and a marker, for use in methods of treatment or diagnosis of gliomas.

Description

[0001] The present invention relates to a method comprising + Targeted delivery system for separation of leukocytes, which is used in the treatment method or diagnosis method of glioma, wherein the CD45 + The leukocytes contain a complex of the iron-binding protein with the pharmaceutically active substance and / or the marker. Background Art

[0002] The prognosis of glioma patients is poor. For glioblastoma (GBM), whether or not treated, most patients can only survive for a few months, and the median survival period is 11 to 12 months. Only 3% to 8% of patients can survive for more than 3 years. These tumors are diffuse and infiltrative growths, and radical resection is usually not possible and is ineffective for radiotherapy. Blood-brain barrier inhibition includes most compounds of modern treatments (such as monoclonal antibodies, CAR-T) penetrating into the brain. It is challenging to safely and effectively deliver drugs locally to the brain. In addition, even if applied locally, the active ingredients often cannot reach the deep sites of gliomas and penetrate the entire tumor mass.

[0003] The inventors found that they contained CD45 + The targeted delivery system for the separation of leukocytes can specifically target glioma cells in the brain and exert a therapeutic effect. + The leukocytes contain a complex of the iron-binding protein and the active ingredient. In an animal model of glioblastoma, the inventors showed that administration of their isolated targeted delivery system containing an anticancer drug significantly prolonged survival.

[0004] The targeted delivery system of the present invention is used in the treatment or diagnosis method of glioma, and compared with the prior art, it has the following advantages, among others: (i) targeted delivery of active ingredients that usually cannot reach the brain to gliomas; (ii) targeted delivery of active ingredients to glioma cells, (iii) improved penetration of active ingredients into deep sites of glioma masses, (iv) improved distribution of active ingredients within glioma masses, (v) protection of active ingredients from inactivation in blood circulation or clearance from the body, (vi) delivery of active ingredients with poor pharmacokinetics to the brain, (vii) reduced toxicity of active ingredients due to direct targeted delivery, (viii) higher therapeutic effects with lower doses of active ingredients due to direct targeted delivery and local deposition in glioma cells; (ix) improved efficacy of systemic administration of active ingredients, thereby preventing the risk of tissue damage during local delivery to the brain; and / or (x) improved therapeutic effects of alkylating drugs and (xi) improved therapeutic effects of temozolomide (TMZ)-resistant tumors. Summary of the invention

[0005] The present invention relates to a method comprising+ A targeted delivery system for separation of leukocytes, which is used in a method for treating or diagnosing gliomas. The cells contain one or more iron-binding proteins and a pharmaceutically active substance, a marker, or a complex of a pharmaceutically active substance and a marker.

[0006] Specific implementation methods of the preferred embodiment

[0007] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methods, schemes and reagents described herein, because these methods, schemes and reagents can vary. It should also be understood that the terms used herein are only used to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, the meanings of all technical terms and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art.

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

[0009] The term "amino acid" encompasses naturally occurring amino acids and 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). The amino acid identified with the letter X corresponds to any amino acid. The amino acid identified with the letter B corresponds to D (asparagine) or N (aspartic acid). The amino acid identified with the letter Z corresponds to E (glutamine) or Q (glutamic acid).

[0010] In this article, the terms "polynucleotide" and "nucleic acid" are used interchangeably, and are understood to be polymeric macromolecules or oligomeric macromolecules composed of nucleotide monomers. Nucleotide monomers are composed of bases, pentose (including but not limited to ribose or 2'-deoxyribose) and one to three phosphate groups. Generally, polynucleotides are formed by phosphodiester bonds between single nucleotide monomers. In the context of the present invention, the nucleic acid molecules mentioned include but are not limited to ribonucleic acid (RNA) and its various forms (such as but not limited to ssRNA, LNA, etc.), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be synthesized by, for example, chemical methods, for example, according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). "Aptamers" are nucleic acids that bind to polypeptides with high affinity. Aptamers can be isolated from large libraries of different single-stranded RNA molecules by selection methods such as SELEmir146-a (see, for example, Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981). Aptamers can also be synthesized and selected in their mirror image form, for example as L-ribonucleotides (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The form isolated in this way has the advantage that it will not be degraded by naturally occurring ribonucleases and therefore has better stability.

[0011] The term "identity" is used throughout this specification for comparison of polypeptide sequences and nucleotide sequences. In the case of comparing two sequences and not specifying a reference sequence for calculating the percentage of sequence identity, if not otherwise specified, the longer of the two sequences to be compared should be used as a reference to calculate the sequence identity. If a reference sequence is specified, if not otherwise specified, the sequence identity will be determined based on the full length of the reference sequence indicated by the SEQ ID. For example, a polypeptide sequence consisting of 200 amino acids can present a maximum sequence identity percentage of 66.6% (200 / 300) compared to a reference polypeptide sequence of 300 amino acids in length, while a sequence of 150 amino acids in length can present a maximum sequence identity percentage of 50% (150 / 300). If 15 of these 150 amino acids are different from the corresponding amino acids in the reference sequence of 300 amino acids in length, the level of sequence identity is reduced to 45%. The similarity of nucleotide sequences and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. This alignment can be performed by several algorithms known in the art, preferably using the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or using the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), which can be obtained, for example, at http: / / www.ebi.ac.uk / Tools / clustalw / or at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or at 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 rank of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). BLAST protein searches are performed using the BLASTP program with score = 50 and word length = 3.To obtain gapped alignments for comparison purposes, Gapped BLAST was used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program were used. Sequence matching analysis can be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. Using information from sequence database searches, available homologs with 3D structures and user-defined constraints can also be used to compare multiple protein sequences and / or protein structures based on structure (Pei J, Grishin NV: PROMALS: toward accurate multiple sequence alignments of distantly related proteins. Bioinformatics 2007, 23: 802-808; 3DCoffee@igs: a webserver for combining sequences and structures into a multiple sequence alignment. Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul 1; 32: W37-40.). When referring to percentages of sequence identity in this application, if not otherwise specified, these percentages are calculated based on the full length of the longer sequence.

[0012] In the context of the present invention, the term "antibody" refers to a glycoprotein belonging to the immunoglobulin superfamily; the term antibody and the term immunoglobulin are often used interchangeably. Antibodies refer to protein molecules produced by plasma cells and are used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. Antibodies recognize unique parts of a foreign target, i.e., its antigens.

[0013] As used herein, the term "antibody fragment" refers to a fragment of one or more than one antibody that retains the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antibody fragment" include antigen binding fragments (Fab), Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAb), single chain variable region fragments (scFv), variable region fragments (Fv), VH domains, VL domains, single domain antibodies, nanobodies, IgNAR (immunoglobulin new antigen receptor), double scFv, bispecific T cell engager (BITE), dual affinity redirection (DART) molecules, three antibodies (triple body), double antibodies, single chain double antibodies, alternative scaffold proteins (alternative scaffold protein) and fusion proteins thereof.

[0014] The term "antigen" is used to refer to a substance, preferably an immunogenic peptide comprising at least one epitope, preferably said epitope is capable of eliciting a B cell response or a T cell response or both a B cell response and a T cell response.

[0015] An "epitope", also called an antigenic determinant, is a portion of a substance, such as a portion of an immunogenic polypeptide, that is recognized by the immune system. Preferably, this recognition is mediated by the binding of antibodies, B cells or T cells to the epitope in question. In this context, the term "binding" is preferably associated with specific binding. Epitopes usually consist of chemically active surface groups 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 conformational epitopes and non-conformational epitopes. The difference between conformational epitopes and non-conformational epitopes is that the binding to the former is lost in the presence of denaturing solvents, while the binding to the latter is not lost.

[0016] According to the present invention, the immunogenic polypeptide is preferably derived from a pathogen selected from the group consisting of viruses, bacteria and protozoa. However, in an alternative embodiment of the present invention, the immunogenic polypeptide is a tumor antigen, ie a polypeptide or polypeptide fragment specifically expressed by cancer.

[0017] Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether preceding or following, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to such disclosure in advance due to prior invention.

[0018] In practicing the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry and recombinant DNA techniques will be employed as explained in the literature in the field (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 claims that follow, unless the context requires otherwise, the words "comprises / comprising" and variations thereof shall be understood to mean the inclusion of a stated integer or step or group of integers or groups of steps, but not the exclusion of any other integer or step or group of integers or groups of steps. As used in this specification and the claims that follow, the word "includes" without a quantifier preceding a noun refers to a plurality unless the context clearly indicates otherwise.

[0020] The elements of the present invention will be described below. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any quantity to create other embodiments. The various embodiments and preferred embodiments described should not be construed as limiting the present invention to only the embodiments clearly described. This description should be understood to support and include those embodiments that combine the embodiments clearly described with any number of disclosed elements and / or preferred elements. In addition, unless the context otherwise indicates, it should be considered that the description of the present application discloses any arrangement and combination of all elements described in the present application.

[0021] Targeted delivery system

[0022] The present invention relates to a method comprising + A targeted delivery system for separation of leukocytes, which is used in a method for treating or diagnosing gliomas. The cells contain one or more iron-binding proteins and a pharmaceutically active substance, a marker, or a complex of a pharmaceutically active substance and a marker.

[0023] As used herein, the term "treatment" includes all types of preventive and / or therapeutic interventions medically permitted for purposes such as cure, temporary relief, prevention, etc., for example, for different purposes including delaying or preventing disease progression, causing lesions to regress or disappear, preventing onset or inhibiting recurrence. In the case where the provided separation targeted delivery system is used for a method for treating glioma, it contains a pharmaceutically active substance. In the case where the provided separation targeted delivery system is used for a method for diagnosing glioma, it contains a marker. The diagnostic method is an in vivo diagnostic method. In a preferred embodiment, the provided separation targeted delivery system is used for a method for treating glioma.

[0024] The term "glioma" refers to a cancer of the glial cells surrounding the nerve endings of the brain. In a preferred embodiment, the glioma is a glioblastoma. A glioma or glioblastoma may be a primary tumor or a secondary tumor.

[0025] In some embodiments, the glioma is resistant to chemotherapy. In some embodiments, the glioma is resistant to chemotherapy using an alkylating agent. In some embodiments, the glioma is a temozolomide (TMZ)-resistant glioma. In some embodiments, the glioma expresses O-6-methylguanine-DNA methyltransferase (MGMT).

[0026] The therapeutic effect of TMZ depends on its ability to alkylate / methylate DNA, most often at the N-7 or O-6 position of guanine residues. However, some tumor cells are able to repair such DNA damage by synthesizing a protein encoded by the O-6-methylguanine-DNA methyltransferase (MGMT) gene, thereby attenuating the efficacy of TMZ.

[0027] Surprisingly, the inventors have demonstrated that treatment with the isolated targeted delivery system of the present invention, particularly comprising a ferritin-drug conjugate, more particularly comprising a ferritin-MMAE conjugate, and even more particularly comprising HFt-vcMMAE, can significantly prolong the survival of TMZ-resistant gliomas in in vivo models.

[0028] 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.

[0029] In some embodiments, the isolated targeted delivery system is used in a method of treating glioma in a human individual who is experiencing disease progression or recurrence during or after treatment with an alkylating agent, particularly TMZ.

[0030] In the context of this specification, the term "active ingredient" is used to refer to at least one pharmaceutically active substance and / or at least one marker. Preferably, the active ingredient is a pharmaceutically active substance.

[0031] The term "targeted delivery" refers to the delivery of a therapeutic agent or diagnostic agent (also collectively referred to herein as "active ingredient") to an object such as a patient, particularly to a cell, more particularly directly to a cell in the patient's body. Targeted delivery increases the concentration of the active ingredient in a specific area of ​​the body compared to the administration of the active ingredient alone, the administration of a complex of an iron-binding protein and an active ingredient, or the administration of other delivery systems. In particular, targeted delivery increases the concentration of the active ingredient in tumor tissue, particularly in glioma tissue, and more particularly in glioma cells, compared to the administration of the active ingredient alone, the administration of a complex of an iron-binding protein and an active ingredient, or the administration of other delivery systems. Targeted delivery also includes "targeted diagnostic and therapeutic delivery", which means that the therapeutic agent and the diagnostic agent are delivered simultaneously, preferably to the lesion area, so that treatment and diagnosis and / or therapeutic monitoring can be performed simultaneously.

[0032] In a preferred embodiment, the active ingredient is delivered directly into the glioma cells, preferably from the CD45 + Leukocytes transfer directly to glioma cells (direct cell-cell transfer). Direct transfer is preferably by a mechanism involving cell-cell contact and / or cell membrane fusion.

[0033] In some embodiments, the targeted delivery system is administered via intratumoral injection. In some embodiments, the targeted delivery system is administered via intravenous injection.

[0034] The term "targeted pharmaceutically active substance delivery system" used in the present application refers to a system capable of delivering pharmaceutically active substances to a target area, that is, a system capable of targeted delivery in a patient's body, preferably to a lesion area.

[0035] The term "targeted marker delivery system" used in the present application refers to a system capable of delivering a marker to a target area, that is, a system capable of targeted delivery in a patient's body, preferably to a lesion area.

[0036] The term "targeted diagnostic and therapeutic delivery system" used in this application refers to a system that can simultaneously deliver a drug active substance complex and a marker to a target area, that is, a system that can perform targeted delivery within the patient's body, preferably to the diseased area, thereby allowing simultaneous treatment and diagnosis and / or treatment monitoring.

[0037] The term "targeted delivery system" is generally used to refer to "targeted pharmaceutical active substance delivery system", "targeted marker delivery system" and "targeted diagnostic and therapeutic delivery system". Targeted delivery systems have been described in WO2016207257A1, WO2016207256A1 and WO2017222398A1, all of which are incorporated herein by reference.

[0038] Composites & Connectors

[0039] In the isolated targeted delivery system of the present invention, the active ingredient can be covalently bound or non-covalently bound to the iron-binding protein, or can also be encapsulated by the iron-binding protein or its polymer. The term "complex" also encompasses that the active ingredient is enclosed in the iron-binding protein or its polymer, especially the ferritin polymer encapsulation forming a "ferritin cage". In the case where the active ingredient is encapsulated by the iron-binding protein or its polymer, the encapsulation can occur in the presence or absence of covalent bonds or non-covalent bonds. In some embodiments, the association / dissociation characteristics of the ferritin macromolecule itself can be utilized to encapsulate the active ingredient in the internal cavity of the ferritin oligomer (physical confinement). In these embodiments, the active ingredient is fixed in place by non-covalent interactions with the amino acid residues on the internal surface of the cavity. Hemoglobin macromolecules also provide the possibility of non-covalent binding to selected pharmaceutically active substances and / or marker molecules that can be accommodated in the heme binding pocket of hemoglobin itself. The heme in the pocket can be replaced and substituted by pharmaceutically active substances and / or markers with suitable hydrophobicity.

[0040] When CD45 + When leukocytes internalize iron-binding proteins, a complex is formed that allows the active ingredient to be transported to CD45 + In leukocytes. Therefore, it is preferred that the active ingredient is bound to the iron binding protein in a manner that does not interfere with the transport mechanism. A person skilled in the art can easily test this using an uptake assay known in the art and described in WO2016207257A1, WO2016207256A1 and WO2017222398A1. If the complex containing the active ingredient is bound to the iron binding protein by CD45 + If the complex is taken up by leukocytes and transported to target cells in the body, then the complex is preferably sufficiently stable to survive intracellular transport to the target area in the body. Therefore, it is preferred to deliver the complex rather than the active ingredient alone, preferably to the target cells in the target area. This property also reduces the active ingredient's ability to deliver the active ingredient to the CD45 + Possible harmful effects on leukocytes or other non-target cells in the body, such as cytotoxicity.

[0041] In a preferred embodiment, the active ingredient is covalently and / or non-covalently linked to the iron-binding protein, preferably covalently linked. If the active ingredient is covalently linked to the iron-binding protein, then the coupling is preferably achieved via amino acid residues located in an unrelated surface region of the iron-binding protein that binds to receptors involved in endocytosis.

[0042] In case the iron binding protein is covalently linked to the active ingredient, they may be directly linked or via a linker, ie indirectly linked. In a preferred embodiment, the iron binding protein is covalently linked to the active ingredient via a linker.

[0043] Linkers are known to those skilled in the art, such as polyalanine, polyglycine, carbohydrates, (CH 2 ) n The linker may be a biodegradable or non-biodegradable linker, preferably a biodegradable linker. In a preferred embodiment, the linker is cleavable. The linker may be a peptide, a disulfide, or a hydrazone linker or a linker comprising a carbohydrate. Preferably, the linker is a peptide linker. In some embodiments, the peptide linker may be cleaved by a protease. The linker comprising a carbohydrate may be cleaved by beta-glucuronidase. The hydrazone linker may be cleaved by acid hydrolysis. The disulfide linker may be cleaved by cytoplasmic reduction cleavage. In a preferred embodiment, the linker, preferably a peptide linker, may be cleaved by a lysosomal protease, more preferably a lysosomal cysteine ​​protease, and even more preferably a cathepsin.

[0044] In some embodiments, the joint comprises an active group, which, when activated by a specific stimulus, causes the breaking of the covalent bond inside the joint. Suitable active groups are, for example, photoactivatable groups (i.e., groups that can be activated by UV light, such as 3-amino-3-(-2-nitro)phenyl-propionic acid or its photoactivatable structural equivalent), dithionite activatable groups (such as azobenzene moieties), or periodate activatable groups (such as 1,2-dihydroxy moieties, 1-amino-2-hydroxy moieties, or 4-amino-4-deoxy-L-threonic acid). In other embodiments, the joint can be cut by pH changes.

[0045] Pharmaceutically active substances or labels can also be covalently bound to the amino acid side chains (lysine or cysteine) of iron-binding proteins by appropriate selection of phenylhydrazone, succinimide or maleimide activated drugs. Phenylhydrazone derivatives can cleave and release the drug from the iron-binding protein, lysine-bound derivatives can be activated after the entire protein is degraded into amino acids, or cysteine-bound derivatives can be released intracellularly by reductive hydrolysis of the maleimide thioether linkage.

[0046] 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.

[0047] In some embodiments, the linker is a hetero-bifunctional cross-linker containing an N-hydroxysuccinimide (NHS) ester and a maleimide group, which allows the covalent binding of an amine-containing molecule to a sulfhydryl-containing molecule. In some embodiments, the linker is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or sulfo-SMCC.

[0048] Preferably, the cleavable linker is cleaved within the lysosomal lumen.

[0049] In a most preferred embodiment, the linker is a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB) linker.

[0050] In some embodiments, the iron-binding protein is coupled to the active ingredient or linker via a cysteine ​​residue or a lysine residue, preferably a cysteine ​​residue. To form a covalent bond, the relevant sulfhydryl, amino or carboxyl groups of the iron-binding protein are used to covalently couple the active ingredient that can react with the sulfhydryl or amino group to the iron-binding protein directly or indirectly. The active ingredient can be modified by a specific active part, i.e., a linker.

[0051] Thus, ferritin or hemoglobin can be linked to cysteine ​​thiol-reactive pharmaceutically active substances and / or markers with peptide-based cleavable linkers (e.g., cathepsin-sensitive valine-citrulline sequences and p-aminobenzylcarbamate spacers). The antimitotic agent monomethyl auristatin E (MMAE) has been used as a notable example. The peptidyl linker binds the protein to the cytotoxic compound in a stable manner so that the drug is not easily released from the protein under physiological conditions, which helps prevent toxicity to healthy cells and ensures dose efficiency. The iron-binding protein pharmaceutically active substance and / or marker adducts thus generated are capable of attaching to selected receptor types, such as CD163 for hemoglobin and TfR for transferrin, respectively. Once bound, the iron-binding protein pharmaceutically active substance and / or marker adduct will be internalized by endocytosis and thus selectively taken up by the cell. The vesicles containing the active ingredient fuse with lysosomes and lysosomal cysteine ​​proteases, in particular cathepsin B, begin to cleave the cleavable peptide linkers, in particular the valine-citrulline linker, and the active ingredient, in particular MMAE, is no longer bound to the iron-binding protein but is released directly into the tumor environment.

[0052] Alternatively, DM1-SMCC is an effective mertansine derivative with a linker that specifically binds to lysine residues, forming a covalent complex with ferritin, hemoglobin or transferrin in the reaction of the successfully described antibodies. In particular, hemoglobin, ferritin or transferrin can react with DM1-SMCC to provide a covalent protein-drug adduct that can be cleaved intracellularly and release the active drug in a time-dependent manner. DM1's inhibition of microtubule dynamics can lead to mitotic arrest and cell death.

[0053] Methods for preparing complexes of iron-binding proteins and active ingredients are described in WO2016207257A1, WO2016207256A1 and WO2017222398A1.

[0054] In the context of the present invention, the term "full load" is used to refer to the amount of + The maximum amount of an iron-binding protein, preferably ferritin, complexed with a pharmaceutically active substance, a marker or a pharmaceutically active substance and a marker, which is taken up by leukocytes, preferably macrophages, more preferably activated macrophages.

[0055] It is also conceivable that different active ingredients are included in separate targeted delivery systems. For example, one type of active ingredient can be covalently bound to a ferritin polypeptide, while another type of active ingredient is encapsulated in the complex. This approach takes advantage of the different release rates of the active ingredients from the complex once delivered to the target tissue and / or target cell. For example, by exploiting the reactivity of the associated sulfhydryl, amino or carboxyl groups, the active ingredient can be covalently attached to the 24-mer surface or internal cavity of the ferritin molecule. This type of useful reaction is well known in the art and can be easily applied to a specific active ingredient by one skilled in the art 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.

[0056] In diagnostic and therapeutic applications, i.e., when the complex contains both a label and a pharmaceutically active substance, it is preferred that the label is covalently attached to the iron-binding protein, while the pharmaceutically active substance is non-covalently attached to the iron-binding protein and / or encapsulated in the internal cavity formed when the ferritin polypeptide polymer is assembled.

[0057] Iron-binding protein

[0058] In some embodiments, the iron-binding protein is selected from ferritin, preferably heavy (H) ferritin, light (L) ferritin and / or mitochondrial ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemochromatin, transferrin and lactoferrin.

[0059] Human transferrin and ferritin have been considered as effective carriers for delivering small molecules or toxin conjugates to specific targeted cancer cells. So far, despite considerable efforts, no successful transferrin or ferritin drug complex has entered the clinical stage (Luck AN et al., 2013, Adv Drug Deliv Rev 65(8):1012-9).

[0060] Ferritin is a hollow spherical protein complex composed of 24 ferritin monomer subunits assembled into a cage-like structure. Ferritin is the main intracellular iron storage protein. Almost all organisms can produce ferritin, and it is present in every cell type. Ferritin genes are highly conserved between species. In vertebrates, there are two ferritin monomers: light (L) chain type and heavy (H) chain type, with molecular weights of 19kDa or 21kDa, respectively. The 24-mer of vertebrate ferritin can be a homo-oligomer composed of L chain or H chain, or a hetero-oligomer composed of L chain and H chain (Theil EC, 1987, Annual Review of Biochemistry. 56 (1): 289-315): The inner diameter and outer diameter of the typical ferritin complex are about 8nm and 1 to about 2nm, respectively. It has been shown that ferritin is internalized by endocytosis after binding to CD71. The interaction of ferritin with CD71 is mediated by the ferritin-H chain (Li L et al., Proc. Natl. Acad. Sci. USA 107(8)(2010)3505-3510). Ferritin is not abundant in plasma, but can be easily produced in high yield as a recombinant protein in common protein expression systems such as Escherichia coli cells.

[0061] Purified transferrin can be efficiently coupled to a variety of molecules including anticancer drugs through covalent linkers that are appropriately released intracellularly (Beyer U et al. 1998, J Med Chem 41(15):2701-2708). For transferrin, only the lysine groups on the surface of the protein are readily available for covalent attachment.

[0062] Hemoglobin has been considered as a possible drug carrier due to its diversity of chemical coupling with drugs, its abundance in blood and its relative stability (Somatogen, 1993, WO1993008842 A1). However, due to the lack of receptor targeting properties, its biomedical applications have not been promoted except as a blood substitute or anti-sickling cell agent. In fact, Hb can only be recognized by the CD163 (haptoglobin / hemoglobin receptor) epitope on leukocytes, especially monocyte-macrophages. The CD45-based + Leukocyte, and in particular macrophage-based protein delivery, has brought hemoglobin to center stage as a targeted specific carrier of pharmaceutically active substances and / or markers. Hemoglobin can be easily covalently linked to suitable pharmaceutically active substances and / or markers, hydrophobic pharmaceutically active substances and / or markers accommodated in the heme binding pocket, or even transport small molecules, such as cytotoxic molecules linked to the heme iron. Hb can be easily modified by selectively attaching suitable 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 cross-linking drug pyrrolobenzodiazepine dimer (PBD), are the most notable examples of extremely potent cytotoxic agents that can be easily and specifically attached to the relevant cysβ93 residue.

[0063] Alternatively, the lysine residues on the Hb surface (each Hb tetramer has at least 10 titratable lysine residues) can be easily coupled to drugs by cleavable succinimide linkers. Hemoglobin also has the unique ability to release the heme group of non-covalent binding at acidic pH values. The unbound heme hemoglobin thus obtained (apo-haemoglobin) 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 for markers with fluorescent properties (e.g., chlorin e6, hypericin, derivatives of phthalocyanine) (Dong J et al. J Photochem Photobiol B 2014, 140: 163-172).

[0064] In a preferred embodiment of the targeted delivery system for use according to the present invention, the iron-binding protein is selected from ferritin, preferably heavy (H) ferritin, light (L) ferritin and / or mitochondrial ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemochromatin, transferrin; and lactoferrin. The terms ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemoglobin, 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%, more preferably at least 100% of the ability of the corresponding wild-type protein to bind one or more iron ions. The iron-binding protein used in the context of the present invention is preferably from a mammal, more preferably from a mouse, rat, dog, ape, in particular a chimpanzee, or a human, and most preferably from a human. WO2016207257A1 discloses the consensus sequence of preferred iron-binding proteins and preferred structural variants.

[0065] In a preferred embodiment, the iron-binding protein is ferritin, preferably mammalian ferritin. The mammalian ferritin may be mouse, rat, dog, ape, in particular 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.

[0066] Amino acid substitutions in iron-binding proteins are preferably selected in a manner that does not excessively alter the conformation of the polypeptide. For example, a "small amino acid" should be replaced by another small amino acid. In the context of the present invention, a "small amino acid" is preferably an amino acid with a molecular weight of less than 125 Daltons. In the context of the present invention, preferred small amino acids are selected from the amino acids glycine, alanine, serine, cysteine, threonine and valine, or derivatives thereof. Another example is that an amino acid with a hydrophobic side chain should be replaced by another amino acid with a hydrophobic side chain.

[0067] Any ferritin used in the isolated targeted delivery system for use according to the present invention must retain the properties of wild-type ferritin with respect to complex formation (cage-like structure composed of 24 ferritin monomer subunits) and iron uptake.

[0068] In some embodiments, the ferritin comprises an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 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%, more preferably at least 95% of the ability of wild-type ferritin, particularly human wild-type ferritin, to bind one or more iron ions and / or to form ferritin 24-mers. 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, it represents any amino acid, preferably Ile; X at position 29 can be any naturally occurring amino acid, preferably Tyr; X at position 81 can be any naturally occurring amino acid, preferably Phe; X at position 83 can be any naturally occurring amino acid, preferably Gln; X at position 105 can be any naturally occurring amino acid, preferably His; X at position 144 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala; X at position 180 is absent or is any naturally occurring amino acid, preferably Asn; X at position 181 is absent or is any naturally occurring amino acid, preferably Glu; X at position 182 is absent or is any naturally occurring amino acid, preferably Ser. 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, it represents any amino acid, preferably Ile; X at position 22 represents any amino acid, preferably Asn; X at position 30 can be any naturally occurring amino acid, preferably Tyr; X at position 40 can be any naturally occurring amino acid, preferably Tyr or Cys, more preferably Tyr; X at position 82 can be any naturally occurring amino acid, preferably Phe; X at position 84 can be any naturally occurring amino acid, preferably Gln; X at position 91 can be any naturally occurring amino acid, preferably Arg or Cys, more preferably Cys; X at position 106 can be any naturally occurring amino acid, preferably His; X at position 110 can be any naturally occurring amino acid; The X at position 137 may be any naturally occurring amino acid, preferably His or Tyr, more preferably His; the X at position 140 may be any naturally occurring amino acid, preferably Asn or Ser, more preferably Asn; the X at position 145 may be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala; the X at position 164 may be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ser; the X at position 166 may be any naturally occurring amino acid, preferably Met or Leu, more preferably Leu; the X at position 178 may be any naturally occurring amino acid, preferably Asp or His, more preferably Asp; the X at position 181 may be deleted or any naturally occurring amino acid, preferably Asn; the X at position 182 may be deleted or any naturally occurring amino acid, preferably Glu; the X at position 183 may be deleted 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 comprising 1 to 5, 1 to 10, 1 to 15, 1 to 20 or 1 to 25 amino acid mutations outside position 54, position 72, position 87 and / or position 144, in particular outside position 54, and has 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 to bind one or more iron ions and / or to form ferritin 24-mers, in particular the ability to form ferritin 24-mers, of wild-type ferritin, in particular human wild-type ferritin.

[0069] Drugs and markers

[0070] In the context of the present invention, the terms "drug" or "pharmaceutically active substance" are used synonymously to refer to any compound that is capable of altering or modulating cellular activity, or to a compound that can be activated to alter or modulate cellular activity, i.e. a prodrug, preferably to exert its effect in the patient's body. Examples of such active ingredients include so-called "small molecules" and peptides. In the context of the present invention, the term "small molecule" refers to a hydrocarbon or a pharmaceutically active radioisotope having a molecular weight of less than 1.500 g / mol.

[0071] In a preferred embodiment, the pharmaceutically active substance is an anticancer drug selected from proteins, peptides, nucleic acids, non-proteinaceous non-nucleic acid compounds having a molecular weight of less than 1.5 kD, photosensitive compounds, viruses and pharmaceutically active radioisotopes.

[0072] Preferred pharmaceutically active radioisotopes are radioisotopes that emit alpha or beta radiation, which also emit cell-damaging amounts of gamma radiation, selected from lutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, cesium-131, palladium-103, radium-223, iodine-125 and boron-10, or cell-damaging amounts of alpha radiation, preferably selected from actinium-225, bismuth-213, lead-212 and polonium-212. It is also preferred that the complex of the above-mentioned compound with the isotope is linked to nanoparticles (e.g., gold, silver, graphene) or these nanoparticles.

[0073] If the pharmaceutically active substance is a virus, it is preferably an oncolytic virus.

[0074] If the drug is a nucleic acid, it is preferably miRNA, siRNA, chemically modified RNA, LNA, ssRNA, DNA enzyme, or a nucleic acid encoding a pharmaceutically active protein such as an antibody, antibody mimetic, cytokine, prodrug converting enzyme, immunogenic peptide, etc.

[0075] In a preferred embodiment, the anticancer drug is a cytostatic drug, a cytotoxic drug, or a prodrug thereof.

[0076] Preferred anticancer drugs are selected from apoptosis-inducing drugs / autophagy-inducing drugs or necrosis-inducing drugs. Apoptosis-inducing drugs / autophagy-inducing drugs or necrosis-inducing drugs can be any drugs 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 than one ferritin.

[0077] In a preferred embodiment, the anticancer drug is selected from apoptosis-inducing drugs, alkylating substances, antimetabolites, antibiotics, antimitotic agents, DNA modifying drugs, DNA minor groove interstrand crosslinking drugs, DNA synthesis inhibitors, RNA synthesis inhibitors, epothilones, nuclear receptor agonists and nuclear receptor antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, cyclin-dependent kinase (CDK) inhibitors, cyclooxygenase inhibitors and / or lipoxygenase inhibitors, biogenic fatty acids, biogenic fatty acid derivatives including prostaglandins and leukotrienes, protein kinase inhibitors, protein phosphatase inhibitors, lipid kinase inhibitors, platinum coordination complexes. complex), ethyleneimine, methylmelamine, triazine, vinca alkaloids, pyrimidine analogs, purine analogs, alkyl sulfonates, folic acid analogs, anthraquinones, substituted ureas, and methylhydrazine derivatives, enediyne antibiotics, maytansinoids, auristatin derivatives, immune checkpoint inhibitors, and tumor-specific protein inhibitors or tumor-specific marker inhibitors, preferably Rho-GDP dissociation inhibitors, more preferably Grp94 inhibitors or AXL inhibitors, tubulin inhibitors or topoisomerase inhibitors.

[0078] In a preferred embodiment, the anticancer drug is selected from acediasulfone, aclarubicin, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium leucovorin, carboplatin, capecitabine, carmustine, celecoxib, chaliceamycin, chlorambucil, cisplatin, cladribine, Cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, deruxtecan, dibromide, diethylstilbestrol, docetaxel, doxorubicin, dolastatin 10, dolastatin 15, danemucin A, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate phosphate), estrogen, ethinyl estradiol, etoposide, exotecan derivatives, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fosfestrol, furazolidone, gemcitabine, gonadotropin-releasing hormone analogs, altretamine, hydroxyurea, hydroxymethylnitrofurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, iodoxuridine, ifosfamide, interferon alfa, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, maytansine, nitrogen mustard, medroxyprogesterone acetateacetate), megastrolacetate, melphalan, mepacrine, mercaptopurine, maytansine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, neocazinostatin, nifuratel, nifuroxazide, nifurazine ralazine), nifurtimox, nimustine, nimorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalosulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, pyrrolobenzodiazepine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, sulfasalazine, scriflavinium chloride chloride), semustine, streptozocin, sn-38, sulfonylurea, sulfacetamide, sulfachloropyridazine, sulfadiazine, sulfapentene, sulfadimethoxine, sulfaethidole, sulfisoxazole, sulfaguanidine, sulfaguanol, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, trimethoprim-sulfamethoxazole, ... The invention relates to sulfamoxole, sulfonamide, sulfaperine, sulfaphenazole, sulfathiazole, sulfathiazole, sulfathiopyrimidine, staurosporine, tamoxifen, paclitaxel, teniposide, tertiposide, testosterone lactone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triazinon, trioxan, trimethoprim, clofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine and zorubicin. More preferably, the anticancer drug is selected from auristatin, banoxantrone, bendamustine, chlorambucil, calicheamicin, danemycin A, maytansine, melphalan, maytansine, neocarzinostatin and pyrrolobenzodiazepine.

[0079] Even more preferably, the anticancer drug is an auristatin, in particular monomethyl auristatin E or monomethyl auristatin F, or delutecan.

[0080] In some embodiments, the anticancer drug is an immunomodulatory drug that can activate or inhibit the activation of immune cells. Preferably, the immunomodulatory drug is a ligand or antagonist of a pattern recognition receptor, in particular a ligand or antagonist of a Toll-like receptor, a NOD-like receptor (NLR), a RIG-I-like receptor (RLR), or a stimulator of interferon genes (STING) protein. From a physiological point of view, these receptors can recognize signal categories known as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

[0081] In a preferred embodiment, the anticancer drug is a proliferation inhibitory protein or a proliferation inhibitory peptide, preferably a cell cycle inhibitor, or an antibody or antibody-like binding protein that specifically binds to a proliferation promoting protein, or a nucleic acid, preferably a nucleic acid encoding a proliferation inhibitory protein or an antibody or antibody-like binding protein that specifically binds to a proliferation promoting protein, or siRNA, oligonucleotide, LNA or DNA enzyme.

[0082] In the context of the present invention, the term "prodrug" refers to any active ingredient that is metabolized or otherwise converted into a biologically active or more active ingredient (or drug) for at least one property after administration. Compared with the drug, the prodrug is chemically modified to make it less active or inactive relative to the drug, but this chemical modification enables it to generate the corresponding drug through metabolic processes or other biological processes after the prodrug is administered to the patient. For example, the prodrug can have altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved odor relative to the active drug (see, for example, reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). Prodrugs can be synthesized using reactants other than the corresponding drug.

[0083] In some embodiments, the pharmaceutically active substance is a hypoxia-activated prodrug, preferably selected from benzotriazine N-oxide, apaziquinone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH-302, TH-4000 and AQ4N. The use of active ingredients activated under hypoxic conditions can further increase the specificity of targeting and / or further reduce the adverse reactions of the active ingredient. Therefore, in a particularly preferred embodiment, the active ingredient is a hypoxia-activated prodrug. The main chain of all hypoxia-activated prodrugs is one of five different chemical moieties (nitro, quinine, aromatic N-oxides and aliphatic N-oxides and transition metals), which are enzymatically reduced under hypoxic conditions in tissues. Hypoxia-activated prodrugs are any prodrugs that are less active or inactive relative to the corresponding drug, and comprise a drug and one or more than one bioreducible group. Such hypoxia activated prodrugs include all prodrugs that can be activated by various reducing agents and reductases, including but not limited to single electron transferases (such as cytochrome P450 reductases) and double electron transfer (or hydride transfer) enzymes. According to a preferred embodiment of the present invention, the hypoxia activated prodrug is TH-302. The synthesis method of TH-302 is described in PCT applications WO 07 / 002931 and WO 08 / 083101.

[0084] In some embodiments, the pharmaceutically active substance is an antigen or a nucleic acid encoding an antigen.

[0085] As used herein, the terms "marker" or "diagnostic agent" are used interchangeably and refer to any compound suitable for diagnostic purposes. In a preferred embodiment, the marker is selected from a fluorescent dye, a radioisotope / fluorescence emitting isotope, a detectable polypeptide or a nucleic acid encoding a detectable polypeptide, and a contrast agent, or, wherein the marker comprises a chelating agent that forms a complex with a divalent metal cation or a trivalent metal cation. More preferably, the marker is selected from a fluorescent dye, a radioisotope, and a contrast agent. A contrast agent is a dye or other substance that helps to visualize abnormal areas in the body.

[0086] Preferred fluorescent dyes are selected from the following classes of fluorescent dyes: xanthenes (e.g. fluorescein), acridines (e.g. acridinium yellow), oxazines (e.g. oxazine 1), cyanines (e.g. Cy7 / Cy3), styryl dyes (e.g. dye-28), coumarins (e.g. Alexa Fluor 350), porphins (e.g. chlorophyll B), metal-ligand complexes (e.g. PtOEPK), fluorescent proteins (e.g. APC, R-phycoerythrin), nanocrystals (e.g. QuantumDot 705), perylenes (e.g. Lumogen red F300), and phthalocyanines (e.g. IRDYE TM 700DX), as well as conjugates and combinations of these dye classes.

[0087] Preferred radioactive isotopes / fluorescent emitting isotopes are selected from alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes such as 65Tb, fluorescent emitting isotopes such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 9 7Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag, as well as conjugates and compositions of the above isotopes with proteins, peptides, small molecule inhibitors, antibodies or other compounds (e.g., 18F-FDG, 89Zr-oxide or 64Cu-porphyrin).

[0088] A preferred detectable polypeptide is an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof having an altered absorption and / or emission spectrum.

[0089] Preferred contrast agents are selected from paramagnetic agents such as Gd, Eu, W and Mn, preferably complexed with chelating agents. Other options are superparamagnetic iron (Fe) complexes and superparamagnetic iron (Fe) particles, compounds containing high atomic number atoms, i.e. iodine for computed tomography (CT), microbubbles and carriers containing these contrast agents, such as liposomes. In a preferred embodiment, the marker comprises a chelating agent that forms a complex with a divalent metal cation or a trivalent metal cation.

[0090] Preferred chelating agents are selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetraamine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA) and 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

[0091] leukocyte

[0092] The ability of a given cell or cell population to internalize ferritin depends on the expression of receptors involved in this internalization process. Receptors that lead to ferritin internalization include, for example, TfR, CXCR4, scavenger receptors, CD163 and TIM-2. The skilled person is well aware of how to measure the amount of ferritin absorption, and preferred methods for measuring the amount of absorption will be described in the Examples section below.

[0093] The term "leukocyte" (leukocyte or leukocyte cell) as used in the context of the present invention refers to cells of the immune system that are involved in protecting the body from infectious diseases and foreign invaders. All leukocytes are generated and derived from multipotent cells in the bone marrow, called hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic systems. All leukocytes have a nucleus, which distinguishes them from other blood cells, namely anucleated red blood cells (RBCs) and platelets. Types of leukocytes can be classified in a standard manner. The two broadest pairs of categories classify them by structure (granulocytes or agranulocytes) or by cell division lineage (myeloid cells or lymphoid cells). These broadest categories can be further divided into five major types: neutrophils, eosinophils, basophils, lymphocytes, and leukocytes. Cells and monocytes. These types of cells are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytes. They can also be classified into subtypes; for example, among lymphocytes, there are B cells, T cells and NK cells. Granulocytes are distinguished from agranulocytes by the shape of their nuclei (lobed vs. round, i.e. polymorphonuclear vs. mononuclear) and by their cytoplasmic granules (presence or absence, or more precisely, visible or invisible under a light microscope). Another classification method is by lineage: myeloid cells (neutrophils, monocytes, eosinophils and basophils) are distinguished from lymphoid cells (lymphocytes) by the hematopoietic cell lineage (cell differentiation lineage).

[0094] The expression of CD45 is characteristic of leukocyte subsets, namely monocytes, monocyte-macrophages, lymphocytes, granulocytes, NK cells, which are suitable for the targeted delivery system described in the context of the present invention, in particular because CD45 + Leukocytes are attracted to specific tissues and cells in the body and are capable of delivering one or more iron-binding proteins and a complex with more than one pharmaceutically active substance, marker, or pharmaceutically active substance and marker to or within the cell. This subset of leukocytes is hereinafter referred to as "CD45 +Preferably, the monocyte is not a dendritic cell, and its differentiation is regulated 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-κB subunit)-RELB, Spi-1 proto-oncogene (PU / 1), recombinant hairless binding protein suppressor (RBPJ), IFN-regulatory factor 4 (IRF4) or transcription factor E2-2 (also known as TCF4).

[0095] The skilled person understands that the CD45 + Unless they are of clonal origin, leukocytes are a mixed population of different leukocytes expressing the common property of CD45 surface antigen. Therefore, the CD45 defined above is characterized throughout this specification by further functional characteristics and / or structural characteristics. + The term "CD45 + " indicates that the majority of cells or substantially all cells in a cell population express the CD45 surface antigen.

[0096] In this regard, "expression" means that most cells or substantially all cells in a cell population express the marker (also referred to herein as a surface antigen). In this context and with reference to other cell surface antigens, the term "expression" means that the surface antigen is produced within the cell and can be detected by exposure on the cell surface. The expression level of the surface antigen and the amount of antigen detected on the cell surface can vary greatly between different cells. Generally, for a cell surface antigen, if at least 5 copies, preferably at least 10 copies, of the surface antigen are detectably exposed on the cell surface, the cell is considered positive, i.e., expressed as "positive". + ". The skilled person is familiar with how to detect, quantify and select cells that are positive (or negative) for a given cell surface antigen. The preferred method includes fluorescence activated cell sorting (FACS). In this technology, fluorescently labeled antibodies are used to bind to cell surface antigens in a cell population, and the cells are then separated into single cells. Based on the fluorescence intensity measurement results of the single cells, it is determined whether they are positive or negative for a given cell surface antigen. In some embodiments of the present invention, it is indicated whether the expression level of a given protein is high or low. This means that the protein expressed in both cases can be detected, that is, " +", however, the levels are different. For different proteins, high expression and low expression mean different absolute amounts of the protein per cell, respectively. Thus, a given protein is considered to be expressed at a high level if there are more than 500 detectable copies of the protein per cell, while a given protein is considered to be expressed at a low level if there are between 1 and 50 detectable copies of the protein per cell. However, for another protein, its expression level is considered to be high if there are more than 5000 detectable copies per cell, while its expression level is considered to be low if there are between 1 and 500 detectable copies per cell. It is well known in the art how to use flow cytometry and Becton DickinsonQuantibrite TM The amount of protein expressed or produced by cells can be quantified by microbead method (see, for example, Pannu, KK, 2001, Cytometry. 2001 Dec 1; 45(4): 250-8) or mass spectrometry (see, for example, Milo, R., 2013, Bioessays, 35(12): 1050-1055).

[0097] For the purposes of the present invention, the term "high expression" of a given protein refers to detectable expression of the protein in healthy cell populations, particularly CD45 + The highest expression level found in leukocyte populations is at least 70% of the copy number per cell. The term "low expression" of a given protein means that the detectable expression of the given protein is in healthy cell populations, especially CD45 + The highest expression level found in the leukocyte population is 30% or less of the number of copies per cell. Preferably, the "highest expression level" is determined as the number of copies of healthy cells, especially CD45 +The average value of the highest expression level found in leukocytes. In some embodiments, the preferred cell subpopulation is characterized by "generating" a given protein. This is understood to mean that the protein does not have to be detectable on the cell surface, but can only be present inside the cell. Technicians are familiar with how to detect and / or quantify the generation of intracellular proteins, and / or how to select cells that generate such proteins. Alternatively, cell populations can be defined by the expression of a given transcription factor. It is well known in the art how to determine the expression of a given protein or its encoding mRNA in a cell population or even a single cell, for example, using antibodies for in vivo labeling, FISH determination, using in vivo single-molecule fluorescence microscopy alone (Crawford, R. et al. Biophys J. (2013) 105 (11): 2439) or in combination with fluorescence activated cell sorting (FACS), or by PrimeFlow technology (eBioscience) (Adam S. Venable, et al., (2015) Methods in Molecular Biology).

[0098] In the context of the present invention, the term "differentiated monocytes" is used to refer to monocytes that differentiate from committed precursors called macrophage-DC precursors (MDPs), which are mainly found in the bone marrow (but can also be found in the spleen) and differentiate into dendritic cells or macrophages. In mice, they consist of two major subsets: (i) CD11b with high expression of CX3CR1, low expression of CCR2 and Ly6C- + cells and (ii) with low expression of CX3CR1, CCR2 and Ly6C + High expression of CD11b + After leaving the bone marrow, the mouse Ly6C + Monocytes differentiate into Ly6C-monocytes in the circulation. Similarly, in the differentiation of human monocytes, it is generally believed that CD14 ++ Classical monocytes leave the bone marrow and differentiate into CD14 ++ CD16 + Intermediate monocytes, which then differentiate into CD14 + CD16 ++ Non-classical monocytes (Yang et al. 2014; Biomark Res 2(1) doi.10.1186 / 2050-7771-2-1). Preferably, the differentiated monocytes are not dendritic cells, whose differentiation is controlled by one or more of the following transcription factors: IRF8, NFIL3, BATF3, RELB, PU / 1, RBPJ, IIRF4 and / or TCF4, and more preferably, are not dendritic cells.

[0099] Macrophages are professional phagocytes and antigen presenting cells (APCs) that reside in tissues and differentiate from circulating peripheral blood mononuclear cells (PBMs). The term "activated macrophages" is used in the context of the present invention to refer to any polarized macrophages. The activation of macrophages is usually 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 divided into M1 macrophages, classically activated macrophages (CAMs), and M2 macrophages, alternatively activated macrophages (AAMs). Classically activated M1 macrophages include immune effector cells with an acute inflammatory phenotype. These cells are highly invasive to bacteria and generate a large amount of lymphokines (Murray and Wynn, 2011, J Leukoc Biol, 89 (4): 557-63). Alternatively activated, anti-inflammatory M2 macrophages can be divided into at least three subgroups. These subtypes have various different functions, including immunomodulation, maintenance of tolerance and tissue repair / wound healing. In the context of the present invention, the term "M1 inducer" refers to a compound that can guide PBM differentiation into M1 macrophages. In the context of the present invention, the term "M2 inducer" refers to a compound that can guide PBM differentiation into M2 macrophages. The skilled person knows a large number of methods to promote differentiation into M1 macrophages or M2 macrophages. The term "macrophage phagocytosis" refers to the process by which macrophages engulf solid particles to form internal vesicles called phagosomes. The expression "viral / bacterial / fungal / helminth protein or product" refers to a molecule produced by or derived from a virus, bacteria, fungus or worm during a viral / bacterial / fungal / helminth infection.

[0100] The CD45 contained in the targeted delivery system for use according to the present invention + CD34 + Produced in hematopoietic precursor cells.

[0101] In a preferred embodiment, CD45 + The leukocytes are selected from monocytes, differentiated monocytes, preferably macrophages, lymphocytes and granulocytes. Preferably, CD45 + The leukocytes are macrophages, more preferably activated macrophages.

[0102] For monocytes, it is preferably CD11b + Monocytes, preferably selected from 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.

[0103] For differentiated monocytes, they are preferably selected from macrophages, activated macrophages, preferably CD11b + Macrophages, preferably CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, CD11b + CD68 + Macrophages, preferably CD11b + CD86 + M1 macrophages, which preferably produce iNOS and / or secrete interleukin 12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + 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 (DC), preferably CD11b + CD11c + DC、CD11b + CD80 + DC、CD11c + CD80 + DC、CD11c + CD86 + DC、CD11c + HLA-DR + DC or CD11c + CD123 + DC, preferably differentiated monocyte-macrophages, are not Lox1 + 、CXCR7 + and NRF2 + Foam cells.

[0104] In some embodiments, the macrophage is an undifferentiated macrophage. In some embodiments, the macrophage is a naive macrophage. In some embodiments, the macrophage is an M0 macrophage. In a preferred embodiment, the macrophage is slightly polarized to M2. In some embodiments, the macrophage is an M2 macrophage. Those skilled in the art know the surface markers expressed by M2 macrophages or macrophages slightly polarized to M2.

[0105] Preferably, the differentiated monocytes express at least one chemokine receptor, which is preferably selected from CCR1, CCR2, CXCR4 and CXCR6, or at least one growth factor receptor, which is 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).

[0106] In preferred embodiments, the monocytes or differentiated monocytes:

[0107] (i) Can be expressed by CD34 + Hematopoietic precursor cell generation;

[0108] (ii) can be generated 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;

[0109] (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

[0110] (iv) Possess phagocytic ability.

[0111] Preferably,

[0112] (i) the M1 inducer is selected from LPS, GM-CSF, INF-γ, a protein or product of a virus, or a protein or product of a bacteria;

[0113] (ii) The M2 inducer is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated γ-globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor, cancer conditioned medium, cancer cells, adenosine, and proteins or products of helminths or proteins or products of fungi.

[0114] In preferred embodiments, the activated macrophages:

[0115] (i) can be generated by incubating monocytes or macrophages in vitro with factors that can change markers expressed on macrophages, preferably,

[0116] (a) using at least one M1 inducer,

[0117] (b) using at least one M2 inducer, or

[0118] (c) using factors that can alter the ability of macrophages to secrete cytokines, chemokines and / or produce iNOS, arginase or other immunomodulatory enzymes, preferably IL-10 and IL-12;

[0119] (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;

[0120] (iii) can be generated by incubating monocytes or macrophages in vitro with factors capable of inducing the phagocytic ability of macrophages;

[0121] (iv) characterized by expressing at least one of the following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC chemokine receptor 4 (CXCR4), CD163, and / or showing low expression of HLA-DR;

[0122] (v) possess phagocytic ability; and / or

[0123] (vi) capable of secreting cytokines, preferably IL-12 or IL-10, or producing inducible nitric oxide synthase (iNOS), pro-inflammatory compounds, arginase immunosuppressive compounds or anti-inflammatory compounds.

[0124] Preferably,

[0125] (i) the M1 inducer is selected from LPS, INF-γ, GM-CSF and a protein or product of a virus or a protein or product of a bacterium; or

[0126] (ii) The M2 inducer is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated γ-globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor, adenosine, proteins or products of helminths, or proteins or products of fungi.

[0127] 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 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, CXCR4, 25f9 and / or CXCR1.

[0128] For lymphocytes, they are preferably selected from CD3 + and CD4 + CD8 + T lymphocytes, or CD19 + B lymphocytes, CD20 + B lymphocytes, CD21 + B lymphocytes, CD19 + CD20 + B lymphocytes, CD19 + CD21 + B lymphocytes, CD20 + CD21 +B lymphocytes, or CD19 + CD20 + CD21 + B lymphocytes, and natural killer (NK) cells.

[0129] In a preferred embodiment of the targeted delivery system of the present invention, the lymphocytes:

[0130] (i) can be obtained from blood, spleen or bone marrow, or can be obtained from CD34 + Precursor cell generation, as known to those skilled in the art and described in, for example, 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.;

[0131] (ii) are immunocompetent lymphocytes;

[0132] (iii) express antigen-specific T cell receptors; and / or

[0133] (iv) is characterized by the expression of at least one of the following antigens: (a) CD3 and CD4 or CD8, or (b) CD19, CD20, CD21, CD19 CD20, CD19 CD21, CD20 CD21 or CD19 CD20 CD21 antigens, and is preferably capable of producing immunoglobulins.

[0134] In a particularly preferred embodiment, CD45 + Lymphocytes are NK cells, which

[0135] (i) can be obtained from blood, spleen or bone marrow, or can be obtained from CD34 + Progenitor cell generation; and / or

[0136] (ii) Characterized by lack of CD3 expression and expression of at least one of the following: CD56 + and / or CD94 + 、CD158a + CD158f + CD314 + CD335 + .

[0137] Regarding granulocytes, they are preferably selected from neutrophils, preferably CD66b +Neutrophils, eosinophils and basophils, preferably CD193 + Eosinophils.

[0138] In a preferred embodiment of the targeted delivery system of the present invention, the granulocytes:

[0139] (i) can be obtained from blood, spleen or bone marrow, or can be obtained from CD34 + Precursor cell generation, for example as described in Kuhs et al. 2015, Curr Protoc Immunol 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;

[0140] (ii) characterized by the expression of at least one of the following: CD66b and / or CD193;

[0141] (iii) is a polymorphonuclear leukocyte characterized by the presence of granules in its cytoplasm; and / or

[0142] (iii) Characterized by the expression of at least one of the following: TfR, CD163, TIM-2 and / or CXCR4.

[0143] In a cell population, as long as the majority of cells in the cell population possess the characteristic, even if not every cell possesses the characteristic, the targeted delivery system for use described in the present invention still provides the listed advantages. Therefore, the characteristics of a preferred cell for the targeted delivery system for use described in the present invention are described below.

[0144] In a preferred embodiment, the CD45 + Leukocytes, preferably macrophages, are derived from isolated peripheral blood mononuclear cells (PBMCs). Preferably, the CD45 + Leukocytes, preferably macrophages, are primary cells, i.e. cells directly isolated from human tissues, especially peripheral blood. Preferably, the CD45 + The leukocytes, preferably macrophages, are not cells of an immortalized cell line.

[0145] In some embodiments, the CD45 +The leukocytes are derived from the patient to be treated. In this case, the cells loaded with the complex are autologous to the patient. It is also contemplated that the patient is HLA typed prior to treatment using the targeted delivery system of the present invention, and that the cell type used for a particular patient is HLA matched to the patient. In both preferred embodiments, the cells are primary cells or derived from primary cells through a few differentiation steps. Alternatively, the cells may be from an immortalized cell line, but a non-transformed cell line is preferred.

[0146] For separation of CD45 + The blood of leukocytes is preferably obtained from the patient to be treated or from a healthy donor. Alternatively, the blood can also be obtained from a blood bank. The use of umbilical cord blood is also contemplated herein.

[0147] WO2016207257A1, WO2016207256A1 and WO2017222398A1 describe loading a complex of an iron-binding protein and an active ingredient onto CD45 + Leukocyte method.

[0148] mechanism

[0149] The present invention utilizes CD45 loaded with iron-binding protein linked to a drug / prodrug + Leukocytes, preferably activated macrophages, are used as a delivery system for targeting gliomas. Glioma does not respond well to chemotherapy or is difficult to detect by imaging methods, mainly due to the blood-brain barrier causing reduced permeability of anticancer drugs to tumors. However, the CD45 of the targeted delivery system for treating gliomas of the present invention is + Leukocytes, preferably activated macrophages, can migrate across the blood-brain barrier to the glioma area. In the brain, increased intratumoral interstitial fluid pressure and the blood-tumor barrier hinder the penetration of therapeutic agents into the glioma. The CD45 targeted delivery system for treating gliomas in the present invention is + Leukocytes, preferably activated macrophages, are able to reach sites deeper into the tumor. Compared with drugs administered topically or in complex with iron-binding proteins, the targeted delivery system of the present invention can ensure better distribution of drugs within tumor tissue when administered topically. In summary, the targeted delivery system of the present invention constitutes an effective delivery system for delivering active ingredients to the entire glioma mass.

[0150] The inventors observed that after the isolated targeted delivery system for use according to the present invention was administered intravenously or intratumorally to animals, the loaded CD45 + Leukocytes, preferably activated macrophages, migrate to the glioma site and release the complex of iron-binding protein and one or more active ingredients into the cancer cells ( Figure 3 , Figure 4and Figure 8 The inventors demonstrated that cell-cell contact is necessary for efficient transfer of the complex, in particular the conjugate, comprising ferritin and the active ingredient from macrophages to cancer cells, and that secretion of the complex, in particular the conjugate, comprising ferritin and the active ingredient by macrophages and subsequent uptake by cancer cells is not sufficient to ensure efficient delivery ( Figure 5 and Figure 6 ). This indicates that the transfer of the targeted delivery system of the present invention is direct transfer between cells and requires CD45 + Cell-cell contact or at least close proximity between leukocytes (macrophages) and cancer cells. The advantage of this direct transfer is that the active agent can be delivered specifically to the target cells without increasing the extracellular concentration outside the tumor cells in the brain, thereby improving the efficacy and reducing side effects. Therefore, the method of the present invention can accurately deliver the active ingredient to the glioma site, especially to the glioma cells.

[0151] Combination therapy

[0152] In some embodiments of the isolated targeted delivery system for use according to the present invention, the treatment or diagnosis of glioma comprises irradiating the glioma prior to administering the isolated targeted delivery system.

[0153] The inventors found that in an aggressive glioma model, a combined treatment including irradiation of glioma and subsequent administration of a targeted delivery system further improved the survival rate of mice (Example 3, Tables 5 and 6). After irradiation, injection of macrophages containing ferritin-drug conjugates significantly prolonged survival and was more effective than injection of ferritin-drug conjugates alone (Example 3, Table 6).

[0154] The beneficial effects of combined irradiation and subsequent administration of a targeted delivery system are not limited to gliomas but are also applicable to other cancers. + A targeted delivery system for separating leukocytes, which is used in a method for treating or diagnosing gliomas, wherein the cells contain one or more iron-binding proteins and a pharmaceutically active substance, a marker, or a complex of a pharmaceutically active substance and a marker, wherein the treatment or diagnosis of gliomas comprises irradiating the gliomas before administering the separated targeted delivery system. + All embodiments of leukocytes, complex formation means, iron-binding proteins, pharmaceutically active substances and markers are also applicable in connection with the treatment or diagnosis of cancer.

[0155] Pharmaceutical composition

[0156] On the other hand, the present invention provides a pharmaceutical composition for treating or diagnosing glioma, comprising the targeted delivery system for separation described in the first aspect of the present invention and a pharmaceutically acceptable carrier and / or suitable one or more than one excipient. All embodiments described in the first aspect of the present invention are also applicable to the pharmaceutical composition.

[0157] Since the isolated targeted delivery system comprises living cells, carriers and excipients are preferably selected that are capable of keeping the cells alive.

[0158] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0159] As used herein, the term "carrier" refers to a pharmaceutically inactive substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer or carrier used together 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 solutions and oils, and the oils include, but are not limited to, oils of petroleum origin, animal origin, plant origin or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous glucose solutions and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. When the pharmaceutical composition is administered intravenously, saline solutions are preferred carriers. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin.

[0160] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol and the like.

[0161] "Surfactants" include anionic surfactants, cationic surfactants and nonionic surfactants, such as but not limited to sodium deoxycholate, sodium dodecyl sulfate, Triton X-100 and polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0162] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease antagonists and / or nuclease antagonists.

[0163] "Physiological buffer" includes, but is not limited to, sodium chloride solution, deionized 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) piperazine] ethanesulfonic acid) or MOPS buffer (3-morpholino-1-propanesulfonic acid). The selection of the corresponding buffer generally depends on the desired buffer molar concentration. For example, phosphate buffer is suitable for injection solutions and infusion solutions.

[0164] Treatment

[0165] 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 the isolated targeted delivery system described in the first aspect of the present invention. All embodiments described in the first aspect of the present invention are also applicable to the method for treating or diagnosing. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 : Shows that in the mouse GL261 glioma model, the survival of tumor-bearing mice was prolonged after intravenous or intratumoral injection of macrophages containing ferritin-drug conjugates.

[0167] Figure 2 : (A) shows that the total number of cancer cells decreased after irradiation. (B) shows that the number of macrophages in tumor masses increased after irradiation.

[0168] Figure 3 : Shows increased macrophage infiltration in brains containing gliomas compared to healthy brains.

[0169] Figure 4 : Shows an increase in ferritin concentration within gliomas following administration of ferritin-containing macrophages compared to administration of ferritin alone.

[0170] Figure 5 : Cell-cell contact is required for the transfer of ferritin from macrophages to cancer cells. Ferritin transfer between cells isolated by Transwell chambers was analyzed. THP-1 macrophages (HFt( +)) were inoculated into Transwell chambers or co-cultured directly with MDA-MB-231 cancer cells grown on 24-well plates for 24 hours. Macrophages not loaded with ferritin (HFt(-)) were used as a control. After 24 hours of co-culture with macrophages, the AF488 fluorescence of cancer cells was analyzed by flow cytometry. The bar graph represents the average geometric mean fluorescence intensity of AF488-positive cancer cells. AF488-positive cancer cells can be detected when co-cultured directly with macrophages, but if separated from macrophages by Transwell chambers, AF488-positive cancer cells cannot be detected, indicating that cell-cell contact is required to deliver the conjugate of ferritin and active agent (marker) to cancer cells.

[0171] Figure 6 : Analyze the role of secretion and cell-cell contact in the transfer of ferritin from macrophages to cancer cells. EMT6 cancer cells are directly cultured with RAW264.7 macrophages loaded with HFt-AF488, or they are cultured with conditioned medium. Conditioned medium is collected from RAW264.7-HFt-AF488 macrophages cultured for 24 hours in basal growth medium, or collected from RAW264.7-HFt-AF488 macrophages cultured for 24 hours in the medium generated by cancer cells, or collected from the co-culture of RAW264.7-HFt-AF488 macrophages and cancer cells. Macrophages not loaded with ferritin are used as controls. (a) representative density plots show the AF488 fluorescence distribution in EMT6 cells. Under all conditions, the density plots on the left are all ferritin-free controls. Bar graphs represent the average (b) geometric mean fluorescence and (c) percentage of AF488 positive cancer cells from three independent experiments. Under all conditions, the bars on the left are all ferritin-free controls. After 4 hours, effective uptake of HFt-AF488 by cancer cells was observed only in direct co-culture, but not in culture with conditioned medium. After 24 hours, uptake of HFt-AF488 by cancer cells was higher and more frequent in direct co-culture compared with culture with conditioned medium. The data suggest that cell-cell contact is required for delivery of the conjugate of ferritin and active ingredient (marker) to cancer cells, and that secretion of the conjugate of ferritin and active ingredient (marker) into the culture medium by macrophages and subsequent uptake by cancer cells is not sufficient to ensure effective delivery of the conjugate of ferritin and active ingredient (marker) to cancer cells.

[0172] Figure 7 : Shows that in the ZH-161 glioma model, the survival of tumor-bearing mice was prolonged after intratumoral injection of macrophages containing ferritin-drug conjugates.

[0173] Figure 8: Shows the systemic distribution of macrophages containing ferritin-drug conjugate after intratumoral administration.

[0174] Examples

[0175] Example 1 - BMDM-Ft-vcMMAE Treatment of Mouse Glioma GL261 Tumors

[0176] In the mouse GL261 glioma model, bone marrow-derived macrophages (BMDM) containing ferritin-drug conjugate (BMDM-Ft-vcMMAE) were injected intratumorally and intravenously.

[0177] The ferritin-drug conjugate Ft-vcMMAE was generated by covalently linking the anticancer drug monomethyl auristatin E (MMAE) to ferritin using a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB) linker. Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (vcMMAE) was obtained from MedChem Express (Princeton, NJ). The conjugate was prepared as follows: 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 coupled 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) sulfhydryl groups at pH values ​​of 6.5 to 7.5 to form a stable thioether bond. Excess vcMMAE was purified using a PM 100 ultrafiltration concentrator and buffer exchanged with D-PBS. The yield of the coupling was approximately 80% of total cysteine. The formation of the Ft-vcMMAE conjugate was confirmed by LC-MS analysis and titration of the residual free sulfhydryl groups with para-chloromercury benzoate. The concentration of the Ft-vcMMAE conjugate was determined by UV-visible spectroscopy.

[0178] Under standard culture conditions, macrophages were incubated in a Ft-vcMMAE solution at a concentration of 0.5 mg / ml to 0.75 mg / ml for 1 to 4 hours to load Ft-vcMMAE to the macrophages.

[0179] As controls for injection, PBS, macrophages without ferritin-drug conjugates, and ferritin-drug conjugates without macrophages were used. The group receiving intravenous treatment was administered four times, 5 million macrophages containing ferritin-drug conjugates each time. The group receiving intratumoral administration treatment received only two doses, 2 million macrophages loaded with ferritin-drug conjugates each time. From Tables 1 and 2 below and Figure 1 A and Figure 1As can be seen in B, injection of macrophages containing ferritin-drug conjugate can significantly prolong survival after tumor implantation and is more effective than injection of ferritin-drug conjugate alone or injection of macrophages alone.

[0180] Table 1 - Intratumoral treatment of GL-261 glioma-bearing mice using BMDM-HFt-vcMMAE

[0181]

[0182] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0183] p-value BMDM Ft-vcMMAE BMDM-Ft-vcMMAE PBS 0.385 0018＊ 0.014* BMDM - 0.027＊ 0014* Ft-vcMMAE - - 0.010＊

[0184] Table 2 - Intravenous treatment of GL-261 glioma-bearing mice with BMDM-HFt-vcMMAE

[0185]

[0186] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0187] p-value BMDM Ft-vcMMAE BMDM-Ft-vcMMAE PBS 0.1535 0.4335 0.0734 BMDM - 0.0511 <![CDATA[0.0063 ＊＊ ]]> Ft-vcMMAE - - 0.1535

[0188] Example 2 - BMDM-Ft-vcMMAE Treatment of Mouse Glioma CT-2A Tumors

[0189] As described in Example 1, intratumoral and intravenous injections of macrophages (BMDM-Ft-vcMMAE) containing ferritin-drug conjugates were performed in the CT-2A glioma model of mice. As can be seen from Tables 3 and 4 below, injection of macrophages containing ferritin-drug conjugates can significantly prolong the survival period after tumor implantation and is more effective than injection of ferritin-drug conjugates or macrophages alone.

[0190] Table 3 - Intratumoral treatment of CT-2A glioma-bearing mice using BMDM-HFt-vcMMAE

[0191]

[0192] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0193] p-value BMDM Ft-vcMMAE BMDM-Ft-vcMMAE PBS <![CDATA[0.0449 ＊ ]]> 0.7052 <![CDATA[0.0024 ＊＊ ]]> BMDM - 0.4077 <![CDATA[0.0024 ＊＊ ]]> Ft-vcMMAE - - <![CDATA[0.0031 ＊＊ ]]>

[0194] Table 4 - Intravenous treatment of CT-2A glioma-bearing mice with BMDM-HFt-vcMMAE

[0195]

[0196] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0197] p-value BMDM Ft-vcMMAE BMDM-Ft-vcMMAE PBS 0.28 0.28 0.26 BMDM - 0.14 0.14 Ft-vcMMAE - - 0.33

[0198] Example 3 - Radiation promotes tumor infiltration of BMDM-Ft-vcMMAE

[0199] Low-dose irradiation (2 Gy of gamma radiation applied daily for 3 consecutive days before the first of two administrations of the targeted delivery system: 3×2 Gy) or high-dose irradiation (4 Gy of gamma radiation applied one day before each of two administrations of the targeted delivery system: 2×4 Gy) (IR treatment) was performed in the GL261 glioma model in mice. Subsequently, macrophages were administered intravenously. These macrophages were differentiated from the bone marrow of a donor mouse strain carrying the CD45.1 variant of the CD45 marker. For experiments including in vivo imaging, macrophages were stained with a far-red fluorescent marker membrane dye NIR790 staining.

[0200] After IR treatment combined with macrophage injection, brains were harvested, enzymatically dissociated, and stained for flow cytometry analysis. Anti-CD45.1 antibody was used to distinguish injected macrophages from endogenous CD45 cells.

[0201] The inventors found that after irradiation, the number of CD45.1 cells (macrophages administered intravenously) within the tumor mass increased ( Figure 2 B), while IR treatment resulted in a decrease in tumor cells ( Figure 2 A).

[0202] Example 4 - Low-dose radiation promotes tumor infiltration and proliferation of GL261 glioma cells treated with intravenous BMDM-Ft-vcMMAE Efficacy

[0203] In the GL261 glioma model in mice, low-dose radiation (2 Gy of gamma radiation applied 3 consecutive days before the first of two administrations of the targeted delivery system: 3×2 Gy) was performed. Subsequently, macrophages containing the ferritin-drug conjugate (BMDM-Ft-vcMMAE) were administered intravenously.

[0204] As shown in Tables 5 and 6 below, irradiation prior to treatment with ferritin-drug conjugate-containing macrophages can also prolong survival. Without wishing to be bound by theory, the inventors hypothesize that irradiation increases tumor inflammation relative to the absence of irradiation, thereby attracting more ferritin-drug conjugate-containing macrophages (BMDM-Ft-vcMMAE) to infiltrate the tumor.

[0205] Furthermore, in the group receiving irradiation (Table 6), injection of macrophages containing ferritin-drug conjugate was more effective than injection of ferritin-drug conjugate alone or injection of macrophages alone.

[0206] Table 5 - Intravenous treatment of GL-261 glioma-bearing mice with BMDM-Ft-vcMMAE

[0207]

[0208] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0209] p-value BMDM Ft-vcMMAE BMDM-Ft-vcMMAE PBS 0.952 0.952 <![CDATA[0.043 ＊ ]]> BMDM - 0.952 <![CDATA[0.043 ＊ ]]> Ft-vcMMAE - - <![CDATA[0.043 ＊ ]]>

[0210] Table 6 - Combined intravenous treatment of GL-261 glioma-bearing mice with BMDM-Ft-vcMMAE and low-dose irradiation (radiotherapy)

[0211]

[0212] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0213]

[0214] Example 5 - Relative infiltration of BMDM into the diseased brain

[0215] Healthy mice and glioma-bearing mice were intravenously injected with membrane dyes marked with far-red fluorescent markers. NIR790-stained macrophages. Infiltration in the brain was examined by measuring in vivo far-infrared fluorescence in the brain relative to other body parts. Infiltration in the brain was increased in glioma-bearing mice ( Figure 3 ).

[0216] Example 6-Penetration of glioma by BMDM-Ft

[0217] In a mouse glioma model, a conjugate of ferritin and Alexa Fluor 488 (Ft-AF488) or macrophages containing the conjugate (BMDM-Ft-AF488) were injected intratumorally. These macrophages were differentiated from the bone marrow of a donor mouse strain of the CD45.1 variant. After 24 hours, the mice were sacrificed and brain sections were analyzed. To visualize the macrophages, CD45.1 immunostaining was performed. Injection of BMDM-Ft-AF488 resulted in the localization of macrophages and the ferritin conjugate within tumor tissue, but not within the healthy brain ( Figure 4In contrast, injection of Ft-AF488 did not result in localization of ferritin conjugates within tumor tissue. Some ferritin conjugates were detectable in healthy brain.

[0218] Example 7 - HMDM-Ft-vcMMAE Treatment of Human Glioma ZH-161 Tumors

[0219] In the ZH-161 glioma model, human ZH-161 tumor cells were injected into mice. The ZH-161 glioma model is known for having an unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter, MGMT expression, and resistance to temozolomide (TMZ), as described by 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). As described in Example 1, intratumoral injection of macrophages (MDM-Ft-vcMMAE) containing ferritin-drug conjugates was performed in the ZH-161 glioma model. From Table 7 and Figure 7 As can be seen, injection of macrophages containing ferritin-drug conjugates can significantly prolong survival after tumor implantation and is more effective than injection of ferritin-drug conjugates or macrophages alone.

[0220] Table 7 - Intratumoral treatment of ZH-161 glioma-bearing mice using MDM-HFt-vcMMAE

[0221]

[0222] Statistical analysis of survival data (multiple log-rank test with Bonferroni-Hochberg corrected p-values):

[0223] p-value MDM Ft-vcMMAE MDM-Ft-vcMMAE PBS 0.230 0.180 <![CDATA[0.002 ＊＊ ]]> MDM - 0.403 <![CDATA[0.002 ＊＊ ]]> Ft-vcMMAE - - <![CDATA[0.002 ＊＊ ]]>

[0224] Example 8 - Biodistribution

[0225] In the biodistribution study, the systemic distribution of macrophages containing the ferritin-drug conjugate after intratumoral therapeutic administration was evaluated. Notably, the results showed that the therapeutic agent was localized within the brain tissue ( Figure 8). The administered dose remained largely confined to the brain, with only trace amounts, if any, detected in other major organs and tissues. In addition, the amount of drug detected in brain tissue remained nearly constant for up to 72 hours after injection, emphasizing its persistent presence and potential long-term therapeutic effect. This unique property not only highlights the targeted efficacy of macrophages containing ferritin-drug conjugates against gliomas, but also suggests that it reduces the potential for off-target side effects and systemic toxicity.

[0226] sequence

[0227] SEQ ID NO: 1—Human ferritin heavy chain

[0228]

[0229] SEQ ID NO:2—Mouse ferritin heavy chain

[0230]

[0231] SEQ ID NO.3 - Mammalian common ferritin

[0232]

[0233] SEQ ID NO.4-FT_2

[0234]

[0235] SEQ ID NO.5 - Alternative mammalian consensus ferritin

[0236]

Claims

1. A CD45 + A targeted delivery system for separation of leukocytes, which is used in a method for treating or diagnosing gliomas. The cells contain one or more iron-binding proteins and a pharmaceutically active substance, a marker, or a complex of a pharmaceutically active substance and a marker.

2. The isolated targeted delivery system for use according to claim 1, wherein (i) the iron-binding protein and the pharmaceutically active substance or marker are covalently and / or non-covalently linked, and / or (ii) The pharmaceutically active substance or marker is encapsulated by the iron-binding protein or its polymer.

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

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

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

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

7. The isolated targeted delivery system for use according to claim 6, wherein (i) The monocytes are CD11b + Monocytes, preferably selected from 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 selected from macrophages, activated macrophages, preferably CD11b + Macrophages, more preferably CD11b + CD16 + Macrophages, CD11b + CD32 + Macrophages, CD11b + CD64 + Macrophages, CD11b + CD68 + Macrophages, preferably CD11b + CD86 + M1 macrophages, which preferably produce iNOS and / or secrete interleukin 12 (IL-12), or preferably CD11b + CCR2 + M2 macrophages, CD11b + CD204 + M2 macrophages, CD11b + CD206 + M2 macrophages, CD11b + CD204 + CD206 + M2 macrophages, CD11b + 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 (DC), preferably CD11b + CD11c + DC、CD11b + CD80 + DC、CD11c + CD80 + DC、CD11c + CD86 + DC、CD11c + HLA-DR + DC or CD11c + CD123 + DC, preferably differentiated monocyte-macrophages, are not Lox1 + 、CXCR7 + and NRF2 + Foam cells; (iii) the lymphocytes are selected from CD3 + and CD4 + or CD8 + T lymphocytes, or CD19 + B lymphocytes, CD20 + B lymphocytes, CD21 + B lymphocytes, CD19 + CD20 + B lymphocytes, CD19 + CD21 + B lymphocytes, CD20 + CD21 + B lymphocytes, or CD19 + CD20 + CD21 + B lymphocytes, and natural killer (NK) cells; or (iv) The granulocytes are selected from neutrophils, preferably CD66b + Neutrophils, eosinophils and basophils, preferably CD193 + Eosinophils.

8. The isolated targeted delivery system for use according to any one of claims 6 to 9, wherein the monocytes or differentiated monocytes: (i) Can be expressed by CD34 + Hematopoietic precursor cell generation; (ii) can be generated 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) It is characterized by expresses at least one of the following antigens: TfR, CD163, CD14, CD16, CD33, CXCR4, 25f9, HLA-DR and / or CD115; and / or (iv) possess phagocytic ability, Preferably, wherein (i) the M1 inducer is selected from LPS, GM-CSF, INF-γ, viral proteins or products, or bacterial proteins or products; (ii) the M2 inducer is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated γ-globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor, cancer conditioned medium, cancer cells, adenosine, and proteins or products of worms or proteins or products of fungi.

9. The isolated targeted delivery system for use according to claim 7, wherein the activated macrophages: (i) can be generated by incubating monocytes or macrophages in vitro with factors that can change the markers expressed on macrophages, preferably (a) using at least one M1 inducer, (b) using at least one M2 inducer, or (c) using factors that can change the ability of macrophages to secrete cytokines, chemokines and / or produce iNOS, arginase or other immunomodulatory enzymes, preferably IL-10 and IL-12; (ii) It is characterized by express at least one of the following antigens: CD64, CD86, CD16, CD32, HLA-DR, and / or produce iNOS and / or IL-12; (iii) can be generated by incubating monocytes or macrophages in vitro with factors capable of inducing the phagocytic ability of macrophages; (iv) characterized by expressing at least one of the following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC chemokine receptor 4 (CXCR4), CD163, and / or showing low expression of HLA-DR; (v) possess phagocytic ability; and / or (vi) capable of secreting cytokines, preferably IL-12 or IL-10, or producing inducible nitric oxide synthase (iNOS), pro-inflammatory compounds, arginase immunosuppressive compounds or anti-inflammatory compounds, Preferably, wherein (i) the M1 inducer is selected from LPS, INF-γ, GM-CSF, and viral proteins or products or bacterial proteins or products; or (ii) the M2 inducer is selected from IL-4, IL-10, IL-13, immune complexes of antigens and antibodies, IgG, heat-activated γ-globulin, glucocorticoids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonists, leukocyte inhibitory factor, adenosine, proteins or products of helminths, or proteins or products of fungi.

10. An isolated targeted delivery system for use according to any one of claims 1 to 9, wherein the iron-binding protein is selected from ferritin, preferably heavy (H) ferritin, light (L) ferritin and / or mitochondrial ferritin; hemoglobin, preferably hemoglobin A, hemoglobin AS, hemoglobin SC, hemoglobin C, hemoglobin D, hemoglobin E, hemoglobin F, hemoglobin H; hemoglobin-haptoglobin complex, hemochromatin, transferrin and lactoferrin.

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

12. The isolated targeted delivery system for use according to claim 11, wherein the anticancer drug - selected from apoptosis-inducing drugs, alkylating substances, antimetabolites, antibiotics, antimitotic agents, DNA-modifying drugs, DNA minor groove interstrand crosslinking drugs, DNA synthesis inhibitors, RNA synthesis inhibitors, epothilones, nuclear receptor agonists and nuclear receptor antagonists, antiandrogens, antiestrogens, platinum compounds, hormones, antihormones, interferons, cyclin-dependent kinase (CDK) inhibitors, cyclooxygenase enzyme inhibitors and / or lipoxygenase inhibitors, bio-fatty acids, bio-fatty acid derivatives including prostaglandins and leukotrienes, protein kinase inhibitors, proteins White phosphatase inhibitors, lipid kinase inhibitors, platinum coordination complexes, ethyleneimine, methylmelamine, triazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkyl sulfonates, folic acid analogs, anthraquinones, substituted urea and methylhydrazine derivatives, enediyne antibiotics, maytansinoids, auristatin derivatives, immune checkpoint inhibitors, and tumor-specific protein inhibitors or tumor-specific marker inhibitors, preferably Rho-GDP dissociation inhibitors, more preferably Grp94 inhibitors or AXL inhibitors, tubulin inhibitors or topoisomerase inhibitors; - selected from the group consisting of acetosulfone, aclarubicin, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium leucovorin, carboplatin, capecitabine, carmustine, celecoxib, calicheamicin, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, dapsone, daunorubicin, derutecan, dibromide, diethylstilbestrol, docetaxel, doxorubicin, dolastatin 10, dolastatin 15, danemycin A, enediynes, epirubicin, epothilone B, epothilone D, estramustine phosphate, estrogen, ethinyl estradiol, etoposide glycosides, exotecan derivatives, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fosfostil, furazolidone, gemcitabine, gonadotropin-releasing hormone analogs, hexamethylmelamine, hydroxyurea, hydroxymethylfurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, iodine, ifosfamide, interferon α, irinotecan, leuprolide, lomustine, lortotecan, sulfamethoxazole sulfonamide, maytansine, nitrogen mustard, medroxyprogesterone acetate, megestrol acetate, melphalan, imipaline, mercaptopurine, maytansine, methotrexate, metronidazole, mitomycin C, mitozazide, mitotane, mitoxantrone, mithramycin, nalidixic acid, neocarcinomatidylcholine, nifuratel , nifuroxide, furazolidone, nifurtimox, nimustine, nimorazole, nitrofurantoin, nitrogen mustards, olomocin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalosulfathiazole, pipobroman, prednimustine, prednisone, presin, procarbazine, pyrimethamine, pyrrolobenzodiazepine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, sulfasalazine, scrivane chloride, semustine, streptozotocin, sn-38, sulfonylurea, sulfacetamide, sulfachloropyridazine, sulfadiazine, sulfapentene, sulfadimethoxine, sulfadiazole, sulfisoxazole, sulfaguanidine, sulfaguanol, sulfamethoxazole, sulfamethoxazole, compound new Normine, sulfamethoxazole, sulfamethoxazole, sulfadimethoxine, sulfa, sulfapyrine, sulfaphenazole, sulfathiazole, sulfathiazole, staurosporine, tamoxifen, paclitaxel, teniposide, terteposide, testosterone lactone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triazinon, trioxosulfan, trimethoprim, clofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine and daunorubicin; preferably selected from auristatin, banoxantrone, bendamustine, chlorambucil, calicheamicin, danemycin A, maytansine, melphalan, maytansine, neocarcinomatine and pyrrolobenzodiazepine; - is an immunomodulatory drug that can activate or inhibit the activation of immune cells, preferably, the immunomodulatory drug is a ligand or antagonist of a pattern recognition receptor, in particular a ligand or antagonist of 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 -is a proliferation inhibitory protein or a proliferation inhibitory peptide, preferably a cell cycle inhibitor, or an antibody or antibody-like binding protein that specifically binds to a proliferation promoting protein, or a nucleic acid, preferably a nucleic acid encoding a proliferation inhibitory protein or an antibody or antibody-like binding protein that specifically binds to a proliferation promoting protein, or siRNA, oligonucleotide, LNA or DNA enzyme.

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

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

15. The isolated targeted delivery system for use according to any one of claims 1 to 14, wherein the label is selected from fluorescent dyes, fluorescent emitting isotopes, radioactive isotopes, detectable polypeptides or nucleic acids encoding detectable polypeptides and contrast agents, or wherein the label comprises a chelating agent that forms a complex with a divalent metal cation or a trivalent metal cation, preferably wherein - the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetraamine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA) and 6-hydrazinopyridine-3-carboxylic acid (HYNIC); - the contrast agent comprises a paramagnetic agent, preferably selected from Gd, Eu, W and Mn, or ferrihydrite; - The radioactive isotope / fluorescent emitting isotope is selected from alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65 Tb, fluorescent isotopes such as 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 Such as 109 Pd, 105 Rh, 101m15 Rh, 119 Sb, 128 8. 123 I. 124 I. 131 I. 197 Hg, 211 At 169 Eu, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au and 199 Ag, and conjugates and compositions of the above isotopes with proteins, peptides, small molecule inhibitors, antibodies or other compounds; - the fluorescent dye is selected from the following classes of fluorescent dyes: xanthenes, acridines, oxazines, cyanines, styryl dyes, coumarins, porphins, metal-ligand complexes, fluorescent proteins, nanocrystals, perylenes and phthalocyanines, as well as conjugates and combinations of dyes of these classes; and / or - The detectable polypeptide is an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof having an altered absorption spectrum and / or emission spectrum.

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