Isolated targeted delivery system for the treatment of head and neck cancer

Abstract

The present invention relates to an isolated targeted delivery system comprising a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a pharmaceutically active substance, for use in a method of treating head and neck cancer.

Description

[0001] Cellis AG

[0002] Our Ref.: 976-19 PCT

[0003] ISOLATED TARGETED DELIVERY SYSTEM FOR THE TREATMENT OF HEAD AND

[0004] NECK CANCER

[0005] The present invention relates to an isolated targeted delivery system comprising a CD45+leukocyte cell comprising a complex of an iron binding protein and a pharmaceutically active substance, for use in a method of treating head and neck cancer.

[0006] BACKGROUND OF THE INVENTION

[0007] Head and neck cancer encompasses a group of solid tumors including cancers of the oral cavity, pharynx, larynx, and salivary glands. Despite the remarkable advances in cancer therapy, solid tumors remain a significant medical challenge due to their heterogeneity and limited blood supply resulting in a poor delivery of anticancer agents. Only a small fraction of systemically administered drugs reaches the tumor, and even within the tumor, drugs are primarily concentrated in well-vascularized areas, leaving hypoxic regions largely untreated. This can result in surviving tumor cells that are highly aggressive and metastatic. Drug stability, bioavailability, and solubility pose additional obstacles. Head and neck cancer poses unique challenges due to the proximity of the cancer to critical structures, and the potential for aggressive progression. Traditional treatment modalities, including surgery, radiation therapy, and chemotherapy, often result in limited efficacy and substantial side effects. Recent advancements in targeted therapies and immunotherapies have shown promise; however, there remains a need for new approaches that enhance treatment efficacy while minimizing adverse effects. Treatment approaches based only on surgery, RT, CT and biotherapeutic antibodies fail to reach satisfactory results in terms both of survival and quality of life for head and neck cancer patients. The inventors have discovered that their targeted delivery system comprising a CD45+ leukocyte cell comprising a complex of an iron binding protein and an active ingredient is capable of specifically targeting and killing cancer cells in an animal model of aggressive head and neck cancer, alone or in combination with immune checkpoint modulator therapy.

[0008] Macrophages are abundant in solid tumor tissues and play a critical role in tissue homeostasis, immunity, and promotion and resolution of inflammation. They are attracted to the site of tissue injury and migrate into the tumor, reaching both vascular and hypoxic regions. Their migration is driven by cytokines and chemokines, such as CCL2 and CSF-18. As the only cell type that actively and abundantly infiltrates such hypoxic regions, macrophages represent an attractive vehicle for the delivery of therapeutic agents to tumors. Macrophage-based therapies are currently emerging as a promising avenue in cancer treatment, attracting increasing interest and research. Loading macrophages with anticancer agents is particularly interesting emerging approach in the development of anticancer therapies. The inventor’s approach involves the use of ferritin complexed with anticancer drugs, in a strategy called macrophage drug conjugate (MDC) therapy. The ferritin nanocage architecture enables effective drug encapsulation and overcomes challenges posed by drug stability, bioavailability, solubility, and toxicity. The inventors have discovered a mechanism, called TRAnsfer of Iron-binding proteiN (TRAIN), by which macrophages can directly transfer the complex of ferritin and anticancer drug to cancer cells. In contrast, for other nanoparticles or complexes that can be engulfed by macrophages, there is no such direct delivery mechanism and controlled release, but instead the compounds are released spontaneously. Thus, the use of ferritin has important advantages over other nanoparticles in macrophage-based approaches. The results shown herein provide critical evidence of the benefits of MDC in reducing tumor burden and increasing survival in head and neck cancer.

[0009] The inventive targeted delivery system for use in a method of treating head and neck cancer provides inter alia the following advantages over the prior art: (i) targeted delivery of active ingredients into head and neck cancer cells, (ii) improved penetration of active ingredients into deep sites of the head and neck cancer mass, (iii) improved distribution of active ingredients within the head and neck cancer mass, (iv) protection of active ingredients from inactivation in the blood circulation or clearance from the body, (v) delivery of active ingredients with poor pharmacokinetics into the head and neck cancer, (vi) reduced toxicity of active ingredients due to direct targeted delivery, (vii) higher treatment efficacy with lower doses of the active ingredient due to direct targeted delivery and local deposition in the head and neck cancer cell; (viii) easy access for intratumoral administration; (ix) reduced toxicity of local administration due to hidden / encapsulated drug; (x) efficient killing of tumor cells; and / or (xi) improved treatment efficacy compared to standard therapies for head and neck cancer.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention relates to an isolated targeted delivery system comprising a CD45+leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a pharmaceutically active substance, for use in a method of treating head and neck cancer.

[0012] The invention also relates to said isolated targeted delivery system and an immune checkpoint regulator for use in a method of treating head and neck cancer.

[0013] DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0014] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) and as described in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary- 20, published by the United States Pharmacopeial Convention, Inc., Rockville Md., 2001.

[0015] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0016] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0017] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise.

[0018] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0019] Targeted delivery system

[0020] The present invention relates to an isolated targeted delivery system comprising a CD45+leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a pharmaceutically active substance for use in a method of treating head and neck cancer.

[0021] The preferred way of administration is intratumoral administration. The term “targeted delivery” refers to the delivery of a pharmaceutically active substance (herein also referred to as “active ingredient”) to a subject, e.g. patient, in particular to a cell, more particularly directly into a cell within the body of a patient. The targeted delivery results in an increased concentration of the active ingredient in a particular region of the body when compared to administration of the active ingredient alone, administration of a complex of an iron binding protein and the active ingredient, or administration of other delivery systems. In particular, the targeted delivery results in an increased concentration of the active ingredient in tumor tissue, in particular head and neck cancer tissue, more particularly inside head and neck cancer cells, when compared to administration of the active ingredient alone, administration of a complex of an iron binding protein and the active ingredient, or administration of other delivery systems. Targeted delivery also includes “targeted theragnostic delivery”, meaning that both a therapeutic and a diagnostic agent are delivered concomitantly, preferably to a diseased region, thus allowing simultaneous treatment and diagnosis and / or treatment monitoring.

[0022] In preferred embodiments, the active ingredient is delivered directly into the head and neck cancer cells, preferably via direct transfer from the CD45+leukocyte to the head and neck cancer cell (direct cell-cell transfer). The direct transfer is preferably via a mechanism involving cell-cell contact and / or fusion of cell membranes.

[0023] The term “targeted pharmaceutically active substance delivery system” is used in the present application to refer to a system that is capable of delivering a pharmaceutically active substance to the targeted region, i.e. capable of targeted delivery within the body of a patient, preferably to a diseased region.

[0024] The term “targeted theragnostic delivery system” is used in the present application to refer to a system that is capable of delivering a complex of a pharmaceutically active substance and at the same time a label to the targeted region, i.e. capable of targeted delivery within the body of a patient, preferably to a diseased region and thus allows simultaneous treatment and diagnosis and / or treatment monitoring.

[0025] Targeted delivery systems have been described in WO 2016 / 207257 Al, WO 2016 / 207256 Al and WO 2017 / 222398 Al, which are incorporated herein by reference.

[0026] The isolated targeted delivery system may further comprise a cryoprotectant to enable freezing without harming the cells. The cryoprotectant may comprise dimethyl sulfoxide (DMSO), glucose, dextrose, mannitol, trehalose, salts, glutamine, glycerol, propylene glycol, sodium pyruvate, sodium bicarbonate and / or human serum albumin. Stability studies performed by the inventors with an isolated targeted delivery system comprising a cryoprotectant showed that the isolated targeted delivery system can be safely stored for months, highlighting its potential to become an "off-the-shelf product. This advantage addresses current challenges in cell-based therapies, such as the need for autologous approaches and manufacturing difficulties.

[0027] A preferred embodiment of the isolated targeted delivery system referred to herein is a macrophage comprising ferritin, in particular a ferritin according to SEQ ID NO: 1 or 4, preferably SEQ ID NO:4, linked to MMAE, in particular via a maleimidocaproyl-valine-citrulline-para-aminobenzoyl- oxycarbonyl (mc-vc-PAB) linker. Such an isolated targeted delivery system is also referred to as MDC herein.

[0028] Head and Neck Cancer

[0029] The term "head and neck cancer" is used in the broadest sense and refers to all cancers that start in the neck or in the head. It includes the subtypes laryngeal cancer, pharyngeal cancer, tongue cancer, hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer and oral and oropharyngeal cancer. Head and neck squamous cell carcinoma is a subtype of head and neck cancer which specifically arises from the squamous cells that line the mucosal surfaces of these organs. Head and neck cancer includes the following stages (as defined by the corresponding TNM classification(s) in brackets): stage 0 (Tis, NO, MO), stage I (Tl, NO, MO), stage II (T2, NO, MO), stage III (T3, NO, MO; or Tl to T3, Nl, MO), stage IVA (T4a, NO or Nl, MO; or Tl to T4a, N2, MO), stage IVB (T4b, any N, MO or any T, N3, MO), and stage IVC (any T, any N, Ml). The TNM classification is a staging system for malignant cancer. As used herein the term “TNM classification” refers to the 6th edition of the TNM stage grouping as defined in Sobin et al. (International Union Against Cancer (UICC), TNM Classification of Malignant tumors, 6th ed. New York; Springer, 2002, pp. 191-203).

[0030] In some embodiments, the targeted delivery system of the invention is provided for use in a method of treating head and neck cancer in a subject to which one or more risk factors can be attributed. These risk factors are preferably risk factors as defined by the American Cancer Society generally or for HNC. Examples of risk factors for HNC are: heavy alcohol use (more than 3 or 4 alcohol units a day for men, or more than 2 or 3 alcohol units a day for women; an alcohol unit is defined as 10 ml (8 g) of pure alcohol), tobacco consumption (in particular smoking, but also including smokeless tobacco), infection with cancer-causing types of human papillomavirus (HPV, especially HPV type 16), paan (betel quid) consumption, diet rich in preserved or salted foods during childhood, poor oral hygiene (manifested in, e.g., missing teeth), occupation (exposure to wood dust, nickel dust, asbestos, formaldehyde and / or synthetic fibers; worker in construction, metal, textile, ceramic, logging, and food industries), radiation exposure, Epstein-Barr virus infection, ethnicity (in particular Chinese), male gender, gastroesophageal reflux disease, Barrett’s esophagus, and age of 50 or older (in particular 55 or older).

[0031] Surgical removal represents the first-line treatment for head and neck cancer, complemented by chemotherapy, including cisplatin (CDDP), 5 -fluorouracil (5-FU) and docetaxel, and / or radiotherapy (RT), particularly at advanced stages. In some embodiments, the targeted delivery system is for use in a method of treating head and neck cancer in a human individual who experiences progressive disease or relapse during or following treatment with surgery, chemotherapy, or radiotherapy. In some embodiments, the head and neck cancer is resistant to radiotherapy and / or chemotherapy, in particular chemotherapy with cisplatin, 5 -fluorouracil and / or docetaxel. In some embodiments of the invention, the head and neck cancer is selected from pharyngeal cancer, salivary gland cancer and tongue cancer. Preferably, the head and neck cancer is HNSCC.

[0032] In some embodiments of the invention, the head and neck cancer is poorly immunogenic. It is envisioned that the head and neck cancer is resistant to immunotherapy alone. In some embodiments, the head and neck cancer, in particular HNSCC, is resistant to monotherapy with an immune checkpoint modulator, in particular to monotherapy with an anti-PD-1 antibody.

[0033] In some embodiments of the invention, the head and neck cancer is recurrent or refractive. In some embodiments of the invention, the head and neck cancer is metastatic. Surprisingly, the inventors found that treatment with the targeted delivery system, in particular MDC as described above, prolongs survival and reduces tumor volume in a mouse model of head and neck cancer, in particular HNSCC. HNSCC is one of the most common types of malignancies with poor prognosis. The HNSCC mouse model used by the inventors (SCC7) is highly aggressive and effects on tumour growth and survival shown using this model are highly relevant for HNSCC treatment. The effect was particularly pronounced in case of a combination therapy with the targeted delivery system and an anti-PD-1 antibody. This is particularly surprising since the efficacy of anti-PD-1 treatment in recurrent / metastatic HNSCC was described to be very limited, with response rates ranging from 13 to 20%. The mouse model used by the inventors was previously described to be immunologically cold and immunotherapyresistant (Mao et al., Br J Cancer. 2023 Jun; 128(11):2126-2139). The inventors observed an unexpected synergistic effect of the combination treatment with the targeted delivery system and anti-PD-1 (Fig. 2- 5).

[0034] Complex & Linker

[0035] Within the isolated targeted delivery system of the invention, the active ingredient may be covalently or non-covalently bound to the iron binding protein or may be encapsulated by the iron binding protein or multimers thereof. The term “complex” also encompasses the enclosure of an active ingredient within an iron binding protein or multimers thereof, in particular multimers of ferritin forming a “ferritin cage”. In instances where the active ingredient is encapsulated within an iron binding protein or multimers thereof, the encapsulation may occur in the presence or absence of a covalent or non- covalent bond. In some embodiments, active ingredients can be encapsulated within the internal cavity of a ferritin oligomer (physical confinement) by exploiting the association / dissociation properties of the ferritin macromolecule itself. In such embodiments, the active ingredients are held in place by non- covalent interactions with amino acid residues within the cavity internal surface. Haemoglobin macromolecules also offer the possibility of non-covalent binding of selected pharmaceutically active substances that may be hosted within the heme binding pocket of haemoglobin itself. The heme in the pocket can be displaced and be replaced by pharmaceutically active substances and / or labels with appropriate hydrophobicity profile.

[0036] The formation of the complex allows the transport of the active ingredients into the CD45+leukocyte when the CD45+leukocyte is internalizing the iron binding protein. Thus, it is preferred that the active ingredients are bound to the iron binding protein in a way that does not interfere with the transport mechanism. This can be easily tested by the skilled person using uptake assays known in the art and described in WO 2016207257 Al, WO 2016207256 Al and WO 2017222398 Al. If the complex comprising an active ingredient is taken up by a CD45+leukocyte and transported to a target cell within the body, it is preferred that the complex is sufficiently stable to survive the transport within the cell to the target region within the body. Thus, it is preferred that the complex rather than the active ingredient alone is delivered to, preferably into the target cells in the target region. This property also reduces possible deleterious effects, e.g. cytotoxicity, of the active ingredient to the CD45+leukocyte delivering the active ingredient or to other cells of the body that are not the target cells.

[0037] In preferred embodiments, the active ingredient and the iron binding protein are covalently and / or non-covalently linked, preferably covalently linked. If active ingredients are covalently linked to the iron binding proteins such coupling is preferably through amino acids residues known to be located in surface areas that are not involved in binding of the iron binding protein to a receptor involved in endocytosis.

[0038] In instances where the iron binding protein and the active ingredient are covalently linked, they may be linked directly or via a linker, i.e. indirectly. In preferred embodiments, the iron binding protein and the active ingredient are covalently linked via a linker.

[0039] Suitable linkers are known to the skilled artisan, such as polyalanine, polyglycin, carbohydrates, (CH2)ngroups or polypeptide linkers. The linker may be biodegradable or non-biodegradable, preferably biodegradable. In preferred embodiments, the linker is cleavable. The linker may be a peptide, disulfide, or hydrazone linker or a linker comprising carbohydrates. It is preferred that the linker is a peptide linker. In some embodiments, the peptide linker is cleavable by a protease. A linker comprising carbohydrates may be cleavable by P-glucuronidase. A hydrazone linker may be cleaved by acid hydrolysis. A disulfide linker may be cleaved by cytosolic reductive cleavage. In preferred embodiments, the linker is a peptide linker and is cleavable by lysosomal proteases, more preferably lysosomal cysteine proteases, even more preferably cathepsins.

[0040] In some embodiments, the linker comprises a reactive group that, when activated by a defined stimulus, effects cleavage of a covalent bond within linker. Suitable reactive groups are e.g. light- activatable groups (i.e. groups that can be activated by UV light, such as 3-amino-3-(-2-nitro)phenyl- propionic acid or a light-activatable structural equivalent thereof), dithionite-activatable groups (such as an azobenzene moiety), or periodate-activatable groups (such as a 1,2-dihydroxy moiety, a l-amino-2- hydroxy moiety or 4-amino-4-deoxy-L-threonic acid). In other embodiments, the linker is cleavable via a pH shift.

[0041] Pharmaceutically active substances may also be covalently bound to iron binding protein amino acid side chains (lysines or cysteines) by appropriate choice of phenylhydrazone, succinimide or maleimide activated drugs. A phenylhydrazone derivative may break and liberate the drug from the iron binding protein, a lysine bound derivative may become active after full protein degradation into aminoacids, or a cysteine bound derivative may be liberated within the cell through reductive hydrolysis of the maleimede thioether link.

[0042] In some embodiments, the linker is a dipeptide linker, in particular Val-Cit, Vai-Ala, or Ala-Ala, or a tripeptide and tetrapeptide linker.

[0043] In some embodiments, the linker is a hetero-bifunctional crosslinker that contains N- hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent conjugation of amine- and sulfhydryl-containing molecules. In some embodiments, the linker is succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC) or Sulfo-SMCC.

[0044] It is preferred that the cleavable linker is cleaved within the lysosomal compartment.

[0045] In most preferred embodiments, the linker is a maleimidocaproyl-valine-citrulline-para- aminobenzoyloxycarbonyl (mc-vc-PAB) linker.

[0046] In some embodiments, the iron binding protein is conjugated to the active ingredient or linker via a cysteine residue or a lysine residue, preferably a cysteine residue. For the formation of covalent bonds, relevant thiol, amino or carboxyl groups of the iron binding proteins are used to covalently couple active ingredients reactive towards thiol or amino groups directly or indirectly to the iron binding protein. The active ingredients may be modified by specific active moieties, i.e. linkers.

[0047] As such, ferritins or haemoglobin may be linked to cysteine thiol reactive pharmaceutically active substances and / or labels bearing a peptide based cleavable linker (e.g. cathepsin sensitive valinecitrulline sequence and para-aminobenzylcarbamate spacer). As a notable example, the antimitotic agent monomethyl auristatin E (MMAE) has been used. The peptide-based linker binds the protein to the cytotoxic compound in a stable manner so the drug is not easily released from the protein under physiologic conditions, which helps prevent toxicity to healthy cells and ensures dosage efficiency. The iron binding protein pharmaceutically active substance and / or label adduct thus generated is capable of attaching to the selected receptor types, e.g. CD 163 for haemoglobin and TfR for transferrin, respectively. Once bound, the iron binding protein pharmaceutically active substance and / or label adduct is internalised by endocytosis and thus selectively taken up by the cells. The vesicle containing the active ingredient is fused with lysosomes and lysosomal cysteine proteases, particularly cathepsin B, start to break down the cleavable peptide linker, in particular the valine-citrulline linker, and the active ingredient, in particular MMAE, is no longer bound to the iron binding protein and is released directly into the tumour environment.

[0048] Alternatively, DM1 -SMCC is an efficient mertansine derivative bearing a linker that specifically binds to lysine residues generating a covalent complex with ferritin, haemoglobin or transferrin in a reaction that has been successfully described for antibodies. In particular, haemoglobin, ferritin or transferrin can be reacted with DM1 -SMCC thus providing a covalent protein-drug adduct that can be cleaved inside cells and releases the active drug in a time-dependent manner. The suppression of microtubule dynamics by DM1 induces mitotic arrest and cell death. Ways of preparing complexes of an iron binding protein and an active ingredient are described in WO 2016 / 207257 Al, WO 2016 / 207256 Al and WO 2017 / 222398 Al.

[0049] The term “full load” is used in the context of the present invention to refer to the maximum amount of iron binding protein, preferably ferritin, complexed with a pharmaceutically active substance that can be taken up by the CD45+leukocyte cell, preferably macrophage more preferably activated macrophage.

[0050] It is also envisioned that different active ingredients are comprised in the isolated targeted delivery system. For example, one type of active ingredient may be covalently bound to a ferritin polypeptide, while another type is encapsulated in the complex. This approach utilizes different release rates of the active ingredients from the complex once delivered to the targeted tissue and / or cells. For example, an active ingredient can be covalently attached to a ferritin molecule either on the surface of the 24-mer or within the internal cavity by exploiting the reactivity of relevant thiol, amino or carboxyl groups. The types of such useful reactions are well known in the art and can be adopted by the person skilled in the art to the particular active ingredient 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(l):46-53.

[0051] In theragnostic applications, i.e., applications in which the complex comprises both a label and a pharmaceutically active substance, it is preferred that the label is covalently attached to the iron binding protein and the pharmaceutically active substance is non-covalently bound to the iron binding protein and / or entrapped in the internal cavity formed upon assembly of the multimer of iron binding proteins.

[0052] Iron binding protein

[0053] In some embodiments, the iron binding protein is selected from the group consisting of ferritin, preferably heavy (H) type ferritin, light (L) ferritin and / or mitochondrial ferritin; haemoglobin, preferably haemoglobin A, haemoglobin AS, haemoglobin SC, haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F, haemoglobin H; haemoglobin-haptoglobin complex, haemopexin, transferrin; and lactoferrin. The terms ferritin; haemoglobin, preferably haemoglobin A, haemoglobin AS, haemoglobin SC, haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F, haemoglobin H; haemoglobin-haptoglobin complex, hemopexin, transferrin; and lactoferrin encompass structural variants of the naturally occurring proteins and, thus relates to proteins that have 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 respective wildtype protein to bind iron ion(s). The iron binding proteins used in the context of the present invention are preferably of mammalian, more preferably mouse, rat, dog, ape, in particular, chimpanzee, or human, most preferably of human origin. Consensus sequences of the preferred iron binding proteins and preferred structural variants are disclosed in WO 2016 / 207257 Al.

[0054] Human transferrin and ferritin proteins have been considered as effective carriers for the delivery of small molecules or toxin-conjugates to specifically target cancer cells. To date, in spite of considerable efforts, no successful transferrin or ferritin drug complexes have however reached the clinic (Luck AN et al. 2013, Adv Drug Deliv Rev 65(8): 1012-9).

[0055] Ferritin is a hollow globular protein complex consisting of 24 ferritin monomer subunits assembled into a cage-like structure. Ferritin is the primary intracellular iron-storage protein. It is produced by almost all living organisms and is present in every cell type. Ferritin genes are highly conserved among species. In vertebrates, two ferritin monomers exist: the light (L) chain and the heavy (H) chain type with a molecular weight of 19 kDa or 21 kDa respectively. Vertebrate ferritin 24-mers can be homooligomers consisting of either L or H chains, or hetero-oligomers consisting of both L and H chains (Theil EC, 1987, Annual Review of Biochemistry. 56 (1): 289-315): Typically ferritin complexes have internal and external diameters of about 8 and 12 nm, respectively. Ferritin was shown to be internalized by endocytosis upon binding to CD71. Interaction of ferritin and CD71 is mediated via ferritin-H chains (Li L et al, Proc. Natl. Acad. Sci. USA 107 (8) (2010) 3505-3510). Ferritins are not abundant in plasma, but can be readily produced in high yield as recombinant proteins in common protein expression systems such as Escherichia coli cells.

[0056] Purified transferrin can be efficiently conjugated to various molecules including anticancer drugs through covalent linkers that are appropriately released inside the cells (Beyer U et al. 1998, J Med Chem 41(15):2701-2708). In case of transferrin, only lysine groups on the protein surface are readily available for covalent attachment.

[0057] Haemoglobin (Hb) has been considered in the past as a possible drug carrier, due to its versatility in chemical conjugation with drugs, its abundance and relative stability in the blood (Somatogen, 1993, WO 1993 / 008842 Al). Nevertheless, the lack of receptor targeting properties did not foster biomedical applications other than blood substitutes or antisickling agent. Haemoglobin can only be recognized by CD 163 (haptoglobin / haemoglobin receptor) epitopes on the leukocytes, especially monocytemacrophage origin. The CD45+leukocyte, in particular macrophage-based protein delivery, described in this application moved haemoglobin center stage as a target specific carrier of pharmaceutically active substances and / or labels. Haemoglobin can be readily covalently linked to appropriate pharmaceutically active substances and / or labels, host hydrophobic pharmaceutically active substances and / or labels within the heme binding pocket or even transport small molecules, e.g. cytotoxic molecules linked to the heme iron. Hb can be easily modified by selective attachment of the appropriate drug conjugate to the beta93 cysteine residue, the only titratable cysteine on the protein surface. Maleimido functionalized drugs, such as the tubulin inhibitor Monomethyl auristatin (MMAE) or the DNA crosslinking drug Pyrrolobenzodiazepine dimer (PBD) are most notable examples of extremely potent cytotoxic agents that can be readily and specifically attached to the relevant cys beta93 residue.

[0058] Alternatively, lysine residues on the Haemoglobin surface (at least 10 titratable lysine residues per Hb tetramer) may be easily amenable to drug conjugation through cleavable succinimide linkers. Haemoglobin also offers a unique capability of releasing non-covalently bound heme group at acidic pH values. Apo-haemoglobin thus obtained is capable of hosting several hydrophobic molecules within 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 labels with fluorescent properties (e.g. chlorine e6, hyperycin, phtalocyanine derivatives) (Dong J et al. J PhotochemPhotobiol B 2014, 140: 163-172).

[0059] In a preferred embodiment, the iron binding protein is ferritin, preferably a mammalian ferritin. The mammalian ferritin may be a mouse, rat, dog, ape, in particular chimpanzee, or human ferritin. In a preferred embodiment, the mammalian ferritin is a mouse, rabbit, rat or human ferritin, preferably human ferritin. In an even more preferred embodiment, the human ferritin is a human heavy chain ferritin.

[0060] Amino acid substitutions within the iron binding proteins are preferably selected in a way that they do not unduly change the conformation of the polypeptide. As an example, a “small amino acid” should be substituted with another small amino acid. A “small amino acid” in the context of the present invention is preferably an amino acid having a molecular weight of less than 125 Dalton. Preferably, a small amino acid in the context of the present invention is selected from the group consisting of the amino acids glycine, alanine, serine, cysteine, threonine, and valine, or derivatives thereof. As another example, an amino acid having a hydrophobic side chain should be substituted with another amino acid having a hydrophobic side chain.

[0061] In some embodiments, the ferritin comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NO: 1-4 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, in particular human wild type ferritin, to bind iron ion(s) and / or 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 amino acid, preferably Pro, X at position 14 can be any amino acid, preferably His, X at position 16 can be any amino acid, preferably Asp, X at position 21 is absent or any amino acid, preferably He, X at position 22 can be any amino acid, preferably Asn, X at position 30 can be any amino acid, preferably Tyr, X at position 40 can be any amino acid, preferably Tyr or Cys, more preferably Tyr, X at position 82 can be any amino acid, preferably Phe, X at position 84 can be any amino acid, preferably Gin, X at position 91 can be any amino acid, preferably Arg or Cys, more preferably Cys, X at position 106 can be any amino acid, preferably His, X at position 110 can be any amino acid, preferably Asn or Ser, more preferably Asn, X at position 137 can be any amino acid, preferably His or Tyr, more preferably His X at position 140 can be any amino acid, preferably Asn or Ser, more preferably Asn, X at position 145 can be any amino acid, preferably Ala or Ser, more preferably Ala, X at position 164 can be any amino acid, preferably Ala or Ser, more preferably Ser, X at position 166 can be any amino acid, preferably Met or Leu, preferably Leu, X at position 178 can be any amino acid, preferably Asp or His, more preferably Asp, X at position 181 is absent or any amino acid, preferably Asn, X at position 182 is absent or any amino acid, preferably Glu, and X at position 183 is absent or any amino acid, preferably Ser. If an amino acid position can be any amino acid, it is preferably a naturally occurring amino acid. In some embodiments, the ferritin comprises an amino acid sequence according to SEQ ID NO: 4, optionally comprising 1-5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 54, 72, 87 and / or 144, in particular outside position 54, and having at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the ability of wild-type ferritin, in particular human wild type ferritin, to bind iron ion(s) and / or form ferritin 24-mers, in particular to form ferritin 24-mers.

[0062] Any ferritin used in the isolated targeted delivery system for use according to the invention has to retain the properties of a wild type ferritin with regard to complex formation (cage-like structure consisting of 24 ferritin monomer subunits) and uptake of iron.

[0063] Drugs and Labels

[0064] The inventors successfully generated a variety of stable ferritin-drug complexes, incorporating a wide range of drugs from different pharmacological categories.

[0065] The terms “drug”, “active ingredient”, therapeutic agent” or “pharmaceutically active substance” are used synonymously in the context of the present invention and refer to any compound that modifies or modulates cell activity or is capable of being activated, i.e. a prodrug, to modify or modulate cell activity, preferably in the body of a patient. Examples of such active ingredients include so called “small molecules” and peptides. The term “small molecule” is used in the context of the present invention to refer to a hydrocarbon with a molecular mass of below 1.500 g / mol or to pharmaceutically active radioactive isotopes.

[0066] In preferred embodiments, the pharmaceutically active substance is an anticancer drug selected from the group consisting of a protein, a peptide, a nucleic acid, a non-protein non-nucleic acid compound with a molecular weight of less than 1.5 kD, a photosensitizing compound, a virus, and pharmaceutically active radioactive isotope.

[0067] A preferred pharmaceutically active radioactive isotope is an a or B radiation emitting radioisotope, which also emits a cell damaging amount of y radiation is selected from the group consisting of lutetium- 177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, caesium-131, palladium- 103, radium- 233, iodine- 125, and boron- 10 or a cell damaging amount of a radiation, preferably selected from the group consisting of actinium-225, bismuth-213, lead-212, and polonium-212. Also preferred is a complex of above-mentioned compounds and isotopes linked to the nanoparticles (e.g. gold, argentum, graphen) or these nanoparticles.

[0068] If the pharmaceutically active substance is a virus, it is preferably an oncolytic virus.

[0069] If the drug is a nucleic acid it is preferred that it is a miRNA, siRNA, chemically modified-RNA, LNA, ssRNA, DNAzyme or a nucleic acid encoding a pharmaceutically active protein, e.g. an antibody, an antibody mimetic, a cytokine, a prodrug-converting enzyme, an immunogenic peptide or the like.

[0070] In a preferred embodiment, the anticancer drug is a cytostatic drug, cytotoxic drug or prodrug thereof. Preferred anticancer drugs are selected from an apoptosis / autophagy or necrosis-inducing drug. An apoptosis / autophagy or necrosis-inducing drug can be any drug that is able to induce apoptosis / autophagy or necrosis effectively even in cells having an abnormality in cell proliferation. These drugs are preferably used in complexes with one or more ferritins.

[0071] In preferred embodiments, the anti-cancer drug is selected from the group consisting of an apoptosis-inducing drug, an alkylating substance, anti-metabolites, antibiotics, antimitotic agent, a DNA-modifying drug, a DNA minor groove interstrand crosslinking drug, an inhibitor of DNA synthesis, an inhibitor of RNA synthesis, epothilones, nuclear receptor agonists and antagonists, an anti- androgene, an anti-estrogen, a platinum compound, a hormone, a antihormone, an interferon, an inhibitor of cell cycle-dependent protein kinases (CDKs), an inhibitor of cyclooxygenases and / or lipoxygenases, a biogenic fatty acid, a biogenic fatty acid derivative, including prostanoids and leukotrienes, an inhibitor of protein kinases, an inhibitor of protein phosphatases, an inhibitor of lipid kinases, a platinum coordination complex, an ethyleneimine, a methylmelamine, a triazine, a vinca alkaloid, a pyrimidine analog, a purine analog, an alkylsulfonate, a folic acid analog, an anthracendione, a substituted urea, and a methylhydrazin derivative, an ene-diyne antibiotic, a maytansinoid, an auristatin derivate, an immune check-point inhibitor, and an inhibitor of a tumour-specific protein or marker, preferably a Rho-GDP-dissociation inhibitor, more preferably Grp94 or AXL inhibitor, a tubulin inhibitor, or a topoisomerase inhibitor.

[0072] In preferred embodiments, the anti-cancer drug is selected from the group consisting of acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chaliceamycin, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycindapsone, daunorubicin, deruxtecan, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, dolastatin 10, dolastatin 15, dynemycinA, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogen, ethinylestradiole, etoposide, exatecane derivative, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamidefosfestrol, furazolidone, gemcitabine, gonadotropin releasing hormone analog, hexamethylmelamine, hydroxy carbamide, hydroxymethylnitrofurantoin, hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon a, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, maytansine, mechlorethamine, medroxyprogesterone acetate, megastrolacetate, melphalan, mepacrine, mercaptopurine, mertansine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, neocazinostatin, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, pyrrolobenzodiazepine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustinestreptozocine, sn-38, sulfacarbamide, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafiirazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxazole, co-trimoxazole, sulfamethoxy diazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosteronpropionate, thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine, and zorubicin. More preferably, the anti-cancer drug is selected from the group consisting of auristatin, banoxantrone, bendamustine, chlorambucil, chaliceamycin, dynemycin A, maytansine, melphalan, mertansine, neocazinostatin and pyrrolobenzodiazepine.

[0073] In particularly preferred embodiments, the anti-cancer drug is auristatin, in particular monomethyl auristatin E or monomethyl auristatin F. The examples were performed using the drug monomethyl auristatin E.

[0074] In some embodiments, the anti-cancer drug is an immunomodulatory drug that activates or inhibits an activity of an immune cell, preferably the immunomodulatory drug is a ligand or antagonist of Pattern Recognition Receptors, particularly Toll-like Receptors, NOD-like receptors (NLR), RIG-I-like receptors (RLR) or Stimulator of interferon genes (STING) protein. Physiologically, these receptors recognize classes of signals known as pathogen-associated molecular patterns (PAMPs) and damage- associated molecular patterns (DAMPs).

[0075] In preferred embodiments, the anti-cancer drug is a proliferation inhibiting protein or 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 encoding a proliferation inhibiting protein or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a siRNA, oligonucleotide, LNA, or DNAzyme.

[0076] The term "prodrug" as used in the context of the present invention refers to any active ingredient that, after administration, is metabolized or otherwise converted to a biologically active or more active ingredient (or drug) with respect to at least one property. In comparison to the drug, a prodrug is modified chemically in a manner that makes it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered to the patient. A prodrug may for example have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.

[0077] In some embodiments, the pharmaceutically active substance is a hypoxia-activated prodrug, preferably selected from the group consisting of benzotriazine N-oxides, apaziquone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH-302, TH-4000 and AQ4N.The use of active ingredients, which are activated under hypoxic conditions can add a further specificity to the targeting and / or further reduces adverse effects of the active ingredients. Thus, in particularly preferred embodiments the active ingredient is a hypoxia-activated prodrug. The backbone of all the hypoxia-activated prodrugs is the presence of one of five different chemical moieties (nitro groups, quinines, aromatic and aliphatic N- oxides and transition metals) that are enzymatically reduced under hypoxic conditions in tissue. Hypoxia-activated prodrugs are any prodrug that is less active or inactive, relative to the corresponding drug, and comprises the drug and one or more bioreducible groups. Such hypoxia-activated prodrugs include all prodrugs activated by a variety of reducing agents and reducing enzymes, including without limitation single electron transferring enzymes (such as cytochrome P450 reductases) and two electron transferring (or hydride transferring) enzymes. According to preferred embodiment of the invention hypoxia-activated prodrug is TH-302. Methods of synthesizing TH-302 are described in PCT application WO 07 / 002931 and WO 08 / 083101.

[0078] In some embodiments, the pharmaceutically active substance is an antigen or a nucleic acid encoding an antigen.

[0079] The isolated targeted delivery system may comprise a label in addition to the pharmaceutically active substance.

[0080] The terms “label” or “diagnostic agent” are used interchangeably herein and refer to any kind of compound being suitable for diagnostic purposes. In a preferred embodiment, the label is selected from the group consisting of a fluorescent dye, a radioisotope / fluorescence emitting isotope, a detectable polypeptide or nucleic acid encoding a detectable polypeptide, and a contrast agent or the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations. More preferably, the label is selected from a fluorescent dye, a radioisotope and a contrast agent. A contrast agent is a dye or other substance that helps to show abnormal areas inside the body.

[0081] Preferred fluorescent dyes are selected from the group consisting of the following classes of fluorescent dyes: xanthens (e.g. fluorescein), acridines (e.g. acridine yellow), oxazines (e.g. oxazine 1), cynines (e.g. Cy7 / Cy 3), styryl dyes (e.g. dye-28), coumarines (e.g. Alexa Fluor 350), porphines (e.g. chlorophyll B), metal-ligand-complexes (e.g. PtOEPK), fluorescent proteins (e.g. APC, R- phycoerythrin), nanocrystals (e.g. QuantumDot 705), perylenes (e.g. Lumogen red F300) and phtalocyanines (e.g. IRDYE™700DX) as well as conjugates and combinations of these classes of dyes.

[0082] Preferred radioisotopes / fluorescence emitting isotopes are selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65Tb, fluorescence emitting isotopes, such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, U lin, 99mTc, 140La, 175Yb, 153Sm, 166Ho,88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101ml5Rh, 119Sb, 128Ba, 1231, 1241, 1311, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag as well as conjugates and combinations of above with proteins, peptides, small molecular inhibitors, antibodies or other compounds (e.g. 18F-FDG, 89Zr-oxide or 64Cu-porfirin). Preferred detectable polypeptides are an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof with an altered adsorption and / or emission spectrum.

[0083] Preferred contrast agents are selected from paramagnetic agents, e.g. Gd, Eu, W and Mn, preferably complexed with a chelating agent. Further options are superparamagnetic iron (Fe) complexes and particles, compounds containing atoms of high atomic number, i.e. iodine for computer tomography (CT), microbubbles and carriers such as liposomes that contain these contrast agents. In a preferred embodiment, the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations.

[0084] Preferred chelating agents are selected from the group consisting of 1,4,7,10- tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, Diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTP A) and 6-Hydrazinopyridine-3 -carboxylic acid (HYNIC).

[0085] Leukocyte

[0086] The ability of a given cell or of a population thereof to internalize iron binding proteins depends on the expression of receptors involved in this internalization process. Receptors that lead to internalization of ferritin comprise, e.g. TfR, MSR-1, CXCR4, scavenger receptors, CD163, and TIM-2, wherein MSR- 1 is particularly important for the internalization of ferritin by macrophages. Ferritin internalization by CD45+ leukocytes, in particular macrophages, occurs primarily via clathrin-dependent endocytosis.. The skilled person is well aware how to measure the amount of ferritin uptake and preferred methods of measuring the uptake are described in the Example Section below.

[0087] The term “leukocyte” (or “leukocyte cell”) refers to cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All leukocytes are produced and derived from multipotent cells in the bone marrow known as a hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system. All leukocytes have nuclei, which distinguishes them from the other blood cells, the anucleated red blood cells (RBCs) and platelets. Types of leukocyte can be classified in standard ways. Two pairs of the broadest categories classify them either by structure (granulocytes or agranulocytes) or by cell division lineage (myeloid cells or lymphoid cells). These broadest categories can be further divided into the five main types: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These types are distinguished by their physical and functional characteristics. Monocytes and neutrophils are phagocytic. Further subtypes can be classified; for example, among lymphocytes, there are B cells, T cells, and NK cells. Granulocytes are distinguished from agranulocytes by their nucleus shape (lobed versus round, i.e., polymorphonuclear versus mononuclear) and by their cytoplasm granules (present or absent, or more precisely, visible on light microscopy or not thus visible). The other dichotomy is by lineage: Myeloid cells (neutrophils, monocytes, eosinophils and basophils) are distinguished from lymphoid cells (lymphocytes) by hematopoietic lineage (cellular differentiation lineage).

[0088] CD45 expression is characteristic of a subgroup of leukocyte cells, i.e. monocyte, monocytemacrophages, lymphocytes, granulocytes, NK cells that are suitable to be used in the context of the targeted delivery system of the present invention, in particular since CD45+leukocyte cells are attracted to particular tissues and cells within the body and are capable of delivering complexes of one or more iron binding proteins and one or more pharmaceutically active substances, labels or pharmaceutically active substances and labels to or into cells. This subgroup of leukocytes is in the following referred to as “CD45+leukocyte cells” or “CD45+leukocytes”. Preferably the monocyte is not a dendritic cell which differentiation is controlled by one or more of the following transcription factors: IFN-regulatory factor 8 (IRF8), nuclear factor interleukin (IL) -3 -regulated protein (NFIL3), basic leucine zipper transcriptional factor ATF -like 3 (BATF3) or Transcription Factor RelB (NF -KB Subunit) - RELB, Spi- 1 Proto-Oncogene (PU / 1), recombining binding protein suppressor of hairless (RBPJ), IFN-regulatory factor 4 (IRF4) or transcription factor E2-2 (also known as (TCF4).

[0089] It is understood by the skilled person that CD45+leukocyte cells as defined above unless of clonal origin are a mixed population of different leukocytes which share the common property of expressing CD45 surface antigen. Accordingly, subpopulations of cells within the diverse group of CD45+leukocyte cells as defined above are characterized throughout the specification by further functional and / or structural characteristics. The term “CD45+” indicates that the majority of cells within a population of cells or essentially all cells express the CD45 surface antigen.

[0090] “Expressing” means in this respect that the majority of cells within a population of cells or essentially all cells express the marker (also called surface antigen herein). In this context and also with reference to other cellular surface antigens, the term “expresses” indicates that the surface antigen is produced within the cell and detectably exposed on the surface of a cell. The level of expression and, thus the number of surface antigens detectably exposed on the surface of a cell can vary greatly among different cells. Generally, a cell is considered to be positive, i.e. is indicated to be “+”, for a cellular surface antigen, if at least 5, preferably at least 10 copies of the surface antigen are detectably exposed on the surface of the cell. The skilled person is well aware of how to detect, quantify and select for cells, which are positive (or negative) for a given cellular surface antigen. Preferred methods include Fluorescence Activated Cell Sorting (FACS). In this technology, fluorescently labelled antibodies are used to bind to cellular surface antigens of a population of cells, the cells are subsequently isolated into single cells and based on fluorescence intensity measured for the single cell, characterized as being positive or negative for the given cellular surface antigen. In some embodiments of the present invention it is indicated that the expression of a given protein is high or low. This means that the protein is detectably expressed in both instances, i.e. is “+”, however, at different levels. High and low expression, respectively, will mean different absolute numbers of proteins per cell for different proteins. Thus, a given protein may be considered to be expressed at high levels if there are more than 500 detectable copies of that protein per cell and to be expressed at low levels if there are between 1 to 50 detectable copies of that protein per cell. However, another protein may be considered to be expressed at high levels, if there are more than 5000 detectable copies and expressed at low levels, if there are between 1 to 500 detectable copies per cell. It is well known in the art how to quantify the number of proteins expressed or produced in a cell using flow cytometry and Becton Dickinson Quantibrite™ bead method (see e.g. Pannu, K.K., 2001, Cytometry. 2001 Dec l;45(4):250-8) or mass spectrometry (see, e.g. Milo, R„ 2013, Bioessays, 35(12): 1050-1055).

[0091] For the purpose of the present invention the term “high expression” of a given protein refers to detectable expression of that protein that is at least 70% of the highest expression level found, i.e. number of copies per cell, in a population of healthy cells, in particular CD45+leukocytes. The term “low expression” of a given protein refers to detectable expression of that protein that is 30% or less of the highest expression level found, i.e. number of copies of that protein per cell, in a population of healthy cells, in particular CD45+leukocytes. Preferably, the “highest expression level” is determined as the average of the highest expression levels found in healthy cells, in particular CD45+leukocytes of different subjects. In some embodiments preferred subpopulations of cells are characterized as “producing” a given protein. This is understood to mean that the protein is not necessarily detectable on the surface of the cell but may only be present inside the cell. The skilled person is well aware how to detect and / or quantify production of a protein inside a cell and / or select cells producing such proteins. Alternatively, cell populations can be defined by expression of specific transcription factors. It is well known in the art how to determine expression of a given protein or its encoding mRNA in a population of cells or even in single cells, e.g. using in vivo labeling with antibodies, FISH assays, in vivo single molecule fluorescent microscopy (Crawford, R. et al. Biophys J. (2013) 105(11): 2439) alone or in combination with Fluorescent Activated Cell Sorting (FACS), or by the PrimeFlow technique (e Bioscience), (Adam S. Venable, et. al., (2015) Methods in Molecular Biology).

[0092] The CD45+leukocyte cell comprised in the targeted delivery system for use according to the invention is producible from a CD34+hematopoietic precursor cell. In some embodiments, the CD45+leukocyte is selected from the group consisting of a monocyte, a differentiated monocyte, preferably a macrophage, a lymphocyte and a granulocyte.

[0093] In the isolated targeted delivery system of the present invention for use in the treatment of head and neck cancer preferably comprises a CD45+leukocyte cell selected from a monocyte and a macrophage. Unless dictated otherwise, the term “CD45+leukocyte cell” herein preferably refers to a monocyte or a macrophage, most preferably a macrophage.

[0094] In some embodiments, the CD45+leukocyte cell is a monocyte. The monocyte may be an activated monocyte. The monocyte may be a differentiated monocyte.

[0095] The term “differentiated monocyte” is used in the context of the present invention to refer to a monocyte differentiated from the committed precursor termed macrophage-DC precursor (MDP) mainly resident in bone marrow (but could be also in the spleen) and differentiate into either dendritic cells or macrophages. In mice they consist of two main subpopulations: (i) CD1 lb+cell with high expression of CX3CR1, low expression of CCR2 and Ly6C- and (ii) CDl lb+cell with low expression of CX3CR1, high expression of CCR2 and Ly6C+. After leaving the bone marrow, mouse Ly6C+monocytes differentiate into Ly6C- monocytes in circulation. Similarly, in human monocyte differentiation, it is accepted that CD 14++classical monocytes leave bone marrow and differentiate into CD 14++CD16+intermediate monocytes and sequentially to CD14+CD16++non-classical monocytes in peripheral blood circulation (Yang et al. 2014; Biomark Res 2(1) doi. 10.1186 / 2050-7771-2-1). Preferably the differentiated monocyte is not a dendritic cell, which 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 is not a dendritic cell. A preferred embodiment of a differentiated monocyte is a macrophage.

[0096] Macrophages are tissue-resident professional phagocytes and antigen-presenting cells (APC), which differentiate from circulating peripheral blood monocytes (PBMs). The term “activated macrophage” is used in the context of the present invention to refer to any macrophage that is polarized. Unpolarized macrophages are referred to as M0 macrophages herein. Macrophage activation is in general achieved by incubation with interleukins, cytokines and / or growth factors. In particular IL-4 and M-CSF can be used as activating agents. Activated macrophages of different phenotypes are classified into Ml -macrophages, classically activated macrophages (CAM) and M2 -macrophages, alternatively activated macrophages (AAM). The classically activated Ml -macrophages comprise immune effector cells with an acute inflammatory phenotype. These are highly aggressive against bacteria and produce large amounts of lymphokines (Murray, and Wynn, 2011, J LeukocBiol, 89(4):557-63). The alternatively activated, anti-inflammatory M2-macrophages can be separated into at least three subgroups. These subtypes have various different functions, including regulation of immunity, maintenance of tolerance and tissue repair / wound healing. The term “Ml inducer” is used in the context of the present invention to refer to a compound that directs differentiation of PBMs to macrophages of the Ml type. The term “M2 inducer” is used in the context of the present invention to refer to a compound that directs differentiation of PBMs to macrophages of the M2 type. The skilled person is aware of a large number of ways to promote differentiation into either Ml or M2 macrophages. The term “phagocytosis by macrophages” is the process by which a macrophage engulfs a solid particle to form an internal vesicle known as a phagosome. The expressions “viral / bacterial / fimgal / helminth proteins or products” refers to molecules produced by or originating from viruses, bacteria, fungi or helminths during a viral / bacterial / fungal / hehninth infection.

[0097] If the CD45+leukocyte cell is a monocyte, it is preferred that it is a CD 1 lb+monocyte, preferably selected from the group consisting of a CDl lb+CD14+monocyte, a CDl lb+CD16+monocyte, a CDl lb+CD14+CD16+monocyte, a CDl lb+CD14 HLA-DR monocyte, a CDl lb+CD14+CD115+monocyte, a CDl lb+CD14+monocyte, a CDl lb+CD16+monocyte, a CDl lb+CCR1+monocyte, a CDl lb+CCR2+monocyte, a CDl lb+CX3CR+monocyte, a CDl lb+CXR4+monocyte, a CDl lb+CXR6+monocyte and a CD1 lb+CD14+CD33+monocyte.

[0098] With respect to the differentiated monocyte, it is preferred that it is selected from the group consisting of a macrophage, an activated macrophage, preferably a CDl lb+macrophage, more preferably a CD1 lb+CD16+macrophage, a CD1 lb+CD32+macrophage, a CD1 lb+CD64+macrophage, a CDl lb+CD68+macrophage, preferably a CDl lb+CD86+Ml macrophage, preferably producing iNOS and / or secreting interleukin 12 (IL-12) or preferably a CD1 lb+CCR2+M2 macrophage, a CD1 lb+CD204+M2 macrophage, a CDl lb+CD206+M2 macrophage, a CDl lb+CD204+CD206+M2 macrophage, a CDl lb+HLA-DR+M2 macrophage, a CDl lb+CD200R+M2 macrophage, a CDl lb+CD163+M2 macrophage or an activated macrophage producing arginase and / or secreting interleukin 10 (IL-10); and a dendritic cell (DC), preferably a CDl lb+CDl lc+DC, CDl lb+CD80+DC, CDl lc+CD80 DC, CDl lc+CD86 DC, CD1 Ic HLA-DR DC or CD1 lc+CD123 DC, preferably the differentiated monocyte-macrophage is not a Loxl+, CXCR7+and NRF2+foam cell.

[0099] In preferred embodiments, the CD45+leukocyte cell is a monocyte / macrophage. Macrophages are differentiated (matured) monocytes and the term monocyte / macrophage is meant to refer to monocytes, macrophages and intermediate cell types, i.e. monocytes that are partly differentiated into macrophages. Monocytes and macrophages share important features (such as the ability to phagocytose) and markers (such as CD 14). The direct delivery into tumor cells that is important for the present invention is achieved with monocytes and macrophages as well as intermediate monocyte / macrophage cells.

[0100] In preferred embodiments, the CD45+leukocyte cell is a macrophage. In some embodiments, the macrophage is a differentiated macrophage, in particular an Ml macrophage or an M2 macrophage. 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 unpolarized macrophage. In preferred embodiments, the macrophage is an MO macrophage or a macrophage that is mildly polarized towards Ml or M2. In some embodiments, the macrophage is an Ml or M2 macrophage. The skilled person is aware of surface markers expressed by Ml or M2 macrophages or macrophages that are mildly polarized towards Ml or M2.

[0101] Preferably, the differentiated monocyte expresses at least one chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CXCR4, and CXCR6, or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony-stimulating factor receptor (CD115), granulocyte colony-stimulating factor receptor (CD114), and granulocytemacrophage colony stimulating factor receptor (CD116 and CD 131).

[0102] In preferred embodiments, the monocyte or differentiated monocyte:

[0103] (i) is producible from a CD34+hematopoietic precursor cell;

[0104] (ii) is producible by in vitro incubation of monocytes with at least one inducer, preferably Ml or M2 inducer, more preferably at least one M2 inducer; (iii) is characterized by 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 / orCXCR4, 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

[0105] (iv) has the ability to phagocytose.

[0106] Preferably,

[0107] (i) the Ml inducer is selected from the group consisting of LPS, GM-CSF, INF-y, viral or bacterial proteins or products;

[0108] (ii) the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, an immune complex of an antigen and antibody, IgG, heat activated gamma-globulins, glucocorticosteroids, TGF- , IL-1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, cancer-conditioned medium, cancer cells, adenosine and helminth or fungal proteins or products.

[0109] In preferred embodiments, the activated macrophage:

[0110] (i) is producible by in vitro incubation of a monocyte or macrophage with a factor capable of altering expression markers on macrophages, preferably

[0111] (a) with at least one Ml inducer,

[0112] (b) with at least one M2 inducer,

[0113] (c) or with a factor capable of altering the macrophages’ ability to secrete cytokines, preferably IL-10 and IL-12, chemokines and / or to produce iNOS, arginase or other immunomodulating enzymes;

[0114] (ii) is characterized by expression of at least one of following antigens: CD64, CD86, CD16, CD32 HLA-DR, and / or production of iNOS and / or IL- 12;

[0115] (iii) is producible by in vitro incubation of a monocyte or macrophage with a factor capable of inducing the ability of the macrophage to phagocytose;

[0116] (iv) is characterized by expression of at least one of following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4 (CXCR4), CD 163, and / or show low expression of HLA-DR;

[0117] (v) has the ability to phagocytose; and / or

[0118] (vi) is capable of cytokine secretion, preferably of IL- 12, or IL- 10, or production of inducible nitric oxide synthetase (iNOS), pro-inflammatory compounds, arginase immunosuppressive compounds or anti-inflammatory compounds.

[0119] Preferably,

[0120] (i) the Ml inducer is selected from the group consisting of LPS, INF-y, GM-CSF, and viral or bacterial proteins or products; or

[0121] (ii) the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody, IgG, heat activated gamma-globulin, glucocorticosteroid, TGF-p, IL- 1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, adenosine, helminth or fungal proteins or products.

[0122] It is preferred that the differentiated monocyte, preferably macrophage, is characterized by expression of at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven or all of TfR, CD163, CD14, CD16, CD33, 25f9, CD172a and / or CD115 or at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven or all of TfR, CD163, CD14, CD16, CXCR4, 25f9, and / or CXCR1.

[0123] If the CD45+leukocyte cell is a lymphocyte, it is preferred that it is selected from the group consisting of a CD3+and CD4+or CD8+T lymphocyte, or a CD19+, CD20+, CD21+, CD19+CD20+, CD19+CD21+, CD20+CD21+, or CD19+CD20+CD21+B lymphocyte, and a natural killer (NK) cell.

[0124] In a preferred embodiment of the targeted delivery system of the present invention the lymphocyte:

[0125] (i) is obtainable from blood, spleen, or bone marrow or is producible from a CD34+precursor cell as known to the skilled person and also described in, e.g., 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.;

[0126] (ii) is an immunologically competent lymphocyte;

[0127] (iii) expresses antigen specific T cell receptors; and / or

[0128] (iv) is characterized by expression of at least one of the following antigens: (a) CD3 and CD4 or CD8 or (b): CD19, CD20, CD21, CD19 CD20, CD19 CD21, CD20 CD21, or CD19 CD20 CD21 antigen, and is preferably capable of producing immunoglobulins

[0129] In a some embodiment the CD45+lymphocytes is a NK cell, which

[0130] (i) is obtainable from blood, spleen or bone marrow or producible from a CD34+precursor cell; and / or

[0131] (ii) is characterized by the lack of CD3 expression and expression of at least one of the following CD56+and / or CD94+, CD158a+CD158L CD314+CD335+.

[0132] If the CD45+leukocyte cell is a granulocyte, it is preferred that it is selected from the group consisting of a neutrophil, preferably a CD66b+neutrophil, an eosinophil and a basophil, preferably a CD193+eosinophil.

[0133] In a preferred embodiment of the targeted delivery system of the present invention the granulocyte:

[0134] (i) is obtainable from blood, spleen or bone marrow or producible from a CD34+precursor cell as described, e.g. in Kuhs et al. 2015, CurrProtocImmunol 111:7.23-1-7.23.16; Coquery et al. 2012, Cytometry A 81(9): 806-814; Swemydas and Lionakis 2013, J Vis Exp 77: 50586.;

[0135] (ii) is characterized by expression of at least one of the following CD66b and / or CD 193;

[0136] (iii) is a polymorphonuclear leukocyte characterized by the presence of granules in its cytoplasm; and / or

[0137] (iii) is characterized by expression of at least one of the following: TfR, CD163, TIM-2, and / or CXCR4. The targeted delivery system for use of the present invention still provides the outlined advantages, if in a population of cells not every cell has a particular property in as long as the majority of cells within that population has that property. Thus, in the following the property of one preferred cell of the targeted delivery system for use of the present invention is described.

[0138] Ways of isolating, culturing and activating the CD45+ leukocyte cells comprised in the claimed delivery system are described in WO 2016 / 207257 Al, WO 2016 / 207256 Al and WO 2017 / 222398 Al.

[0139] In some embodiments, the CD45+leukocyte cells, preferably macrophages, comprised in the targeted delivery system, are derived from isolated peripheral blood mononuclear cells (PBMCs). Preferably, the CD45+leukocyte cells, preferably macrophages, comprised in the targeted delivery system, are primary cells, i.e. cells isolated directly from human tissues, in particular peripheral blood. It is preferred that the CD45+leukocyte cells, preferably macrophages, comprised in the targeted delivery system, are not cells of an immortalized cell line, in particular not a transformed cell line. Preferably, the CD45+leukocyte cell is a primary cell or derived by a low number of differentiation steps from a primary cell. In most preferred embodiments, the CD45+leukocyte cells are bone marrow derived macrophages (BMDM) or monocyte-derived macrophages (MDM).

[0140] The inventive approach for the treatment of head and neck cancer is successful in both autologous and allogeneic settings. The CD45+leukocyte cell can be autologous and allogeneic with respect to the subject receiving the treatment. Preferably, the CD45+leukocyte cell is allogeneic with respect to the subject receiving the treatment. In an allogeneic setting, the CD45+leukocyte cell (a monocyte) is obtained from a healthy donor, differentiated into a macrophage and incubated with a complex of ferritin and an anticancer drug, leading to the formation of the isolated targeted delivery system (also referred to as macrophage drug conjugate or MDC herein). Subsequently, the isolated targeted delivery system is administered to a patient in need of treatment.

[0141] In an autologous setting, the CD45+leukocyte cells comprised in the targeted delivery system, originate from the patient to be treated. In such case the cell loaded with the complex would be autologous to the patient. It is also envisioned that patients are HLA typed prior to treatment with the targeted delivery system of the present invention and that the cell type used for a given patient is HLA matched to the patient.

[0142] The blood used for isolation of CD45+leukocyte cells is obtained from the patient to be treated or preferably from a healthy donor. Alternatively, the blood can be obtained from the blood bank. Use of umbilical cord blood is also considered herein.

[0143] Ways of loading the CD45+ leukocyte cells with complexes of an iron binding protein and an active ingredient are described in WO 2016 / 207257 Al, WO 2016 / 207256 Al and WO 2017 / 222398 Al.

[0144] Mechanism

[0145] The present invention exploits CD45+leukocytes, preferably macrophages loaded with iron- binding proteins in a complex with a drug / prodrug or a label, as a delivery system to target head and neck cancer. The inventive delivery system delivers the active ingredient directly into the head and neck cancer cells, thereby increasing efficiency and reducing side effects. When administered intratumorally, the CD45+leukocytes, preferably activated macrophages, of the inventive targeted delivery system for use in the treatment of head and neck cancer, are able to migrate deep into the tumor tissue and deliver the active ingredient directly into the cancer cells. The inventive targeted delivery system thus ensures a better drug distribution within the tumor tissue compared to administration of an active ingredient alone or an active ingredient in a complex with an iron binding protein but without a CD45+leukocyte cell. In summary, the inventive targeted delivery system constitutes an effective delivery system of active ingredients to the entire head and neck cancer mass, enabling efficient tumor cell killing with reduced systemic exposure and reduced side effects.

[0146] The inventors observed that upon intratumoral administration of the isolated targeted delivery system for use according to the invention to the animal, loaded CD45+leukocytes, preferably activated macrophages, deliver the complex of iron-binding protein and active ingredient(s) into the head and neck cancer cells. Cell-cell contact is required for efficient transfer of a complex, in particular a conjugate, comprising ferritin and an active agent from macrophages to cancer cells. Secretion of the complex, in particular conjugate, comprising ferritin and an active agent by the macrophages and subsequent uptake by the cancer cells is not sufficient to ensure efficient delivery. The transfer of the targeted delivery system of the invention is a direct transfer between cells, requiring cell-cell contact or at least very close proximity between the CD45+leukocytes (macrophages) and the cancer cells. The inventors elucidated the intricate molecular mechanism underlying the TRAIN process, which involves the formation of an immune synapse-like structure between these two cell types that serves as a conduit for ferritin transfer from macrophages to cancer cells. This direct transfer has the advantage that the active agent is delivered specifically into the target cell without increasing the extracellular concentration outside the tumor cells, thereby increasing efficacy and decreasing side effects. The method of the invention thus allows an advantageously precise administration of the active ingredient(s) to the head and neck cancer cells.

[0147] Once internalized, HFt is trafficked not only to endosomes but also to lysosomes in macrophages. The trafficking to lysosomes is of particular interest because it provides a potential for controlled release of drug payloads within cancer cells.

[0148] Combination Therapy

[0149] In some embodiments of the isolated targeted delivery system for use according to the invention, the treatment of head and neck cancer comprises a combination therapy, wherein the isolated targeted delivery system and an immune checkpoint modulator are administered. Thus, in a second aspect, the invention provides the isolated targeted delivery system according to the first aspect and an immune checkpoint modulator for use in a method of treating head and neck cancer. All embodiments described with respect to the targeted delivery system of the first aspect also apply to the targeted delivery system used in the combination therapy of the second aspect, where applicable.

[0150] The term "immune checkpoint modulator" is used in the broadest sense and refers to compounds that interact with immune checkpoint molecules and promotes an anti-tumor immune response. The term includes agonists of stimulatory immune checkpoints and antagonists of inhibitory immune checkpoints. Immune checkpoints are key regulators of the immune system. Activation of an inhibitory immune checkpoint can dampen the immune response to an immunologic stimulus. On the other hand, activation of a stimulatory immune checkpoint can enhance an immune response. Immune checkpoint modulators include antagonists, agonists, and modulators of PD-1, PD-L1, PD-L2, A2AR, B7-H3 (e.g. MGA271), B7-H4, B7-H7 (HHLA2), TMIGD2, BTLA, CTLA-4, IDO, TDO, KIR, LAG3, TIM-3, TIGIT, DC80, CD86, CD28, ICOS and ICOS Ligand, 4-1BBL, 4-1BB, HVEM, BTLA, CD160, LIGHT, MHC class I and II, LAG3, OX40L, 0X40, CD70, CD27, CD40, CD40L, GITRL, GITR, CD155, DNAM-1, TIGIT, CD96, CD48, 2B4, Galectin-9, Adenosine, Adenosine A2a receptor, CEACAM1, CD47, SIRP alpha, BTN2A1, DC-SIGN, CD200, CD200R, TL1A, DR3 or VISTA. In some embodiments, the immune checkpoint modulator is an antagonist of PD-1, PD-L1, A2AR, B7-H3 (e g. MGA271), B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, TIM-3, TIGIT or VISTA. Preferably, the antagonist is an antagonistic antibody or antibody like protein. Immune checkpoint modulators further include agonists of tumor necrosis factor (TNF) receptor superfamily members and agonists of B7-CD28 superfamily members. The TNF receptor superfamily member may be selected from CD27, CD40, 0X40, GITR and CD137. The B7-CD28 superfamily member may be CD28 or ICOS. Preferably, the agonists of a TNF receptor superfamily member or B7-CD28 superfamily member are (soluble) ligands or an agonistic antibodies or antibody like proteins (e.g. CP-870,893 for CD40).

[0151] In preferred embodiments, the immune checkpoint modulator is selected from an antagonistic antibody specifically binding to PD-1, PD-L1 or CTLA4. In most preferred embodiments, the immune checkpoint modulator is an antagonistic antibody specifically binding to PD-1 and preventing the binding of PD-1 to PD-L1 (also referred to as anti-PD-1 antibody herein).

[0152] Preferably, the isolated targeted delivery system is administered intratumorally and the immune checkpoint modulator is administered intravenously, intraperitoneally or intratumorally. The combination therapy with an immune checkpoint modulator, in particular with an anti-PD-1 antibody provides for a synergistic effect and increases treatment efficacy.

[0153] In some embodiments of the invention, the head and neck cancer is poorly immunogenic. It is envisioned that the head and neck cancer is resistant to immunotherapy alone. In some embodiments, the head and neck cancer, in particular HNSCC, is resistant to monotherapy with an immune checkpoint modulator, in particular to monotherapy with an anti-PD-1 antibody. Pharmaceutical composition

[0154] In another aspect, the invention provides a pharmaceutical composition for the treatment of head and neck cancer comprising the isolated targeted delivery system described with respect to the first aspect of the invention and a pharmaceutically acceptable carrier and / or suitable excipient(s). All embodiments described with respect to the first aspect of the invention also apply to the pharmaceutical composition, where applicable.

[0155] Since the isolated targeted delivery system comprises living cells, it is preferred that carriers and excipients are chosen in such to keep the cells alive.

[0156] “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.

[0157] The term “carrier”, as used herein, refers to a pharmacologically inactive substance such as but not limited to diluents, surfactants, stabilizers, physiological buffer solutions or vehicles with which the pharmaceutically active substance is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carriers include but are not limited to sterile liquids, such as saline solutions in water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0158] Suitable pharmaceutical “excipients” include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0159] “Surfactants” include anionic, cationic, and non-ionic surfactants such as but not limited to sodium deoxycholate, sodium dodecylsulfate, Triton X-100, and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0160] “Stabilizers” include but are not limited to mannitol, sucrose, trehalose, albumin, as well as protease and / or nuclease antagonists.

[0161] “Physiological buffer solution” include but are not limited to sodium chloride solution, demineralized water, as well as suitable organic or inorganic buffer solutions such as but not limited to phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3 morpholino-1 propane sulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer are suitable, for example, for injection and infusion solutions.

[0162] The pharmaceutical composition may further comprise a cryoprotectant to enable freezing of the isolated targeted delivery system. The cryoprotectant may comprise dimethyl sulfoxide (DMSO), glucose, dextrose, mannitol, trehalose, salts, glutamine, glycerol, propylene glycol, sodium pyruvate, sodium bicarbonate and / or human serum albumin.

[0163] Methods of treatment

[0164] In another aspect, the invention provides a method of treatment or diagnosis of head and neck cancer comprising administration of the isolated targeted delivery system described with respect to the first aspect of the invention in an effective amount to a patient in need thereof.

[0165] In further aspect, the invention provides a method of treating head and neck cancer comprising administration of the isolated targeted delivery system of the first aspect of the invention and an immune checkpoint inhibitor in effective amounts to a patient in need thereof.

[0166] All embodiments described with respect to the first aspect of the invention or with respect the combination therapy also apply to the methods of treatment of head and neck cancer, where applicable.

[0167] Definitions

[0168] The specification uses a variety of terms and phrases, which have certain meanings as defined below. Preferred meanings are to be construed as preferred embodiments of the aspects of the invention described herein. As such, they and also further embodiments described in the following can be combined with any embodiment of the aspects of the invention and in particular any preferred embodiment of the aspects of the invention described above.

[0169] The terms “peptide” or “polypeptide” are used interchangeably in the context of the present invention to refer to a chain of at least two amino acids linked by peptide bonds. Thus, the term “polypeptide” in the context of the present invention is also used to refer to amino acid chains with more than 50, more than 100 or more than 150 amino acids.

[0170] The term “amino acid” encompasses naturally occurring amino acids as well as amino acid derivatives. In the context of the present specification, amino acids are identified using the 1-letter code (Hausman RE, Cooper GM (2004). The cell: a molecular approach. Washington, D.C: ASM Press, p. 51. ISBN 978-0-87893-214-6). An amino acid identified with the letter X corresponds to any amino acid. An amino acid identified with the letter B corresponds to either D (asparagine) or N (aspartic acid). An amino acid identified with the letter Z corresponds to either E (glutamine) or Q (glutamic acid).

[0171] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention referred to nucleic acid molecules include but are not limited to ribonucleic acid (RNA) and its various forms (e.g. but not limited to ssRNA, LNA etc.), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA- DNA hybrids. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). "Aptamers" are nucleic acids which bind with high affinity to a polypeptide. Aptamers can be isolated by selection methods such as SELEmirl46-a (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981) from a large pool of different single-stranded RNA molecules. Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1116- 9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms which have been isolated in this way enjoy the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, possess greater stability.

[0172] The term "about" when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value (each limit preferably 4%, 3%, 2% or 1% instead of 5%).

[0173] The term “identity” is used throughout the specification with regard to polypeptide and nucleotide sequence comparisons. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise. For example, a polypeptide sequence consisting of 200 amino acids compared to a reference 300 amino acid long polypeptide sequence may exhibit a maximum percentage of sequence identity of 66.6 % (200 / 300) while a sequence with a length of 150 amino acids may exhibit a maximum percentage of sequence identity of 50 % (150 / 300). If 15 out of those 150 amino acids are different from the respective amino acids of the 300 amino acid long reference sequence, the level of sequence identity decreases to 45 %. The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin&Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http: / / www.ebi.ac.uk / Tools / clustalw / or on http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or on http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. Structure based alignments for multiple protein sequences and / or structures using information from sequence database searches, available homologs with 3D structures and user-defined constraints may also be used (Pei J, Grishin NV: PROMALS: towards accurate multiple sequence alignments of distantly related proteins. Bioinformatics 2007, 23:802-808; 3DCoffee@igs: a web server for combining sequences and structures into a multiple sequence alignment. Poirot O, Suhre K, Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul l;32:W37-40.). When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

[0174] The term "variant" refers, with respect to a polypeptide, generally to a modified version of the polypeptide, e.g., a mutation, so one or more amino acids of the polypeptide may be deleted, inserted, modified and / or substituted. Generally, the variant is a functional variant, meaning that it preserves the function of the parent polypeptide. More specific functions are defined herein and have precedence over the general definition. A "mutation" or "amino acid mutation" can be an amino acid substitution, deletion and / or insertion ("and" may apply if there is more than one mutation). Preferably, it is a substitution (i.e., a conservative or non-conservative amino acid substitution), more preferably a conservative amino acid substitution. In some embodiments, a substitution also includes the exchange of a naturally occurring amino acid with a not naturally occurring amino acid. A conservative substitution comprises the substitution of an amino acid with another amino acid having a chemical property similar to the amino acid that is substituted. Preferably, the conservative substitution is a substitution selected from the group consisting of:

[0175] (i) a substitution of a basic amino acid with another, different basic amino acid;

[0176] (ii)a substitution of an acidic amino acid with another, different acidic amino acid;

[0177] (iii) a substitution of an aromatic amino acid with another, different aromatic amino acid;

[0178] (iv) a substitution of a non-polar, aliphatic amino acid with another, different non-polar, aliphatic amino acid; and

[0179] (v) a substitution of a polar, uncharged amino acid with another, different polar, uncharged amino acid.

[0180] A basic amino acid is preferably selected from the group consisting of arginine, histidine, and lysine. An acidic amino acid is preferably aspartate or glutamate. An aromatic amino acid is preferably selected from the group consisting of phenylalanine, tyrosine and tryptophane. An aliphatic uncharged amino acid is preferably selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine. A hydrophilic uncharged amino acid is preferably selected from the group consisting of serine, threonine, asparagine, and glutamine. A non-polar uncharged amino acid is preferably selected from the group consisting of cysteine, methionine, proline. In contrast to a conservative amino acid substitution, a non-conservative amino acid substitution is the exchange of one amino acid with any amino acid that does not fall under the above-outlined conservative substitutions (i) through (v).

[0181] Amino acids of a protein may also be modified, e.g., chemically modified. For example, the side chain or a free amino or carboxy-terminus of an amino acid of the protein or polypeptide may be modified by e.g., glycosylation, amidation, phosphorylation, ubiquitination, etc. The chemical modification can also take place in vivo, e.g., in a host-cell, as is well known in the art. For example, a suitable chemical modification motif, e.g., glycosylation sequence motif present in the amino acid sequence of the protein will cause the protein to be glycosylated. Unless a modification leads to a change in identity of a modified amino acid (e.g., a substitution or deletion), a modified polypeptide is within the scope of polypeptide as mentioned with respect to a certain SEQ ID NO, i.e., it is not a variant as defined herein.

[0182] Preferably the degree of identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence.

[0183] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation or with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.

[0184] The term "variant" refers, with respect to a polynucleotide, generally to a modified version of the polynucleotide, e.g., a mutation, so one or more nucleotides of the polynucleotide may be deleted, inserted, modified and / or substituted. Generally, the variant is a functional variant, meaning that it preserves the function of the parent polynucleotide, such as specific binding ability to the target (e.g. CXCR4 or ICOS). More specific functions are defined herein and have precedence over the general definition. A "mutation" can be a nucleotide substitution, deletion and / or insertion ("and" may apply if there is more than one mutation). Preferably, it is a substitution, more preferably it causes an amino acid substitution, most preferably a conservative amino acid substitution. Preferably the degree of identity between a given nucleic acid sequence and a nucleic acid sequence which is a variant of said given nucleic acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for a nucleic acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleic acids, the degree of identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleic acids, in some embodiments, continuous nucleic acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference a nucleic acid sequence.

[0185] The term "functional variant" of an amino acid sequence includes "functional" fragments of said amino acid sequence. Characteristics of functional variants, e.g., binding characteristics, can be analysed by known methods, e.g., using an ELISA-assay.

[0186] The term “antibody” as used in the context of the present invention refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. An antibody refers to a protein molecule produced by plasma cells and is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, its antigen.

[0187] The term “antibody fragment” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.

[0188] As used herein, an "antagonist" is a ligand which competitively binds to a receptor at the same site as an agonist, but does not activate an intracellular response initiated by an active form of a receptor, and thereby inhibits the intracellular response induced by an agonist, by at least 10%, at least 15-25%, at least 25-50%, or at least 50-100%, as compared to the intracellular response in the presence of an agonist and in the absence of the antagonist.

[0189] As used herein, an "agonist" refers to a ligand that activates an intracellular response when it binds to the target receptor. An agonist according to the invention may increase the intracellular response mediated by the target receptor by at least 2-fold, at least 5 -fold, at least 10-fold, or at least 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold), as compared to the intracellular response in the absence of the agonist. As used herein, an "inverse agonist" refers to a ligand which decreases a constitutive activity of a cell surface receptor when it binds to a receptor but does not competitively bind to the receptor at the same site as an agonist. An inverse agonist according to the invention may decrease the constitutive intracellular response mediated by a receptor by at least 2-fold, at least 5-fold, at least 10-fold, or at least 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold), as compared to the intracellular response in the absence of the inverse agonist. As used herein, an "inverse antagonist" refers to a ligand which decreases a constitutive activity of a cell surface receptor when it binds to a receptor and competitively binds to the receptor at the same site as an agonist. An inverse antagonist according to the invention may decrease the constitutive intracellular response mediated by a receptor by at least 2-fold, at least 5 -fold, at least 10-fold, or at least 100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold), as compared to the intracellular response in the absence of the inverse antagonist.

[0190] The term “treatment” as used herein includes all types of preventive and / or therapeutic interventions medically allowed for the purpose of cure, temporary remission, prevention, etc. for different purposes including delaying or stopping the progress of a disease, making a lesion regress or disappear, preventing onset, or inhibiting recurrence. Thus, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof. In some embodiments, the treatment comprises or consists of the killing of cancer cells. In some embodiments, the treatment comprises or consists of the prevention of metastatic spread.

[0191] BRIEF DESCRIPTION OF DRAWINGS

[0192] Fig. 1: Bodyweight of female C3H / HeN mice bearing SCC7 tumours. Values shown are mean ±SD; n=10 for all groups.

[0193] Fig. 2 : Volume of SCC7 tumours implanted onto the flank of female C3H / HeN mice. Values shown are mean ±SEM; n=10 for all groups.

[0194] Fig. 3: Mean volume of SCC7 tumours implanted onto the flank of female C3H / HeN mice on Day 14. Values shown are mean ±SD. n=10 for all groups.

[0195] Fig. 4: Volume of SCC7 tumours implanted onto the flank of female C3H / HeN mice (individual animal data).

[0196] Fig. 5: Kaplan Meier survival curve of female C3H / HeN mice implanted on the flank with SCC7 tumours. n=10 for all groups.

[0197] Fig. 6: Co-culture of different head and neck cancer cells with MDC leads to cancer cell death. Each graph shows the mean (± SD) number of live cancer cells after 72 hours of co-culture with MDC at a 1 : 1 ratio (indicated by "MDC" bars), relative to the mean (± SD) number of live cancer cells in monoculture (indicated by "Control" bars). Statistical analysis was performed using the t-test, where **** denotes p < 0.0001. Cell lines used include Detroit 562 (ATCC CCL-138), FaDu (ATCC HTB-43), A-253 (ATCC HTB-41), SCC-9 (ATCC CRL-1629), and SCC-25 (ATCC CRL-1628). Fig. 7: Generation of macrophage drug conjugates and therapeutic approach: (1) isolation of monocytes from the peripheral blood, (2) ex vivo differentiation of monocytes into macrophages, (3) complexation of the desired drug with ferritin cage, (4) loading of the drug-encapsulated ferritin into the macrophages to form the macrophage drug conjugates, and (5) administration of the MDCs to the patient, followed by targeting of the tumor by the macrophages and transfer of the drug form the macrophages into the cancer cells, leading to their death.

[0198] EXAMPLE SECTION

[0199] Example 1 - Material and Methods

[0200] Production of macrophage ferritin drug conjugate

[0201] A ferritin-drug (Ft-drug) conjugate was produced by covalently linking the anti-cancer drug to ferritin using a maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl (mc-vc-PAB) linker. The anti-cancer drug comprising a mc-vc-PAB linker was obtained from MedChem Express (Princeton, NJ). The conjugate was prepared as follows. Human ferritin (according to SEQ ID NO: 4) solution was adjusted to a concentration of 120 pM with reaction buffer (50 mM phosphate buffer pH 6.8, containing 0.1 mM EDTA) and conjugated with 10-fold molar excess of the anti-cancer drug comprising a mc-vc-PAB linker in the presence of 20% v / v acetonitrile solution at 4°C overnight. Maleimide groups react efficiently and specifically with free (reduced) sulfhydryls at pH 6.5-7.5 to form stable thioether bonds. The excess drug was purified and buffer-exchanged with D-PBS using PM 100 ultrafiltration concentrator. The yield of conjugation was approximately 80% of the total cysteines. Formation of the Ft-drug conjugate was confirmed by LC-MS analysis and by titration of residual free thiol group with p-chloromercuribenzoate. The concentrations of Ft-drug conjugates were determined by UV-vis spectroscopy analysis. Stability tests showed that Ft-drug conjugates remained stable for at least six months at -80 °C.

[0202] Ft-drug was loaded into macrophages by incubating bone marrow derived macrophages (BMDM) or monocyte-derived macrophages (MDM) for 1-4 hours in Ft-drug solution having a concentration from 0.5 mg / ml to 1 mg / ml in standard culture conditions. The macrophages were M0 macrophages or macrophages mildly polarized towards M2 cultured during differentiation period with M-CSF1. Macrophages comprising a ferritin-drug conjugate are also referred to as “macrophage ferritin drug conjugate”, “macrophage drug conjugate” or MDC herein.

[0203] Changes in cell viability and phenotype were evaluated in frozen MDC product that was thawed at selected time points. The product remained pharmacologically active in the potency assay, showing similar cancer cell killing activity as the fresh product. All investigated parameters were within expected values, the loading, freezing, and thawing steps did not alter the macrophage phenotype and viability of MDC for 6 months stored in liquid nitrogen which, according to pharmacopeia allows to claim a stability of 12 months.

[0204] Animals

[0205] A total of 60 female C3H / HeN mice aged 5-8 weeks weighing approximately 22-30 g were used for the study. These were purchased from Charles River and subject to a 7-day acclimation period prior to any work being carried out on them. Animals were housed in IVC cages (up to 5 per cage) with individual mice identified by tail mark. All animals were allowed free access to a standard certified commercial diet and sanitised water during the study. The holding room was maintained under standard conditions: 20-24°C, 30-70% humidity and a 12 h light / dark cycle.

[0206] All protocols used in this study have been approved by the Axis Bioservices Animal Welfare and Ethical Review Committee, and all procedures were carried out under the guidelines of the Animal (Scientific Procedures) Act 1986.

[0207] Efficacy study: Tumour cell implantation: SCC7 cells (5x105) were implanted onto the flank of female C3H / HeN mice using a 25-gauge needle. Animals were under inhalant anaesthesia (isoflurane) during the procedure. Seven (7) days after tumour cell implant when tumour volume was 10-30 mm3animals were randomly assigned to the following treatment groups to ensure an equal spread of tumour volume.

[0208] Table 1

[0209] Animals had tumour measured three times per week (Monday, Wednesday and Friday; days: 7, 10, 12, 14, 17, 19, 21, 24, 26 and 28 post-tumour cell implantation) using electronic callipers and tumour volume calculated using a 3D equation.

[0210] Immuno-modulatory study: SCC7 cells (5xl05) were implanted onto both flanks of female C3H / HeN mice. Mice were under inhalant anesthesia (isoflurane) during the procedure. Seven (7) days after implant animals were randomly assigned to the following treatment groups to ensure an equal spread of tumour volume: Table 2

[0211] Animals had tumour measured three times per week (Monday, Wednesday and Friday; days: 7, 10, 12, 14, 17, 19, 21, 24, 26 and 28 post-tumour cell implantation) using electronic callipers and tumour volume calculated using a 3D equation.

[0212] Toxicology study: An additional 3 animals (non-tumour-bearing) were treated with MDC (s.c. injection) and culled after 28 days. Skin surrounding injection site, lungs, liver, and spleen were resected, and processed to formalin-fixed paraffin-embedded (FFPE) samples. Haematoxylin & Eosin (H&E) staining was carried out to identify any toxicity by a pathologist.

[0213] Antibodies aPD-1 Ab was received from supplier (BioXcell) at a concentration of 6.9 mg / ml. Product was diluted in PBS to a final concentration of 1 mg / ml and a dosing volume of 10 ml / kg was applied to individual animal bodyweight to deliver a dose level of 10 mg / kg.

[0214] Isotype control rat IgG2a (BioXcell) was received from supplier at a concentration of 9.15 mg / ml. Product was diluted in PBS to a final concentration of 1 mg / ml and a dosing volume of 10 ml / kg was applied to individual animal bodyweight to deliver a dose level of 10 mg / kg.

[0215] Example 2 - Efficacy Study

[0216] All treatments were well tolerated (Figure 1) with mean bodyweights increasing gradually throughout the study.

[0217] The volume of SCC7 tumours on vehicle treated animals grew rapidly during the study as expected (Figure 2). All treatments slowed the growth of SCC7 tumours. Large error bars are normal for syngeneic tumour studies where the response is generally heterogeneous.

[0218] Day 14 was chosen as the time point for statistical analyses as this was the last time point when all vehicle treated animals remained on study.

[0219] Table 3 - Comparison of all treatments with vehicle controls in SCC7 tumour bearing female C3H / HeN mice. Statistical analysis was carried out using unpaired t-test with Welsh’s correction. Animals were sacrificed when tumour volume reached maximal allowed and that day was recorded. All treatments resulted in a right-shift of the survival curve associated with significant increased survival (Figure 5; Table 4).

[0220] Table 4 - Comparison of all treatments with vehicle controls in SCC7 tumour bearing female C3H / HeN mice. Statistical analysis was carried out using Log Rank (Mantel-Cox) test.

[0221] Example 3 - Toxicology Study

[0222] Three (3) non-tumour-bearing animals were dosed subcutaneously with mouse MDC (4xl06in 40 pl) and 28 days later animals were euthanised, and liver, lung, spleen and skin tissue resected and processed to FFPE. 4 pM sections were sectioned and H&E stained.

[0223] No adverse health effects were reported during the in-life phase; this was confirmed by histological examination of the tissue sections where nothing pathological was observed.

[0224] All treatments were generally well tolerated with no evidence of bodyweight loss or any other effects on animal appearance or behaviour recorded. Considering overall survival, all treatments resulted in a significant increase in survival compared to vehicle treated controls with p-values of 0.0218 for mouse MDC alone, 0.0126 for aPD-1 Ab alone and <0.001 for the combination group. There was no evidence of an immune-modulatory effect in this tumour model. In the toxicology arm, there were no reports of any effects on animal health during the in-life phase and histological examination of liver, lung, spleen and skin indicated that there were no pathological abnormalities. It is worth considering that the mouse head and neck cancer model SCC7 is extremely aggressive and any effects on tumour growth and survival are highly significant.

[0225] Example 4 - Co-culture of MDC with different head and neck cancer cells

[0226] Co-culture of different head and neck cancer cells with MDC leads to cancer cell death (Figure 6).

[0227] In summary, in the SCC7 head and neck cancer model treatment with MDC administered locally (intratumorally) significantly improved survival and reduced tumor volume, especially when combined with checkpoint inhibition, indicating its potential for synergistic effects in the treatment of head and neck cancer. The treatment was well tolerated, as reflected by the weight of the mice. A histopathological analysis of the skin and macrophage -enriched organs (lungs, liver and spleen) after subcutaneous administration of MDC showed that the anatomy of the analyzed organs was unaffected (data not shown).

[0228] SEQUENCES

[0229] SEQ ID NO: 1 - human ferritin heavy chain

[0230] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHE

[0231] EREHAEKLMKLQNQRGGRI FLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATD

[0232] KNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0233] SEQ ID NO: 2 - mouse ferritin heavy chain

[0234] MTTASPSQVRQNYHQDAEAAINRQINLELYASYVYLSMSCYFDRDDVALKNFAKYFLHQSHE

[0235] EREHAEKLMKLQNQRGGRI FLQDIKKPDRDDWESGLNAMECALHLEKSVNQSLLELHKLATD

[0236] KNDPHLCDFIETYYLSEQVKS IKELGDHVTNLRKMGAPEAGMAEYLFDKHTLGHGDES

[0237] SEQ ID NO. 3 - mammalian consensus ferritin

[0238] MTTASXSQVRQNYXQXSEAAXXRQINLELXASYVYLSMSXYFDRDDVALKNFAKYFLHQSHE

[0239] EREHAEKLMKLQNQRGGRIXLXDIKKPDXDDWESGLNAMECALXLEKXVNQSLLELHKLATD

[0240] KNDPHLCDFIETXYLXEQVKXIKELGDHVTNLRKMGAPEXGXAEYLFDKHTLGXSDXXX

[0241] SEQ ID NO. 4 - FT_2

[0242] MTTASTSQVRENYHEDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAEYFLHQSHE

[0243] EREHAEKLMELQNQRGGRI FLQDIQKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATD

[0244] KNDPHLCDFIETHYLNEQVEAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

Claims

38CLAIMS1. An isolated targeted delivery system comprising a CD45+leukocyte cell comprising within said cell a complex of one or more iron binding proteins and a pharmaceutically active substance, for use in a method of treating head and neck cancer.

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

3. The isolated targeted delivery system for use according to claim 1 or 2, wherein the iron binding protein is directly or indirectly conjugated to the pharmaceutically active substance via a cysteine residue or a lysine residue, preferably a cysteine residue.

4. The isolated targeted delivery system for use according to any of claims 1 to 3, wherein the CD45+leukocyte is a monocyte or a macrophage, preferably a macrophage.

5. The isolated targeted delivery system for use according to any of claims 1 to 4, wherein the CD45+leukocyte is a macrophage, preferably an MO macrophage or a macrophage that is mildly polarized towards M2.

6. The isolated targeted delivery system for use according to any of claims 1 to 5, wherein the iron binding protein is ferritin or transferrin, preferably ferritin.

7. The isolated targeted delivery system for use according to any of claims 1 to 6, wherein the iron binding protein is a ferritin comprising an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NO: 1-5.

8. The isolated targeted delivery system for use according to any of claims 1 to 7, wherein the pharmaceutically active substance is an anti-cancer drug selected from the group consisting of a protein, a peptide, a nucleic acid, a non-protein non-nucleic acid compound with a molecular weight of less than 1.5 kD, a photosensitizing compound, a virus, and pharmaceutically active radioactive isotope.

9. The isolated targeted delivery system for use according to claim 8, wherein the anti-cancer drug is auristatin, in particular monomethyl auristatin E or monomethyl auristatin F.

10. The isolated targeted delivery system for use according to any one of claims 1 to 9, wherein the isolated targeted delivery system is administered intratumorally.

11. An isolated targeted delivery system according to any of claims 1 to 10 and an immune checkpoint modulator for use in a method of treating head and neck cancer.

12. The isolated targeted delivery system and immune checkpoint modulator for use according to claim 11, wherein the immune checkpoint modulator is an antibody specifically binding to PD-1.

13. The isolated targeted delivery system and immune checkpoint modulator for use according to claim 11 or 12, wherein the isolated targeted delivery system is administered intratumorally and the immune checkpoint modulator is administered intravenously, intraperitoneally or intratumorally.

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