Targeted drug delivery of a quassinoid

Abstract

The present invention relates to targeted drug delivery of a quassinoid. To this end, the present invention provides a magnetic iron oxide nanoparticle coated with a carboxylated sugar or carboxylated polymer and comprising a targeting moiety and a quassinoid. The present invention further provides a method of producing such a nanoparticle, a composition comprising the nanoparticle, and the use of the nanoparticle of composition comprising the nanoparticle in the treatment of cancer.

Description

TARGETED DRUG DELIVERY OF A QUASSINOIDTECHNICAL FIELD

[0001] The present invention relates to targeted drug delivery of a quassinoid. To this end, the present invention provides a magnetic iron oxide nanoparticle coated with a carboxylated sugar or carboxylated polymer and comprising a targeting moiety and a quassinoid. The present invention further provides a method of producing such a nanoparticle, a composition comprising the nanoparticle, and the use of the nanoparticle of composition comprising the nanoparticle in the treatment of cancer.BACKGROUND OF THE INVENTION

[0002] The development of aggressive lymphoid malignancies results from malignant transformation and clonal expansion of immature or mature B cells in the bone marrow or in the germinal center of secondary lymphoid follicles (Sambade et al. APMIS : acta pathologica, microbiologica, at immunologica Scandinavica 1997, 105 (12), 895-903; Inaba et al. The Lancet 2013, 381 (9881 ), 1943-1955) According to the World Health Organization (WHO) classification that is mainly based on the stage when B cell transformation occurs, aggressive lymphoid malignancies can be classified as precursor B cell or mature B cell phenotype (Swerdlow et al Blood 2016, 127 (20), 2375-2390)

[0003] When malignant transformation occurs at a more mature B cell stage, these types of neoplasms are classified as a mature B cell phenotype. The most common types in adults are aggressive B cell lymphomas (Swerdlow et al. Blood 2016, 127 (20), 2375-2390; Alaggio et al. Leukemia 2022, 36 (7), 1720-1748) and consist of Burkitt’s lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma grade 3 (FL III), and mantle cell lymphoma (MCL) (Armitage and Weisenburger Journal of Clinical Oncology 1998, 16 (8), 2780-2795). DLBCL is the most common subtype accounting for 30-40% of all lymphomas in adults. High molecular and genetic heterogeneity of DLBCL results in patients differing in clinical disease presentation and treatment outcome with a 5-year survival rate of around 50% (Jong and Balague Ponz The Journal of pathology 2011 , 223 (2), 274-282). An important advance in the treatment of DLBCL has been the understanding of MYC mutations, as this gene is rearranged in 5- 15% of DLBCL and is often associated with BCL2 translocation or, to a lesser extent, BCL6 in so-called 'double-hit' or 'triple-hit' lymphomas (Swerdlow et al. Blood 2016, 127 (20), 2375-2390; Prochazka et al. British journal of haematology 2018, 183 (1 ), 142-146). With standard chemotherapy, aggressive B cell lymphoma, even at an advanced stage, is a curable disease in many instances (Susanibar-Adaniya and Barta American journal of hematology 2021 , 96 (5), 617-629). Nevertheless, despite improvements in the available therapy, approximately one third of patients with advanced-stage DLBCL are still refractory to treatment or will relapse (Nowakowski et al. Blood cancer journal 2019, 9 (6), 48; Said Modern pathology ; an official journal of the United States and Canadian Academy of Pathology, / nc 2013, 26 Suppl 1 (0 1 ), S42-56.9). The incidence of aggressive lymphomas has been on the rise in recent years (De Jong, D. & Balague Ponz, O. The molecular background of aggressive B cell lymphomas as a basis for targeted therapy. J. Pathol. 223, 275-283 (2011 )).

[0004] In the case of relapse, third-line therapy uses immunotherapy in which genetically modified T cells express synthetic receptors on the cell surface to detect and eliminate tumour cells by identifying specific tumour antigens, so- called ‘chimeric antigen receptor T (CAR-T) therapy (Inaba et al. The Lancet 2013, 381 (9881 ), 1943-1955; Chen et al. Cancers 2023, 15 (3)). Although this novel therapeutic approach results in high response rate (approximately 80%), a high relapse rate (up to 60%) is also observed (Chen et al Cancers 2023, 15 (3); Tian et al. Frontiers in immunology 2022, 13, 1041177; Shadman et al. Blood 2022, 139 (9), 1330-1339) Moreover, the high cost (prices range from USD 373,000 to USD 475,000 per therapy) makes this therapy a difficult to afford product. All theseobservations indicate the need for a novel therapeutic strategy to increase treatment efficacy and reduce the adverse side effects of current chemotherapy regimens for aggressive lymphomas

[0005] WO 2002 / 43708A2 discloses the use of magnetic particles for tumour treatment. Here, magnetic particles covered with a polymer layer are used for drug delivery of mitonxantron bound through amino groups to phosphate groups on the polymer have been used for tumour treatment EP 2 508 171 A1 discloses magnetic particles for magnetic targeting of drugs. Here, fatty acid-covered magnetic iron oxide particles with a covalently bound drug are used to show that magnetic targeting as well as monitoring is possible for these particles. A combination of dextran- coated SPIONs with hyaluronic acid and cisplatin is another approach for magnetic drug targeting in the treatment of cancer (Zschiesche et al. Nanomaterials (Basel, Switzerland) 2022, 13 (1 )).

[0006] Biocompatibility assessments have explored diverse coatings for nanoparticles, with surfactant and polymer variants taking the forefront Commonly employed coatings encompass polysaccharides like chitosan (Grenha Journal of drug targeting 2012, 20 (4), 291-300), starch (Cole et al. Biomaterials 2011 , 32 (8), 2183-2193), dextran (Chao, et al. Advances in experimental medicine and biology 2012, 733, 115-123), and dextran derivatives (Han et al. Eur. J. Inorg. Chem. 2010, 2010 (34), 5455-5461 ), alongside synthetic polymers such as polyethylene oxide (Phadatare et al Journal of Magnetism and Magnetic Materials 2012, 324 (5), 770-772; Hafeli et al Molecular pharmaceutics 2009, 6 (5), 1417-1428). Dextran, notably, has emerged as a frontrunner, furnishing the requisite stability sans any reported toxicity concerns (Ravikumar et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2012, 403, 1-6). Notably, dextran-coated iron oxide nanoparticles, marketed as Feridex IV.® and Ferumoxytol, have gained endorsement from the U S Food and Drug Administration, finding utility as MRI contrast agents and for treating iron deficiency anemia, respectively (Ayala et al Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology 2013, 15 (8), 1874; Santosh et al. NDT plus 2010, 3 (4), 341-342; Chickera et al. Contrast media & molecular imaging 2011 , 6 (4), 314-327)

[0007] In the realm of biosensors, carboxymethyl-substituted dextran (CMD), an anionic dextran derivative, has gained prominence, both in research and commercial domains. CMD stands out due to its elevated carboxymethyl group density, facilitating chemical conjugations (Ning et al. Soft Maffer2011 , 7 (19), 9394). Impressively, CMD has exhibited a delayed clearance from circulation compared to its unmodified counterpart (Ning et al. Soft Matter 2011 , 7 (19), 9394) coupled with noteworthy low-fouling characteristics (Dubiel et al. Colloids and surfaces. Biointerfaces 2012, 89, 117-125). Doxorubicin (DOX)-loaded CMD-coated liposomes displayed a sustained and pH-responsive drug release profile (Ning et al. Soft Matter 2011 , 7 (19), 9394). The presence of carboxymethyl groups further enables covalent bonding of the polymer onto the nanoparticle surface, exerting a favorable influence on colloidal stability (Ayala et al. Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology 2013, 15 (8), 1874; Creixell et al. ACS nano 2011 , 5 (9), 7124-7129).

[0008] Carboxymethyl-dextran (CMD)-coated iron oxide nanoparticles (lONs) have garnered significant attention within the field of nanomedicine, particularly for their potential applications in drug delivery. The creation of a magnetically controlled drug delivery system involves careful consideration of multiple factors, including composition, surface properties, size, agglomeration, magnetization, cytocompatibility, and drug efficacy. Given their straightforward and cost-effective synthesis, strong biocompatibility, substantial surface area to volume ratio, and superparamagnetic characteristics, modified iron oxide nanoparticles (lONs) hold promise for diverse nanomedicine applications. These attributes make them well-suited for functions such as T2 contrast agents in magnetic resonance imaging (MRI), cancer therapies like hyperthermia treatment or inhibitory factor supplementation, and even as carriers in magnetically regulated drug delivery systems (Turrina et al. International journal of molecular sciences 2022, 23 (23)).

[0009] Carboxymethyl dextran (CMD) emerges as a noteworthy coating for drug delivery applications due to several compelling reasons. The synthesis of CMD-IONs can be easily achieved through an in situ co-precipitation process (Turrina et al. International journal of molecular sciences 2022, 23 (23); Vasic et al. Reactive and Functional Polymers 2020, 148, 104481 ). The carboxyl groups present in CMD offer effective means for cross-linking particles with primary amines found in therapeutic proteins or peptides. The thickness of the CMD coating has been examinedfor its influence on particle properties, as demonstrated in a co-precipitation synthesis study (Turrina et al. International journal of molecular sciences 2022, 23 (23)). Higher quantities of CMD result in a thicker coating, causing a reduction in the superparamagnetic core size and magnetization. CMD-IONs can also be used for bioseparation through covalent attachment of antibodies (Li et al. Journal of nanoscience and nanotechnology 2011 , 11 (11 ), 10187-10192). CMD-coated particles have also been used as carriers for immobilization of enzyme alcohol dehydrogenase (Vasic et al. Reactive and Functional Polymers 2020, 148, 104481 ) The cell uptake of CMD-coated particles is increased for nanoparticles with greater negative charge (Ayala et al Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology 2013, 15 (8), 1874). Internalization patterns suggest that uptake of the most negatively charged particles occurs via non-specific interactions (Ayala et al. Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology 2013, 15 (8), 1874).SUMMARY OF THE INVENTION

[0010] There is a need for a novel therapeutic strategy to increase treatment efficacy and reduce the adverse side effects of current chemotherapy regimens for cancers, such as aggressive lymphomas In order to facilitate a chemotherapeutical treatment of cancer cells (e.g. Burkitt lymphoma cells) it is important to facilitate a directed / targeted treatment of these cells Other, healthy cells should not be affected by the chemotherapeutics while the cancer cells must be treated. Therefore, targeted delivery is critically important, as treatment without targeted delivery typically results in harmful side effects due to healthy cells also being affected by the chemotherapeutic

[0011] For quassinoids such as brusatol only polymersome- and liposome-based strategies have been described for drug delivery so far. Magnetic iron oxide particles (lONs) coated with a carboxylated sugar (e.g. carboxymethyl dextran (CMD)) or carboxylated polymer have a highly negatively charged (hydrophilic) surface due to their coating and therefore have only been used for the delivery of positively charged (hydrophilic) drugs, which can bind to the negatively charged surface of the ION.

[0012] As an example, studies on conjugates of carboxylic particles with ibuprofen and naproxen, both of which are positively charged, achieved comparably high drug loadings (30-70 wt%) by covalent coupling and show the interaction of negatively charged surface and positively charged drug (Hornig et al (2009) Journal of colloid and interface science 338 (1 ), pp. 56-62). Similarly, negatively charged coated lONs enable the adsorption of the positively charged antimicrobial peptide lasioglassin at remarkable drug loading (62%) and an excellent efficiency (82.3%) obtained by covalent coupling (Turrina et al (2022) International journal of molecular sciences 23 (23))

[0013] The interaction of CMD with positively-charged drugs is utilized not only for solid nanoparticles, but also for micellar drug delivery systems. The micellization of CMD-PEG copolymers and drug delivery aspects of the resulting micelles were evaluated using different cationic (i.e. positively charged) drugs: diminazene (DIM), a model cationic drug, minocycline hydrochloride (MH), a semisynthetic tetracycline antibiotic with promising neuroprotective properties and different aminoglycoside antibiotics (Soliman and Winnik (2008) International journal of pharmaceutics 356 (1-2), pp. 248-258).

[0014] Similarly, over several decades, cation exchange hydrogels have been used for the design and development of supports for controlled delivery of drugs (Jeong and Park (2008) International journal of pharmaceutics 361 (1-2), pp. 26-32; Nitanan et al (2013) International journal of pharmaceutics 450 (1-2), pp. 345-353), and the most important advantage of these hydrogels is their high loading capacity due to electrostatic interactions between the anionic groups of the hydrogel and the oppositely charged drug molecules (Vasiliu et al. (2011 ) Carbohydrate Polymers 85 (2), pp. 376-387). Hydrogels such as cation exchange hydrogels based on a natural polymer (sulfopropyl dextran, SPD, carboxymethyl dextran, CMD, dextran sulfate, SD) or a synthetic matrix (styrene- divinylbenzene) (Vionit CS-3) can be used as support for loading and controlled delivery of the hypoglycemic basic drug buformin. The hydrogels, except dextran sulfate, proved high drug loading capacity and efficiency due to the strong drug / cation exchange hydrogel interactions and good accessibility of the drug to the loading sites (Fundueanu et al. (2017) Journal of nanoscience and nanotechnology 17 (7), pp. 4643-4648).

[0015] This strong interaction between cationic drugs and anionic nanomaterials is also used for charge converting nanomaterials. The very first charge converting nanomaterials for oral drug delivery were formed by coacervation of polyethylenimine-6-phosphogluconic acid conjugate and a chitosanphosphotyrosine conjugate with carboxymethylcellulose (Perera et al. (2015) European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e. V 97 (Pt A), pp 250-256; Bonengel et al. (2015) International journal of pharmaceutics 483 (1-2), pp. 19-25). Although not all phosphate groups were cleaved because of limited access for phosphatase such NCs could nonetheless convert their zeta potential from negative to positive values. Polyethylenimine-6- phosphogluconic acid NCs were shown to shift their zeta potential from -6 to +3 mV (Bonengel et al (2015) International journal of pharmaceutics 483 (1-2), pp. 19-25; Spleis et al (2023) Advances in colloid and interface science 313, p. 102848).

[0016] Following the same principle, lONs coated with a carboxylated sugar or carboxylated polymer, e g CMD- lONs, are considered unsuitable for delivery of uncharged (e.g. hydrophobic) or negatively charged drugs in the art due to their negative surface charge Quassinoids (e.g brusatol) are uncharged / hydrophobic. The inventors have however surprisingly found that quassinoids, despite their hydrophobicity, can be bound onto the negatively charged surface of a vehicle based on iron oxide nanoparticles coated with a carboxymethylated sugar or carboxylated polymer via non-specific interactions. These nanoparticles can be targeted to cancer cells by means of a targeting moiety, e.g. a monoclonal antibody, conjugated to the surface of the nanoparticle Advantageously, the quassinoid is released from the nanoparticle upon uptake of the vehicle by the cancer cell.

[0017] In a first aspect, the invention provides a magnetic iron oxide nanoparticle comprising: a targeting moiety that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar or carboxylated polymer. In one such embodiment, the carboxylated sugar is carboxymethyl dextran.

[0018] In an embodiment, the nanoparticle is a superparamagnetic nanoparticle. In one such embodiment, the nanoparticle exhibits a hydrodynamic diameter below 200 nm.

[0019] In an embodiment, the targeting moiety is selected from the group consisting of an antibody and an antigenbinding fragment thereof. In one such embodiment, the antibody is a monoclonal antibody. In another or the same embodiment, the antigen-binding fragment is selected from the group consisting of an F(ab)2 fragment, an Fab fragment, and an scFv fragment.

[0020] In an embodiment, the antibody or antigen-binding fragment thereof binds a protein expressed on the surface of a lymphoma cell. In one such embodiment, the antibody is selected from the group consisting of an anti- CD19 antibody, an anti-CD20 antibody, and a CD79b-antibody.

[0021] In an embodiment, the cancer cell is selected from the group consisting of a pituitary cancer cell, a colorectal cancer cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a pancreatic cancer cell, a melanoma cell, an ovarian cancer cell, an endometrial cancer cell, a breast cancer cell, a nasopharyngeal carcinoma cell, a glioma cell, a head and neck squamous cell carcinoma cell, and a lymphoma cell. In one such embodiment, the lymphoma cell is a B-cell lymphoma cell. In one such embodiment, the B-cell lymphoma cell is an aggressive B-cell lymphoma cell. In one such embodiment, the aggressive B-cell lymphoma cell is selected from the group consisting of a Burkitt’s lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, and a mantle cell lymphoma (MCL) cell

[0022] In an embodiment, the carboxylated sugar is carboxymethyl dextran and the targeting moiety is conjugated to the surface of the nanoparticle by an amide bond, a thioether bond, an ether bond, an acetal bond, an azo bond, a sulfenamide bond, an ester bond, a thiol bond, a thioester bond, a carbon-carbon bond, or a coordinative bond between the targeting moiety and the carboxymethyl dextran.

[0023] In an embodiment, the quassinoid is a bruceolide.

[0024] In an embodiment, the bruceolide is brusatol.

[0025] In a second aspect, the invention provides a composition comprising the nanoparticle of the first aspect.

[0026] In an embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

[0027] In a third aspect, the invention provides the nanoparticle of the first aspect or the composition of the second aspect for use in a method of treating a cancer in a mammal In one such embodiment, the mammal is a human.

[0028] In an embodiment the cancer is selected from the group consisting of pituitary cancer, colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma. In one such embodiment, the lymphoma is a B-cell lymphoma In one such embodiment, the B-cell lymphoma is an aggressive B- cell lymphoma In one such embodiment, the aggressive B-cell lymphoma is selected from the group consisting of a Burkitt’s lymphoma, a diffuse large B cell lymphoma (DLBCL), a follicular lymphoma grade 3 (FL III), and a mantle cell lymphoma (MCL).

[0029] In a fourth aspect, the invention provides a method of manufacturing the nanoparticle of the first aspect, comprising the steps of: a) mixing a quassinoid solution with magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising a targeting moiety conjugated to the surface of the nanoparticle and sonicating to obtain a sonicated mixture; b) shaking the sonicated mixture obtained in step a) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising the targeting moiety conjugated to the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via non-specific interactions; and c) washing the nanoparticles obtained in step b.In one such embodiment, the carboxylated sugar is carboxymethyl dextran.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Schematic illustration of the production of a CMD-ION with a targeting antibody conjugated to its surface and a quassinoid bound to its surface via non-specific interactions.

[0031] Figure 2: Infrared spectra of bare CMD-coated iron oxide nanoparticles, of CM-coated particles modified with antibodies as well as particles modified with brusatol and brusatol as reference

[0032] Figure 3: Annexin staining of BL-2 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0033] Figure 4: Annexin staining of BL-2 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0034] Figure 5: Annexin staining of Raji cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0035] Figure 6: Annexin staining of Raji cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0036] Figure 7: Annexin staining of SU-DHL-4 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0037] Figure 8: Annexin staining of SU-DHL-4 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for

[0038] Figure 9: Annexin staining of SU-DHL-4 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0039] Figure 10: Annexin staining of Karpas-422 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0040] Figure 11 : Annexin staining of Karpas-422 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0041] Figure 12: Annexin staining of Karpas-422 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0042] Figure 13: Annexin staining of U-2932 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0043] Figure 14: Annexin staining of U-2932 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0044] Figure 15: Annexin staining of U-2932 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0045] Figure 16: Annexin staining of Ri-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0046] Figure 17: Annexin staining of Ri-1 incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti- CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0047] Figure 18: Annexin staining of Ri-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0048] Figure 19: Annexin staining of THP-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0049] Figure 20: Annexin staining of THP-1 incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti- CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0050] Figure 21 : Annexin staining of THP-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0051] Figure 22: Annexin staining of Jurkat cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 24h.

[0052] Figure 23: Annexin staining of Jurkat incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti- CD19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 48h.

[0053] Figure 24: Annexin staining of Jurkat incubated with either DMSO, Brusatol, lONs, lONs conjugated to antiCD 19 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD19 antibody, or particles with coumarin for 72h.

[0054] Figure 25: Annexin staining of BL-2 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0055] Figure 26: Annexin staining of Raji cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0056] Figure 27: Annexin staining of SU-DHL-4 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0057] Figure 28: Annexin staining of Karpas-422 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0058] Figure 29: Annexin staining of U-2932 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0059] Figure 30: Annexin staining of Ri-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0060] Figure 31 : Annexin staining of THP-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h.

[0061] Figure 32: Annexin staining of Jurkat cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD20 antibody, lON-brusatol, lON-brusatol conjugated to anti-CD20 antibody, or particles with coumarin for 24, 48, and 72 h

[0062] Figure 33: Annexin staining of BL-2 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0063] Figure 34: Annexin staining of Raji cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0064] Figure 35: Annexin staining of Karpas-422 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0065] Figure 36: Annexin staining of U-2932 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0066] Figure 37: Annexin staining of Ri-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0067] Figure 38: Annexin staining of THP-1 cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0068] Figure 39: Annexin staining of Jurkat cells incubated with either DMSO, Brusatol, lONs, lONs conjugated to anti-CD79b antibody, lON-brusatol, lON-brusatol conjugated to anti-CD79b antibody, or particles with coumarin for 24, 48, and 72 h.

[0069] Figure 40: Annexin staining of untreated SU-DLH-4cells cultured for 24h or 48h and SU-DLH-4 cells treated with either lON-brusatol (“CMD-ION-brusatol [100nM]”), lON-brusatol conjugated to anti-CD19 antibody (“CMD-ION- CD19-brusatol [100nM]”), or the corresponding supernatants (“CMD-ION-brusatol-supernatant” and “CMD-ION- CD19-brusatol-supernatant”, respectively) for 24h or 48hDETAILED DESCRIPTION OF THE INVENTION

[0070] As laid out above, the inventors have however surprisingly found that quassinoids, despite their negative charge, can be bound onto the negatively charged surface of a vehicle based on iron oxide nanoparticles coated with a carboxymethylated sugar or carboxylated polymer via non-specific interactions. These nanoparticles can be targeted to cancer cells by means of a targeting moiety, e.g. a monoclonal antibody, conjugated to the surface of thenanoparticle. Advantageously, the quassinoid may be released from the nanoparticle upon uptake of the vehicle by the cancer cell, for example by lysosome or peroxisome degradation.

[0071] Before the present invention is described in more detail in the example section, the following definitions are introduced.

[0072] As used in the specification and the claims, the singular forms of “a” and “an” also include the corresponding plurals unless the context clearly dictates otherwise.

[0073] The term “about" in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10% and preferably ±5%.

[0074] It needs to be understood that the term “comprising” is not limiting. For the purposes of the pre-sent invention, the term “consisting of is considered to be a preferred embodiment of the term “comprising” If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

[0075] In the following, where a term is defined for one aspect of the invention, the term shall mean the same if used in another aspect of the invention, unless it is explicitly stated otherwiseNanoparticles

[0076] In a first aspect, the invention provides a magnetic iron oxide nanoparticle comprising: a targeting moiety that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar or carboxylated polymer.

[0077] The term “iron oxide nanoparticle” or “ION” as used herein refers to nanoparticles consisting of maghemite (y-Fe2O3) and / or magnetite (Fe3O4) particles. As with other nanoparticles, the surface area to volume ratio increases significantly for lONs This allows a considerably higher binding capacity and excellent dispersibility of lONs in solutions.

[0078] The term “targeting moiety” as used herein refers to a molecule which binds to a protein expressed on the surface of a cancer cell. In a preferred embodiment, the binding to a protein expressed on the surface of a cancer cell is specific When an ION is conjugated to the targeting moiety, the targeting moiety can bring the ION to the cancer cell by binding the protein expressed on the surface of the cancer cell. In this manner, the ION is targeted to the cancer cell Preferably, the protein expressed on the surface of a cancer cell is expressed only on the cancer cell, but not on healthy, non-cancerous cells. In this manner, the targeting moiety targets the ION specifically to the cancer cell, but not to the healthy cells which do not express the protein expressed on the surface of the cancer cell.

[0079] The term “cancer cell” as used herein refers to a cell that divides continually. In a subject, cancer cells form solid tumors or flood the blood or lymph, giving rise to cancer in the subject. Cancer cells can differentially express proteins on their surface that are not expressed in healthy counterpart cells, and which can serve as a marker for cancer and as a target for targeting moieties that bind to these differentially expressed proteins.

[0080] The term “conjugated” or “conjugation" as used herein refers to the overlap of one p-orbital with another across an adjacent o bond (although in transition metals, d-orbitals can be involved). A chemical bond can result. In the lONs of the invention, a bond is formed between the targeting moiety and a carboxyl group of the carboxylated sugar or carboxylated polymer the ION is coated with.

[0081] The term “quassinoid” as used herein refers to degraded triterpene lactones of the Simaroubaceae plant family grouped into C-18, C-19, C-20, C-22 and C-25 types The prototypical member of the group, quassin, was first described in the 19th century from plants of the genus Quassia from which it gets its name. More than 200 quassinoids have been extracted from various Simaroubaceae family species such as, inter alia, Ailanthus excelsa,Ailanthus vilmoriniana, (the fruits of) Brucea javanica, Hannoa klaineana, Pierreodendron kerstingii, Quassia africana, Quassia amara, (the wood of ) Picrasma ailanthoides, Picrasma javanica, Picrolemma pseudocoffea, Simaba guianensis, and Simaruba glauca. Quassinoids isolated from Brucea javanica are termed “bruceolides”. Perhaps the most studied bruceloide is brusatol, which has the chemical formula:

[0082] The term “non-specific interactions” as used herein refers to generic force functions that occur at the interface of many types of atoms, molecules, macromolecules, and surfaces and which do not involve the sharing of electrons.

[0083] The term “carboxylated” as used herein refers to compounds comprising an added carboxyl group generated by carboxylation. Carboxylation is a chemical reaction in which a carboxylic acid is produced by treating a substrate with carbon dioxide That is, a carboxylated sugar is a sugar comprising added carboxyl groups, and a carboxylated polymer is a polymer comprising added carboxyl groups. Examples of carboxylated sugars include, but are not limited to, carboxymethyl dextran (CMD), aldonic acid, uronic acid, aldaric acid Examples of carboxylated polymers include, but are not limited to, polyacrylic acid, carboxymethylcellulose, polysulfates, polysulfonates.

[0084] In one embodiment, the carboxylated sugar is carboxymethyl dextran (CMD). That is, the present invention provides a magnetic iron oxide nanoparticle comprising: a targeting moiety that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with carboxymethyl dextran. An ION coated with CMD is also referred to as a “CMD-ION”.

[0085] In an embodiment, the nanoparticle of the invention is a superparamagnetic nanoparticle. That is, the present invention provides a superparamagnetic iron oxide nanoparticle comprising: a targeting moiety that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0086] “Paramagnetic” as used herein means that the lONs are weakly attracted by an externally applied magnetic field, and form internal, induced magnetic fields in the direction of the applied magnetic field. “Superparamegnetic" as used herein means that the lONs show induced magnetic behavior that follows a Curie type law but with exceptionally large values for the Curie constants Superparamagnetic lONs are characterized by a strong ferromagnetic or ferrimagnetic type of coupling into domains of a limited size that behave independently from one another. Their bulk properties resembles that of a paramagnet, but on a microscopic level they are ordered.

[0087] In an embodiment, the superparamagnetic nanoparticle of the invention exhibits a hydrodynamic diameter below 200 nm, e g of 190, 180, 170, 160, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95,94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64,63, 62, 61 , 60, 59, 58, 57, 56, 55, 54, 5, 52, 51 , 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33,32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9 8, 7, 6, or 5 nm. Preferably,the superparamagnetic nanoparticle of the invention exhibits a hydrodynamic diameter below 150 nm, more preferably below 100 nm. The term “hydrodynamic diameter” as used herein refers to the diameter of a hypothetical hard sphere that diffuses with the same speed as the nanoparticle being measured.

[0088] In an embodiment, the targeting moiety is selected from the group consisting of an antibody and an antigenbinding fragment thereof. That is, the present invention provides a magnetic (e.g superparamagnetic) iron oxide nanoparticle comprising: an antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e g , CMD) or carboxylated polymer.

[0089] The term “antigen-binding fragment" as used herein refers to any fragment of an antibody that comprises at least the variable fragment of the light chain (VL) and variable fragment of the heavy chain (VH) of the antibody and thus maintains binding the antibody’s antigen Antigen-binding fragments include, but are not limited to, Fab (fragment antigen binding), F(ab’)2, chemically linked F(ab’)2, Fab’, scFv, and di-scFv.

[0090] In an embodiment, in any of the inventive lONs, the antibody is a monoclonal antibody.

[0091] In an embodiment, in any of the inventive lONs, the antigen-binding fragment is selected from the group consisting of an F(ab’)2fragment, an Fab fragment, and an scFv fragment

[0092] In an embodiment, in any of the inventive lONs, the antibody is a monoclonal antibody and the antigenbinding fragment is selected from the group consisting of an F(ab’)2fragment, an Fab fragment, and an scFv fragment.

[0093] In an embodiment, in any of the inventive lONs, the (e.g. monoclonal) antibody or antigen-binding fragment (e.g. F(ab’)2fragment, an Fab fragment, and an scFv fragment) thereof binds a protein expressed on the surface of a lymphoma cell That is, the present invention provides a magnetic (e.g. superparamagnetic) iron oxide nanoparticle comprising: an antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a lymphoma cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0094] The term “lymphoma” refers to a cancer that begins in cells of the lymph system. A “lymphoma cell” may be, e g., a cancerous B cell, in which case the lymphoma is a B cell lymphoma.

[0095] In one such embodiment, the antibody is selected from the group consisting of an anti-CD19 antibody, an anti-CD20 antibody, and a CD79b-antibody. That is, the present invention provides a magnetic (e.g superparamagnetic) iron oxide nanoparticle comprising: an anti-CD19 antibody, an anti-CD20 antibody, or a CD79b-antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a lymphoma cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0096] In an embodiment, for any of the inventive lONs, the cancer cell is selected from the group consisting of a pituitary cancer cell, a colorectal cancer cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a pancreatic cancer cell, a melanoma cell, an ovarian cancer cell, an endometrial cancer cell, a breast cancer cell, anasopharyngeal carcinoma cell, a glioma cell, a head and neck squamous cell carcinoma cell, and a lymphoma cell. That is, the present invention provides a magnetic (e.g. superparamagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a pituitary cancer cell, a colorectal cancer cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a pancreatic cancer cell, a melanoma cell, an ovarian cancer cell, an endometrial cancer cell, a breast cancer cell, a nasopharyngeal carcinoma cell, a glioma cell, a head and neck squamous cell carcinoma cell, or a lymphoma cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer

[0097] Brusatol has been shown to anti-tumor properties in pituitary cancer (Wu et al. Oxid Med Cell Longev. 2021 Jun 16;2021 :6696015) and in colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma (Yu et al Chin Herb Med 2020 Aug 19; 12(4): 359-366)

[0098] In one such embodiment, the lymphoma cell is a B-cell lymphoma cell. In one such embodiment, the B-cell lymphoma cell is an aggressive B-cell lymphoma cell. That is, the present invention provides a magnetic (e.g superparamagnetic) iron oxide nanoparticle comprising: a targeting moiety (e g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a B-cell lymphoma cell, preferably an aggressive B-cell lymphoma cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0099] In one such embodiment, the aggressive B-cell lymphoma cell is selected from the group consisting of a Burkitt's lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, and a mantle cell lymphoma (MCL) cell. That is, the present invention provides a magnetic (e.g superparamagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a Burkitt's lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, or a mantle cell lymphoma (MCL) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0100] In one such embodiment that is especially preferred, the present invention provides a magnetic (e.g. superparamagnetic) iron oxide nanoparticle comprising: an anti-CD19 antibody, an anti-CD20 antibody, or a CD79b-antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a Burkitt’s lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, or a mantle cell lymphoma (MCL) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g., CMD) or carboxylated polymer.

[0101] In an embodiment, in any of the inventive lONs, the carboxylated sugar is carboxymethyl dextran and the targeting moiety is conjugated to the surface of the nanoparticle by an amide bond, a thioether bond, an ether bond, an acetal bond, an azo bond, a sulfenamide bond, an ester bond, a thiol bond, a thioester bond, a carbon-carbon bond, or a coordinative bond between the targeting moiety and the carboxymethyl dextran. That is, the present invention provides a magnetic (e g. superparamagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle by an amide bond, a thioether bond, an ether bond, an acetal bond, an azo bond, a sulfenamide bond, an ester bond, a thiol bond, a thioester bond, a carbon-carbon bond, or a coordinative bond between the targeting moiety and carboxymethyl dextran, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with the carboxymethyl dextran (CMD).

[0102] In an embodiment, in any of the inventive lONs, the quassinoid is a bruceolide. That is, the invention provides a magnetic (e g paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a bruceolide, wherein the bruceolide is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e g CMD) or carboxylated polymer.

[0103] In one such embodiment, the bruceolide is brusatol. That is, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0104] In a preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0105] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0106] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a pituitary cancer cell, a colorectal cancer cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a pancreatic cancer cell, a melanoma cell, an ovarian cancer cell, an endometrial cancer cell, a breast cancer cell, anasopharyngeal carcinoma cell, a glioma cell, a head and neck squamous cell carcinoma cell, or a lymphoma cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0107] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: a targeting moiety (e.g an antibody or antigen-binding fragment thereof) that binds a protein expressed on the surface of a B cell lymphoma (e.g an aggressive B cell lymphoma) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0108] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: an anti-CD19 antibody, an anti-CD20 antibody, or a CD79b-antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a B cell lymphoma (e.g. an aggressive B cell lymphoma) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer.

[0109] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: an anti-CD19 antibody, an anti-CD20 antibody, or a CD79b-antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a B cell lymphoma (e.g. an aggressive B cell lymphoma) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with carboxymethyl dextran (CMD).

[0110] In a further preferred embodiment, the invention provides a magnetic (e.g. paramagnetic) iron oxide nanoparticle comprising: an anti-CD19 antibody, an anti-CD20 antibody, or a CD79b-antibody or antigen-binding fragment thereof that binds a protein expressed on the surface of a Burkitt’s lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, or a mantle cell lymphoma (MCL) cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and brusatol, wherein brusatol is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with carboxymethyl dextran (CMD).Compositions

[0111] In a second aspect, the invention provides a composition comprising any nanoparticle of the first aspect.

[0112] In an embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

[0113] The term “pharmaceutically acceptable carrier” as used herein is intended to include, but not be limited to, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the ION of the invention, such media can be used in the compositions described herein. Supplementary activecompounds can also be incorporated into the compositions, e g., an inhibitor of degradation, antibacterial and antifungal agents, etc.

[0114] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic

[0115] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (polyethoxylated castor oil; BASF, Parsippany, N J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0116] Sterile injectable solutions can be prepared by incorporating the composition of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0117] Oral compositions generally include an inert diluent or an edible carrier They can be enclosed in gelatin capsules or compressed into tablets For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL™ (sodium carboxymethyl starch), or corn starch; a lubricant such as magnesium stearate or STEROTES™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0118] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation Such penetrants are generally known, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. T ransmucosal administration can be accomplished through the use of nasal sprays orsuppositories For transdermal administration, the lONs of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.

[0119] In some embodiments, the active lONs of the invention are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0120] The pharmaceutical compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration In one aspect, the pharmaceutical compositions can be included as a part of a kit.

[0121] Generally, the dosage used to administer a pharmaceutical composition facilitates an intended purpose for treatment without undesirable side effects, such as toxicity, irritation, or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the artHuman doses can readily be extrapolated from animal studies. Generally, the dosage required to provide an effective amount of a formulation, which can be adjusted by one skilled in the art, will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s).Uses in methods of treatment of cancer in a mammal

[0122] In a third aspect, the invention provides any nanoparticle of the first aspect or any composition of the second aspect for use in a method of treating a cancer in a mammal.

[0123] The term “treating” or “treatment” as used herein indicate that a cancer is suspected or has been diagnosed in a subject Treatment may lead to a halt in the progression of the cancer or a delay in the progression of the cancer T reatment may also lead to a partial amelioration of symptoms or complete response. Amelioration may, for example, lead to a stop in the progression of the cancer or a delay in the progression of the cancer. Such a partial or complete response may be followed by a relapse.

[0124] The term “mammal” as used herein refers to vertebrate animals of the class Mammalia In a preferred embodiment, the mammal is a human. That is, the invention provides any nanoparticle of the first aspect or any composition of the second aspect for use in a method of treating a cancer in a human.

[0125] In an embodiment, in any of the uses of the invention, the cancer is selected from the group consisting of pituitary cancer, colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma. That is, the invention provides any nanoparticle of the first aspect or any composition of the second aspect for use in a method of treating a pituitary cancer, colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma in a mammal (e.g. a human).

[0126] Brusatol has been shown to anti-tumor properties in pituitary cancer (Wu et al. Oxid Med Cell Longev. 2021 Jun 16;2021 :6696015) and in colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma (Yu et al. Chin Herb Med. 2020 Aug 19; 12(4): 359-366).

[0127] In one such embodiment, the lymphoma is a B-cell lymphoma. In one such embodiment, the B-cell lymphoma is an aggressive B-cell lymphoma. That is, the invention provides any nanoparticle of the first aspect orany composition of the second aspect for use in a method of treating a B cell lymphoma, preferably an aggressive B cell lymphoma in a mammal (e.g a human)

[0128] In one such embodiment, the aggressive B-cell lymphoma is selected from the group consisting of a Burkitt’s lymphoma, a diffuse large B cell lymphoma (DLBCL), a follicular lymphoma grade 3 (FL III), and a mantle cell lymphoma (MCL). That is, the invention provides any nanoparticle of the first aspect or any composition of the second aspect for use in a method of treating Burkitt’s lymphoma, a diffuse large B cell lymphoma (DLBCL), a follicular lymphoma grade 3 (FL III), or a mantle cell lymphoma (MCL) in a mammal (e g. a human).

[0129] The method of treating cancer in a mammal may include administration of any nanoparticle of the first aspect of the invention or any composition of the second aspect of the invention to the mammal (e.g. the human) in a therapeutically effective amount.

[0130] A “therapeutically effective amount” as used herein can be administered by standard methods, for example, by one or more routes of administration, e.g., orally, topically, mucosally, or parenterally, e.g., intravenously or intramuscularly as laid out for the second aspect of the invention. Generally, the dosage used to administer a pharmaceutical composition facilitates an intended purpose for treatment without undesirable side effects, such as toxicity, irritation, or allergic response Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can readily be extrapolated from animal studies Generally, the dosage required to provide an effective amount of a formulation, which can be adjusted by one skilled in the art, will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s).Methods of manufacture

[0131] In a fourth aspect, the invention provides a method of manufacturing any nanoparticle of the first aspect, comprising the steps of: a) mixing a quassinoid solution with magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising a targeting moiety conjugated to the surface of the nanoparticle and sonicating to obtain a sonicated mixture; b) shaking the sonicated mixture obtained in step a) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising the targeting moiety conjugated to the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via non-specific interactions; and c) washing the nanoparticles obtained in step b. This

[0132] Sonication may occur at temperatures from 0 - 60 °C, at powers of 0 1 to 5 W, for 5 seconds to 1 hour.

[0133] Shaking may occur at temperatures from 0 - 60 °C for 5 seconds to 1 hour.

[0134] Washing may occur at temperatures from 0 - 60 °C for 5 seconds to 1 hour with physiological buffers at pH ranges from 4 to 10 for 1 to 20 times.

[0135] In an embodiment, the carboxylated sugar is carboxymethyl dextran. That is, the invention provides a method of manufacturing any nanoparticle of the first aspect, comprising the steps of: a) mixing a quassinoid solution with magnetic iron oxide nanoparticles coated with carboxymethyl dextran and comprising a targeting moiety conjugated to the surface of the nanoparticle and sonicating to obtain a sonicated mixture; b) shaking the sonicated mixture obtained in step a) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with carboxymethyl dextran and comprising the targeting moiety conjugated to the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via non-specific interactions; and c) washing the nanoparticles obtained in step b).

[0136] In an embodiment, in any of the methods of manufacture of the invention, the step of mixing may be preceded by a step of conjugating a targeting moiety (e.g. an antibody or antigen-binding fragment thereof) with magnetic iron oxide nanoparticles coated with a carboxylated sugar (e g. CMD) or carboxylated polymer. That is, the invention provides a method of manufacturing any nanoparticle of the first aspect, comprising the steps of: a) conjugating a targeting moiety (e.g. an antibody or antigen-binding fragment thereof) with magnetic iron oxide nanoparticles coated with a carboxylated sugar (e.g CMD) or carboxylated polymer; b) mixing a quassinoid solution with the magnetic iron oxide nanoparticles coated with coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer and comprising a targeting moiety (e g. an antibody or antigen-binding fragment thereof) conjugated to the surface of the nanoparticle obtained in step a) and sonicating to obtain a sonicated mixture; c) shaking the sonicated mixture obtained in step b) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer and comprising the targeting moiety (e g. an antibody or antigen-binding fragment thereof) conjugated to the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via nonspecific interactions; and d) washing the nanoparticles obtained in step c).

[0137] Such a method of manufacture is schematically illustrated in Figure 1.

[0138] Conjugation in the conjugating step may be achieved by, e g., two-step 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide I N-hydroxylsulfosuccinimide covalent coupling (as described in the art e g , at https: / / www.selectscience.net / application-articles / microsphere-coupling-two-step-edc-sulfo-nhs-covalent-coupling- procedure-for-estapor-carboxyl-modified-dyed-microspheres / ?artid=58381 ); maleimide coupling, glutaraldehyde coupling, diazo coupling, sulfonyl chloride coupling, isocyanate coupling, ROMP coupling (e.g., epoxy), click chemistry (e.g., Suzuki, Heck, Sonogashira, Mannich, Photoclick, etc.), coordinative bonds (e.g., Ni-NTA / Protein A, Streptavidin / avidin, Flag-Tag), or electrostatic bonds (physisorbed molecules) between the targeting moiety (e g. an antibody or antigen-binding fragment thereof) and a carboxy group of the carboxylated sugar (e.g. CMD) or carboxylated polymer. All of these procedures are well known in the art and summarized in Schwaminger et al.(2019) Magnetic Separation in Bioprocessing Beyond the Analytical Scale: From Biotechnology to the Food Industry. Front. Bioeng. Biotechnol. 7:233.

[0139] In an embodiment, in any of the methods of manufacture of the invention, the step of conjugating may be preceded by a step of reacting a salt of a carboxylated sugar (e.g CMD) or carboxylated polymer, an aqueous ammonium hydroxide solution, and an iron (ll / lll) solution to obtain carboxymethyl dextran-coated magnetic iron oxide nanoparticles. That is, the invention provides a method of manufacturing any nanoparticle of the first aspect, comprising the steps of: a) reacting a salt of a carboxylated sugar (e g. CMD) or carboxylated polymer, an aqueous ammonium hydroxide solution, and an iron (ll / lll) solution to obtain carboxymethyl dextran-coated magnetic iron oxide nanoparticles (CMD-IONs); b) conjugating a targeting moiety(e.g. an antibody or antigen-binding fragment thereof) with the magnetic iron oxide nanoparticles coated with a carboxylated sugar (e g. CMD) or carboxylated polymer obtained in step a); c) mixing a quassinoid solution with the magnetic iron oxide nanoparticles coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer and comprising a targeting moiety (e.g. an antibody or antigen-binding fragment thereof) conjugated to the surface of the nanoparticle obtained in step b) and sonicating to obtain a sonicated mixture; d) shaking the sonicated mixture obtained in step c) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with a carboxylated sugar (e.g. CMD) or carboxylated polymer and comprising the targeting moiety (e g. an antibody or antigen-binding fragment thereof) conjugatedto the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via nonspecific interactions; and e) washing the nanoparticles obtained in step d).

[0140] An exemplary reacting step is described in Turrina et al., International journal of molecular sciences 2022, 23 (23) Reacting may occur at temperatures of 0 -60 °C for 1 minute to 24 hours.

[0141] An exemplary method of manufacture of the invention is illustrated in Examples 1 , 2, and 4.Examples

[0142] The following Examples are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention theretoExample 1 - Synthesis of CMD-IONS

[0143] Carboxymethyl dextran (CMD)-coated iron oxide nanoparticles (CMD-IONs) were synthesized in accordance to the method described by Turrina et al., International journal of molecular sciences 2022, 23 (23).

[0144] A total of 20 mL of 25 g L-1(CMD25.0) CMD sodium salt, BioXtra, Sigma Aldrich, Darmstadt, Germany, 39422-83-8); and 2 5 mL of aqueous 25% ammonium hydroxide solution (Aldrich Chemistry) were added to a 100 mL round-bottomed flask in a nitrogen atmosphere.

[0145] The reaction was induced by adding 20 mL of an iron (ll / lll) solution (FeCI24H2O (1 eq., 347 mg 1.75 mmol), EmsureTM; FeCI36H2O (2 eq., 945 mg, 3.50 mmol), Fluka Sigma Aldrich, Darmstadt, Germany) to the reaction mixture and by stirring it uniformly for one hour at a temperature of 85 °C.

[0146] After the completion of the reaction, the synthesized particles were centrifuged and washed with ethanol absolute (2*) and degassed using double-distilled water (ddH2O, 3x) until a conductivity lower than 200 pS cm-1was obtained. The particles were stored in an N2 atmosphere at 4 °C in degassed ddH2O.Example 2 - Conjugation of the monoclonal antibody to the CMD-IONS

[0147] After washing, 24 pL of an aqueous 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution (EDC) was supplemented for 1 mL of 1 g L'1CMD-IONs generated in Example 1 suspended in MES buffer.

[0148] Immediately after the EDC-solution, 240 mL L’1of a 200 mM N-hydroxysulfosuccinimide (sulfo-NHS) solution was added The mixture was incubated for 30 min at room temperature at 600 rpm. After incubation, the supernatant was removed, and the particles washed three times with MES buffer.

[0149] The activated CMD-IONs were then immersed in a 10Oul of 40 g L'1monoclonal antibody (mAb) solution (e.g. anti-CD19 mAb, anti-CD20 mAb, or anti-CD79b-mAb) and incubated at 25 °C for 2.5 h at 600 rpm. After incubation 50 pL ethanolamine were added and incubated for 30 min to stop the reaction. The supernatant was removed, and the CMD-IONs conjugated mAb were immersed in a 50 mM Tris(hydroxymethyl) aminomethane (Tris) 0 5 % (w / v) casein blocking buffer at pH 8.0 and incubated at room temperature overnight at 600 rpm. The supernatant was removed, and the CMD-IONs conjugated mAb (“CMD-ION-mAb”) was washed twice with a blocking buffer.Example 3 - Loading of Brusatol or Coumarin onto CMD-ION

[0150] For the synthesis of immobilised monoclonal antibody CMD-IONs conjugation with Brusatol (“CMD-ION- coumarin"), 5 mg of coumarin was dissolved in 1 mL of 30% ethanol. To the dissolved brusatol, 1 ml of 1 g L’1of the CMD-ION from Example 1 was added and the mixture was sonicated for 10 minutes by an ultrasonic device. The mixture was vigorously shaken at room temperature at least for 4 h. The resulting CMD-ION-coumarin were washed with distilled water three times and were kept at 4 °C.Example 4 - Loading of Brusatol onto the CMD-ION-mAb

[0151] For the synthesis of immobilised monoclonal antibody CMD-IONs conjugation with Brusatol (“CMD-ION- mAb-brusatol”), 5 mg of brusatol was dissolved in 1 mL of 30% ethanol. To the dissolved brusatol, 1 ml of 1 g L1of the CMD-ION-mAbs from Example 2 was added and the mixture was sonicated for 10 minutes by an ultrasonic device. The mixture was vigorously shaken at room temperature at least for 4 h. The resulting CMD-ION-mAb- brusatol were washed with distilled water three times and were kept at 4 °C.Example 5 - Characterization of CMD-ION-mAb-brusatol

[0152] The CMD-ION-mAb-brusatol of Example 4 were characterized with attenuated total reflection Fourier transform infrared spectroscopy with a Perkin-Elmer Spectrum Two. Therefore the CMD-ION-mAb-brusatol were precipitated on the ATR crystal. The CMD-ION-mAb-brusatol were characterized towards their hydrodynamic diameter with DLS (Cordouan Vasco Flex) at pH 7.

[0153] The dynamic light scattering yielded a hydrodynamic diameter of the CMD-ION of 12 nm.

[0154] The infrared spectra show the successful immobilization of CMD on iron oxide, indicated by vibrations corresponding to symmetric and asymmetric C=O vibrations at around 1700 and 1500 cm-1 as well as by C-O-C vibrations around 1100 cm'1(see Figure 2).

[0155] The covalent attachment of mAb can be evidenced by the vibrations corresponding to amide bonds at 1600 and 1400 cm’1(see Figure 2). Brusatol is immobilized on the particles and shows a similar spectrum as the brusatol reference in addition to amide vibrations and vibrations corresponding to CMD (see Figure 2).Example 6 - Screening of Brutasol-CMD-IONs conjugated with a monoclonal anti-CD19 antibody

[0156] Unloaded CMD-IONs (“CMD-IONs”) from Example 1 , CMD-IONs loaded with a monoclonal anti-CD19 antibody (tafasitamab) (“CMD-ION-CD19”) as in Example 2, CMD-IONs loaded with brusatol (“CMD-ION-brusatol”) as in Example 3, CMD-IONs loaded with coumarin (“CMD-ION-coumarin”) as in Example 3, or CMD-IONs loaded with anti-CD19 monoclonal antibody and brusatol (“CMD-ION-CD19-brusatol”) as in Example 4 were incubated with cells from cell lines (BL-2, Raji, SU-DHL-4, Karpas-422, Ri-1 , U-2932, Jurkat, or THP-1 ) for 24, 48, or 72 hours in 37°C, 5% CO2Cells incubated with DMSO or 250 nM brusatol solution served as controls.

[0157] The determination of apoptosis induction after Brusatol treatment was analyzed by Annexin V staining. The cells were collected in duplicates, centrifuged (1000RPM, 5min, room temperature), and resuspended in Annexin V binding buffer (BioLegend, San Diego, California, USA). Next, cells were stained (15min, RT, in the dark) with APC Annexin V (BioLegend), followed by the addition of 7-AAD Viability Staining Solution (BioLegend) and then analyzed on a CytoFLEX S Flow Cytometer (Beckman Coulter, Brea, California, USA). Results were processed using FlowJo v10 8.1 software (BD, Franklin Lakes, New Jersey, USA). The results are shown in Figures 3-24. The results show a high specificity of the antibody-modified iron oxide particles loaded with brusatol. For almost all cells, the amount of live cells is lowest with antibody-labelled and brusatol loaded particles. Brusatol loaded particles and brusatol also show a lowered number of live cells while naked particles as well as antibody-labelled particles and antibodies do not affect the amount of live cells.

[0158] The results show a high specificity of the antibody-modified iron oxide particles loaded with brusatol. For almost all cells, the amount of live cells is lowest with antibody-labelled and brusatol loaded particles Brusatol loaded particles and brusatol also show a lowered number of live cells while naked particles as well as antibody-labelled particles and antibodies do not affect the amount of live cells.Example 7 - Screening of Brutasol-CMD-IONs conjugated with a monoclonal anti-CD20 antibody

[0159] Unloaded CMD-IONs (“CMD-IONs”) from Example 1 , CMD-IONs loaded with a monoclonal anti-CD20 antibody (rituximab) (“CMD-ION-CD20”) as in Example 2, CMD-IONs loaded with brusatol at 0.25 mg / ml (“CMD-ION- brusatol 0.25"), 0.5 mg / ml (“CMD-ION-brusatol 0.5"), or 1 mg / ml (“CMD-ION-brusatol 1") as in Example 3, CMD-IONs loaded with coumarin at 0.25 mg / ml (“CMD-ION-coumarin 0.25”), 0.5 mg / ml (“CMD-ION-coumarin 0.5”), or 1 mg / ml (“CMD-ION-coumarin 1”) as in Example 3, or CMD-IONs loaded with anti-CD20 monoclonal antibody and brusatol at 0 25 mg / ml (“CMD-ION-CD20-brusatol 0.25”), 0.5 mg / ml (“CMD-ION-CD20-brusatol 0 5”), or 1 mg / ml (“CMD-ION- CD20-brusatol 1”) as in Example 4 were incubated with cells from cell lines (BL-2, Raji, SU-DHL-4, Karpas-422, Ri-1 , U-2932, Jurkat, or THP-1 ) for 24, 48, or 72 hours in 37°C, 5% CO2Cells incubated with DMSO or 250 nM brusatol solution served as controls.

[0160] The determination of apoptosis induction after Brusatol treatment was analyzed by Annexin V staining. The cells were collected in duplicates, centrifuged (1000RPM, 5min, room temperature), and resuspended in Annexin V binding buffer (BioLegend, San Diego, California, USA). Next, cells were stained (15min, RT, in the dark) with APC Annexin V (BioLegend), followed by the addition of 7-AAD Viability Staining Solution (BioLegend) and then analyzed on a CytoFLEX S Flow Cytometer (Beckman Coulter, Brea, California, USA). Results were processed using FlowJo v10 8.1 software (BD, Franklin Lakes, New Jersey, USA). The results are shown in Figures 25-32.

[0161] The results show a high specificity of the antibody-modified iron oxide particles loaded with brusatol. For almost all cells, the amount of live cells is lowest with antibody-labelled and brusatol loaded particles Brusatol loaded particles and brusatol also show a lowered number of live cells while naked particles as well as antibody-labelled particles and antibodies do not affect the amount of live cells.Example 8 - Screening of Brutasol-CMD-IONs conjugated with a monoclonal anti-CD79b antibody

[0162] Unloaded CMD-IONs (“CMD-IONs”) from Example 1 , CMD-IONs loaded with a monoclonal anti-CD79b antibody (polatuzumab) (“CMD-ION-CD79b”) as in Example 2, CMD-IONs loaded with brusatol at 0.5 mg / ml (“CMD- ION-brusatol 0.5") or 1 mg / ml (“CMD-ION-brusatol 1”) as in Example 3, CMD-IONs loaded with coumarin at 0.5 mg / ml (“CMD-ION-coumarin 0 5”) or 1 mg / ml (“CMD-ION-coumarin 1 ”) as in Example 3, or CMD-IONs loaded with anti-CD79b monoclonal antibody and brusatol at 0.5 mg / ml (“CMD-ION-CD79b-brusatol 0.5") or 1 mg / ml (“CMD-ION- CD79b-brusatol 1 ”) as in Example 4 were incubated with cells from cell lines (BL-2, Raji, Karpas-422, Ri-1, U-2932, Jurkat, or THP-1 ) for 24, 48, or 72 hours in 37°C, 5% CO2. Cells incubated with DMSO or 250 nM brusatol solution served as controls.

[0163] The determination of apoptosis induction after Brusatol treatment was analyzed by Annexin V staining. The cells were collected in duplicates, centrifuged (1000RPM, 5min, room temperature), and resuspended in Annexin V binding buffer (BioLegend, San Diego, California, USA). Next, cells were stained (15min, RT, in the dark) with APC Annexin V (BioLegend), followed by the addition of 7-AAD Viability Staining Solution (BioLegend) and then analyzed on a CytoFLEX S Flow Cytometer (Beckman Coulter, Brea, California, USA). Results were processed using FlowJo v10 8.1 software (BD, Franklin Lakes, New Jersey, USA). The results are shown in Figures 33-39.

[0164] The results show a high specificity of the antibody-modified iron oxide particles loaded with brusatol. For almost all cells, the amount of live cells is lowest with antibody-labelled and brusatol loaded particles Brusatol loaded particles and brusatol also show a lowered number of live cells while naked particles as well as antibody-labelled particles and antibodies do not affect the amount of live cells.Example 9 - Determining whether during preparation of CMD-IONs loaded with brusatol, all brusatol is bound to the CMD-IONs

[0165] CMD-IONs loaded with brusatol at 100nM (“CMD-ION-brusatol [100nM]) as in Example 3, or CMD-IONs loaded with anti-CD19 monoclonal antibody and brusatol at 100nM (“CMD-ION-CD19-brusatol [100nM]) as inExample 4, or supernatants from these preparations (“CMD-ION-brusatol-supernatant”, “CMD-ION-CD19-brusatol- supernatant”) were incubated with SU-DHL-4 cell line cells for 24 or 48, 5% CO2. Untreated cells at 24 or 48h served as controls.

[0166] The determination of apoptosis induction after Brusatol treatment was analyzed by Annexin V staining. The cells were collected in duplicates, centrifuged (1000RPM, 5min, room temperature), and resuspended in Annexin V binding buffer (BioLegend, San Diego, California, USA). Next, cells were stained (15min, RT, in the dark) with APC Annexin V (BioLegend), followed by the addition of 7-AAD Viability Staining Solution (BioLegend) and then analyzed on a CytoFLEX S Flow Cytometer (Beckman Coulter, Brea, California, USA). Results were processed using FlowJo v10 8.1 software (BD, Franklin Lakes, New Jersey, USA). The results are shown in Figure 40

[0167] SU-DHL-4 lymphoma cells are a model known for its high sensitivity to brusatol The results in Figure 40 show that none of the supernatants affected Annexin V positivity or the percentage of viable cells, indicating no or only a negligible level of brusatol being present In contrast, treatment with CD19-targeted lON-brusatol particles led to increased Annexin V staining and reduced cell viability. These results indicate that during CMD-ION preparation, brusatol is predominantly bound by the delivery system, since only the CMD-IONs showed an effect.

Claims

CLAIMS1 A magnetic iron oxide nanoparticle comprising: a targeting moiety that binds a protein expressed on the surface of a cancer cell, wherein the targeting moiety is conjugated to the surface of the nanoparticle, and a quassinoid, wherein the quassinoid is bound to the surface of the nanoparticle via non-specific interactions, wherein the magnetic iron oxide nanoparticle is coated with a carboxylated sugar or carboxylated polymer; optionally wherein the carboxylated sugar is carboxymethyl dextran.2 The nanoparticle of claim 1 , wherein the nanoparticle is a superparamagnetic nanoparticle, optionally wherein the nanoparticle exhibits a hydrodynamic diameter below 200 nm3 The nanoparticle of claim 1 or claim 2, wherein the targeting moiety is selected from the group consisting of an antibody and an antigen-binding fragment thereof, optionally wherein the antibody is a monoclonal antibody and / or optionally wherein the antigen-binding fragment is selected from the group consisting of an F(ab)2 fragment, an Fab fragment, and an scFv fragment4 The nanoparticle of claim 3, wherein the antibody or antigen-binding fragment thereof binds a protein expressed on the surface of a lymphoma cell, optionally wherein the antibody is selected from the group consisting of an anti- CD19 antibody, an anti-CD20 antibody, and a CD79b-antibody5 The nanoparticle of any one of claims 1-4, wherein the cancer cell is selected from the group consisting of a pituitary cancer cell, a colorectal cancer cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a pancreatic cancer cell, a melanoma cell, an ovarian cancer cell, an endometrial cancer cell, a breast cancer cell, a nasopharyngeal carcinoma cell, a glioma cell, a head and neck squamous cell carcinoma cell, and a lymphoma cell.6 The nanoparticle of claim 5, wherein the lymphoma cell is a B-cell lymphoma cell, optionally wherein the B-cell lymphoma cell is an aggressive B-cell lymphoma cell, further optionally wherein the aggressive B-cell lymphoma cell is selected from the group consisting of a Burkitt’s lymphoma cell, a diffuse large B cell lymphoma (DLBCL) cell, a follicular lymphoma grade 3 (FL III) cell, and a mantle cell lymphoma (MCL) cell.7 The nanoparticle according to any one of claims 1-6, wherein the carboxylated sugar is carboxymethyl dextran and wherein the targeting moiety is conjugated to the surface of the nanoparticle by an amide bond, a thioether bond, an ether bond, an acetal bond, an azo bond, a sulfenamide bond, an ester bond, a thiol bond, a thioester bond, a carbon-carbon bond, or a coordinative bond between the targeting moiety and the carboxymethyl dextran8 The nanoparticle according to any one of claims 1-7, wherein the quassinoid is a bruceolide.9 The nanoparticle according to claim 8, wherein the bruceolide is brusatol.

10. A composition comprising the nanoparticle according to any one of claims 1-911. The composition according to claim 10, wherein the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

12. The nanoparticle according to any one of claims 1-9 or the composition according to claim 10 or 11 for use in amethod of treating a cancer in a mammal, optionally wherein the mammal is a human.

13. The nanoparticle or composition for use according to claim 12, wherein the cancer is selected from the group consisting of pituitary cancer, colorectal cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, melanoma, ovarian cancer, endometrial cancer, breast cancer, nasopharyngeal carcinoma, glioma, head and neck squamous cell carcinoma, and lymphoma.

14. The nanoparticle or composition for use according to claim 13, wherein the lymphoma is a B-cell lymphoma, optionally wherein the B-cell lymphoma is selected from the group consisting of Burkitt’s lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma grade 3 (FL III), and mantle cell lymphoma (MCL)15. Method of manufacturing a nanoparticle according to any one of claims 1-9, comprising the steps of: a) mixing a quassinoid solution with magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising a targeting moiety conjugated to the surface of the nanoparticle and sonicating to obtain a sonicated mixture; b) shaking the sonicated mixture obtained in step a) at room temperature for at least 4 hours to obtain magnetic iron oxide nanoparticles coated with a carboxylated sugar or carboxylated polymer and comprising the targeting moiety conjugated to the surface of the nanoparticle and the quassinoid bound to the surface of the nanoparticle via non-specific interactions; and c) washing the nanoparticles obtained in step b), optionally wherein the carboxylated sugar is carboxymethyl dextran.

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