WO2022124898A1

WO2022124898A1 - Auristatin-loaded liposomes and uses thereof.

Info

Publication number: WO2022124898A1
Application number: PCT/NL2021/050750
Authority: WO
Inventors: Mohamadreza AMIN; Timotheus Lambertus Maria TEN HAGEN
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2020-12-09
Filing date: 2021-12-09
Publication date: 2022-06-16

Abstract

The invention provides a method for producing an auristatin-loaded liposome, comprising the steps of: - providing a liposome that encapsulates a remote loading agent; - generating a concentration gradient of said remote loading agent across the membrane of said liposome; - mixing said liposome with an aqueous medium comprising an auristatin; - loading said auristatin into said liposome; wherein said loading is driven by said concentration gradient; and - optionally, purifying an auristatin-loaded liposome.

Description

Title: Auristatin-loaded liposomes and uses thereof.

FIELD OF THE INVENTION

The present invention is in the field of cancer chemotherapy. More specifically, the invention is in the field of cancer treatment using liposomes loaded with an auristatin such as monomethyl auristatin E (MMAE), optionally co-loaded with one or more further anti-cancer agents and/or one or more immunomodulatory agents. The invention also relates to the auristatin-loaded liposomes and methods for their manufacture.

BACKGROUND OF THE INVENTION

A well-known example of an auristatin is monomethyl auristatin E (MMAE), which is a synthetic analogue of the antineoplastic natural product Dolastatin 10. MMAE is a potent antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. MMAE is 100-1000 times more potent than doxorubicin. However, MMAE cannot be used as a drug itself due to its poor water solubility and ultra-potent cytotoxicity. Other examples of auristatins are monomethyl auristatin F (“MMAF”), also known as desmethyl-auristatin F, and auristatin PE, also known as soblidotin.

The current clinical application of auristatins such as MMAE is restricted to antibody-drug conjugates, in which MMAE is linked to a monoclonal antibody (mAb) that recognizes a specific marker expressed on cancer cells to deliver MMAE to a cancer cell expressing said specific marker. Despite the selectiveness of this approach, in this strategy one mAb can only deliver 3-5 MMAE molecules into a cell. Therefore, to create an effective cytotoxicity several MMAE-mAb conjugates should enter a cell, which requires high administration doses of mAb, making it an extremely expensive approach. There is thus a need for alternative therapeutic strategies that capitahze on the anti-cancer effects of auristatins such as MMAE. SUMMARY OF THE INVENTION

It was unexpectedly found that an auristatin could be beneficially encapsulated into liposomes by a remote loading method at advantageous auristatin encapsulation efficiencies, thereby minimizing auristatin waste during preparation. Such auristatin-loaded liposomes are especially suitable for use as a therapeutic modality in the treatment of cancer since they increase the maximum tolerated dose of auristatins such as MMAE, and make systemic administration of auristatins possible by overcoming water solubility limitations and by retaining the auristatin inside liposomes with low leakage, which restricts MMAE distribution volume in blood and provides for low MMAE exposure to normal, healthy tissue.

Therefore, in a first aspect, the invention provides a method for producing an auristatin-loaded liposome, comprising the steps of: - providing a liposome that encapsulates a remote loading agent; - generating a concentration gradient of said remote loading agent across the membrane of said liposome; - mixing said liposome with an aqueous medium comprising an auristatin; - loading said auristatin into said liposome; preferably wherein said loading is driven by said concentration gradient; and - optionally, purifying an auristatin-loaded liposome.

Hitherto, MMAE in free (unconjugated) form has not been incorporated in any kind of nanoparticle and it has so far only been used as chemically conjugated forms most particularly as an antibody-drug conjugates.

It has now been found that the liposomes produced according to a method as disclosed herein do not exhibit morphological changes upon auristatin loading, even at high drug to lipid ratios (Figure 1, Examples 1- 8). In addition, it was unexpectedly established that advantageous MMAE loading efficiencies could be achieved when loading occurs at an external aqueous medium pH of about 3.5-7, preferably at a pH of about 4.5 (Figures 9 and 10), which was not expected beforehand in view of in silico modelling (Figure 12). It was further established that the liposomes produced by a method as disclosed herein are leakage stable (Figure 2, Example 1). It was further established that the liposomes produced by a method as disclosed herein are cytotoxic against cancer cells (Figure 3, Example 1) and safer than the free form when administered in an in vivo setting (Figure 4, Example 1). Further, it was discovered that auristatin, more specifically MMAE, could be co-loaded into liposomes with a second anti-cancer agent (Figure 6 and 7, Examples 3-4). It was also established that auristatins could be loaded into stable and long-circulating liposomes composed of hydrogenated soy phosphatidylcholine (HSPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG 2000- DSPE) and cholesterol constituting a stable, long-circulating vehicle for MMAE. It was further established that auristatins could also be loaded into liposomes composed of dipalmitoylphosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), and mPEG 2000-DSPE constituting temperature-sensitive MMAE release by application of mild hyperthermia (41-43 °C).

In a preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome that encapsulates a remote loading agent is a liposome that encapsulates a first aqueous medium, wherein said first aqueous medium comprises a remote loading agent; and wherein said aqueous medium comprising an auristatin is a second aqueous medium.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome that encapsulates a remote loading agent is a dried or dehydrated liposome. In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said auristatin is a monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said aqueous medium comprising an auristatin is at a pH of 2-8, preferably 3.5-7 which is a beneficial loading pH.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention (i) said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAE; and wherein said loading is performed at a pH of 3.5-7 or a pH of 2.0-6.0, preferably at a pH of 4.0-4.5, more preferably at a pH of about 4.5; or (ii) wherein said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAF; and wherein said loading is performed at a pH of 1-6, preferably 2-5, more preferably 2.5-4, even more preferably at a pH of about 3.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention (i) said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAE; and wherein said aqueous medium comprising an MMAE is at a pH of 3.5-7, preferably at a pH of 4.0-4.5, more preferably at a pH of about 4.5; or (ii) said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAF; and wherein said aqueous medium comprising an MMAF is at a pH of 2.5-4, preferably at a pH of about 3.

In a preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said remote loading agent is an amine salt and/or a metal salt, for instance amine salts from carboxylate, phosphonate, phosphate or sulfate such as ammonium citrate, ammonium sulphate or triethyl ammonium sulphate (TEAS); or metal salts such as sodium, calcium, magnesium, zinc, copper, potassium salts. In a preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, loading takes place on liposomes when pH gradient or sulfate gradient, ammonium salt gradient, EDTA ion gradient, alkylated ammonium salt gradient, phosphate gradient, phosphonate gradient, citrate gradient, acetate gradient, magnesium gradient, copper gradient, sodium gradient, calcium gradient, zinc gradient, potassium gradient or a combination thereof are established across the interior and exterior of liposomes.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said remote loading agent is an ammonium-based remote loading agent, preferably an ammonium salt such as ammonium sulphate or triethyl ammonium sulphate (TEAS).

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome comprises one or more (vesicle-forming) lipids selected from the group formed by phospholipids, sphingolipids, diglycerides, dialiphatic glycolipids, cholesterol and derivates thereof, and combinations thereof.

In another preferred embodiment of a method for producing an auristatin-loaded liposome, said liposome comprises a lipid bilayer comprising one or more phospholipids, a sterol, and optionally a polymer- modified lipid such as a polyethylene glycol conjugated lipid (PEGylated lipid).

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome comprises one or more lipids selected from egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), dipalmitoylphosphatidylcholme (DPPC), sphingomyelin (SM), cholesterol (Choi), cholesterol sulfate and its salts (OS), cholesterol hemisuccinate and its salts (Chems), cholesterol phosphate and its salts (CP), cholesterol phthalate, cholesterylphosphorylcholine, 3,6,9- trioxaoctan-ol-cholesteryl3e-ol, dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol (DSPG), sterol modified lipids (SML), inversephosphocholine lipids, cationic lipids and zwitterlipids.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome comprises a first (vesicle-forming) phospholipid that is an phosphatidylcholine and a second (vesicle-forming) phospholipid that is an optionally PEGylated phosphatidylethanolamine. Preferably, the phosphatidylethanolamine is PEGylated.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, (i) said phosphatidylcholine is a hydrogenated soy phosphatidylcholine (HSPC), said phosphatidylethanolamine is an optionally PEGgylated 1,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE), and wherein said liposome further comprises a sterol such as cholesterol; or (ii) said phosphatidylcholine is a dipalmitoylphosphatidylcholine (DPPC), said phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and wherein said liposome further comprises a second phosphatidylcholine that is a distearoyl phosphatidylcholine (DSPC). In embodiments, one or more of said phospholipids is PEGylated, for instance said DSPE is PEGylated.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, the HSPC is present in a mole percent of 40-100%, the sterol such as cholesterol in a mole percent of 0-50% and (optionally PEGylated) DSPE in a mole percent of 0-15% calculated over the total mole content of HSPC, cholesterol and (optionally PEGylated) DSPE in said liposome, totaling a mole percent of 100%. Preferably, DSPE is PEGylated. In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, the DPPC is present in a mole percent of 50-95%, DSPC in a mole percent of 0-50% and (optionally PEGylated) DSPE in a mole percent of 0-15% calculated over the total mole content of DPPC, DSPC and (optionally PEGylated) DSPE in said liposome, totaling a mole percent of 100%. Preferably, DSPE is PEGylated.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome that encapsulates a remote loading agent is obtainable by a method comprising the steps of:

- providing an organic solvent comprising one or more vesicle-forming lipids as defined in any one of the aspects and/or embodiments of a method for producing an auristatin-loaded liposome of the invention; - drying said one or more vesicle-forming lipids to thereby provide a dried lipid film; - hydrating said dried lipid film with a first aqueous medium under conditions that allow for the formation of a liposome encapsulating said first aqueous medium, wherein said first aqueous medium comprises a remote loading agent;

- optionally, extruding said liposome through a membrane with pores in order to downsize said liposome; -optionally, drying or dehydrating said liposome encapsulating said first aqueous medium.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said liposome that encapsulates a remote loading agent is formed by a solvent evaporation and hydration method, by detergent removal or by solvent removal (such as with ethanol or ether injection).

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said concentration gradient of said remote loading agent is generated by: - removing non-encapsulated remote loading agent, preferably by dialyzing said liposome against a buffer solution; and/or - mixing said liposome with said aqueous medium comprising an auristatin.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, loading of said auristatin into said liposome is under conditions that allow for remote loading of said auristatin into said liposome.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, the auristatin loading efficiency is at least 40%, more preferably at least 70%, and even more preferably at least 100%.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, the drug (auristatin) to lipid ratio (pg drug/p mol lipid) is in a range of 0.1-300 such as 8-240, 40-160 or 50-300.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, a second anti-cancer agent and/or a first immunomodulatory agent is co-loaded with said auristatin into said liposome.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, a third anti-cancer agent and/or a second immunomodulatory agent is co-loaded with said auristatin into said liposome, wherein said third anti-cancer agent is different from said second anti-cancer agent and/or said second immunomodulatory agent is different from said first immunomodulatory agent.

In another preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said second and/or third anticancer agent is selected from the group formed by - a proteasome inhibitor such as carfilzomib, oprozomib, bortezomib and/or ixazomib; - a tyrosine kinase inhibitor such as imatinib, lapatinib, acalabrutinib, afatinib, alectinib, avapritinib, axitinib, bosutinib, cabozantinib, crizotinib, dacomitinib, dasatinib, entrectinib, erlotinib, gilteritinib, ibrutinib, midostaurin, neratinib, nilotinib, pacritinib, pazopanib, pexidartinib, ponatinib, quizartinib, regorafenib, midostaurine, sorafenib, dasatinib, sunitinib, vandetanib, aflibercept, zanubrutinib and/or ziv-aflibercept; - an anthracycline such as a doxorubicin, daunorubicin, idarubicin, mitoxantrone, valrubicin, epirubicin, pirarubicin, rubidomycin, carcinomycin and/or N-acetyl adriamycin; - an alkylating agent such as busulfan, cyclophosphamide, bendamustine, carboplatin, chlorambucil, cyclophosphamide, cisplatin, temozolomide, melphalan, bendamustine, carmustine, lomustine, lomustine, dacarbazine, oxaliplatin, melphalan, lomustine, ifosfamide, mechlorethamine, thiotepa, trabectedin and/or streptozocin; - an camptothecin such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan and/or rubitecan; - an anti-metabolite such as gemcitabine; - a taxane such as paclitaxel and/or docetaxel; - a targeted anti-cancer agent such as an antibody including for instance trastuzumab, nivolumab and/or bevacizumab; - an anti-cancer agent selected from the group formed by mitomycin C; a plant-derived alkaloid such as vincristine, vinblastine, vinorelbine, vinflunine, vinpocetine, vindesine, ellipticine or 6-3-aminopropyl-ellipticine; 2- diethylaminoethyl-ellipticinium; datelliptium; or orretelliptine; and/or said first and/or second immunomodulatory agent is selected from the group formed by: - an immunomodulatory agent that is a thalidomide, lenalidomide, pomalidomide and/or imiquimod.

In another aspect, the invention provides an auristatin-loaded liposome, wherein the auristatin is loaded as an unconjugated auristatin.

In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome comprises one or more (vesicle-forming) lipids as defined in any one of the aspects and/or embodiments of a method for producing an auristatin-loaded liposome of the invention. In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome is not (for use as) a sustained-release liposome. Preferably, said liposome is not a liposome that allows for sustained-release delivery of said auristatin (during (blood) circulation) when administered systemically such as by intravenous administration. In other words, preferably, said liposome is designed to (substantially) avoid release of said auristatin during blood circulation over an extended period of time.

In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome is (blood serum-)stable.

In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome is stable as characterized by an auristatin release of 0.01-12 mol.% or 0.01-12 wt.%, preferably 0.01-4 or 0.1-1 mol.% or wt.%, of total liposome-loaded auristatin content when incubated at 4°C in HEPES 10 mM buffered sucrose 10% at a pH of 7.4.

In another preferred embodiment of an auristatin-loaded liposome of the invention, the liposome is stable as characterized by an auristatin release of 0.01-12 mol.% or 0.01-12 wt.%, preferably 0.01-4 or 0.1-1 mol.% or wt.%, of total liposome-loaded auristatin content when incubated at 37°C in HEPES 10 mM buffered sucrose 10% at a pH of 7.4.

In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome allows for site-directed delivery of said auristatin, preferably to a tumor site e.g. when administered systemically such as by intravenous administration. In embodiments, said liposome is stable during circulation (i.e. liposomes are not leaky) and preferably accumulates in a tumor tissue of a subject having a tumor over time. Preferably, said liposome is a long-circulating liposome, e.g. a long-circulating PEGylated liposome.

In another preferred embodiment of an auristatin-loaded liposome of the invention, the liposome allows for spatiotemporal and/or externally controlled site-directed delivery of said auristatin, preferably to a tumor site e.g. when administered systemically such as by intravenous administration. In embodiments, said liposome is sensitive to heat and releases said auristatin (payload) inside the tumor (e.g. inside the tumor vasculature or tumor interstitium) upon application of heat to said tumor (e.g. application of heat to said tumor resulting in a tumor tissue temperature of at least 40 °C such as 41-45°C). Thus, in embodiments, the liposome is a controlled- release liposome, preferably a temperature sensitive liposome and optionally a long-circulating temperature sensitive liposomes.

Preferably, the auristatin-loaded liposome of the invention comprises one or more PEGylated lipids and/or a rigid bilayer for instance a rigid bilayer that comprises one or more phospholipids with a high phasetransition temperature (e.g. higher than 40 °C or higher than 55 °C) and optionally one or more of a sterol e.g. at a mol% of 1-10 mol% or 15-42% mol%).

In preferred embodiments of an auristatin-loaded liposome of the invention, the liposome allows for site-directed delivery of said auristatin, preferably to a tumor site e.g. when administered systemically such as by intravenous administration. In embodiments, said liposome comprises a tumor-targeting moiety, such as a cancer-targeting moiety, on its surface that allows for site-directed delivery of said auristatin to a tumor, preferably a cancer, site. Examples of tumor-targeting moieties are small-molecule ligands, peptides and monoclonal antibodies, which selectively, preferentially or specifically bind to a tumor cell, preferably a cancer cell. If said liposome comprises a tumor-specific antibody as said tumor-targeting moiety, said liposome is referred to as an immunoliposome (ILP). In embodiments, said liposome is an immunoliposome that targets (i.e. binds to) tumor cells, preferably cancer cells, and preferably is able to extravasate (from the blood stream into a tumor tissue site).

In another preferred embodiment, said liposome is a liposome that allows for site-directed delivery of said auristatin to a tumor, wherein said liposome is a stable, long-circulating liposome, or a thermosensitive liposome, which may or may not comprise a tumor-targeting moiety, such as a cancer-targeting moiety, on its surface. In medical methods as disclosed herein, release of said auristatin from a thermosensitive liposome is induced by increasing the temperature in a tumor above a temperature threshold value (i.e. the temperature at which said auristatin is released from said liposome). The temperature threshold value is dependent on the composition of the liposome and is routinely set by a person skilled in the art. Exemplary temperature threshold values are 40-45 °C, such as 41, 42, 43 or 44 °C.

In another preferred embodiment, said auristatin-loaded liposome is for tumor-directed (drug) delivery.

In another preferred embodiment said tumor-directed (drug) delivery is by: - passive tumor targeting, preferably wherein the auristatin- loaded liposome is a stable, long-circulating liposome; - active tumor targeting, preferably wherein the auristatin-loaded liposome comprises a tumor-targeting moiety that specifically binds to a tumor cell and/or tumor- associated cell; and/or - controlled or triggered release, preferably wherein said auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C).

In a preferred embodiment of an auristatin-loaded liposome of the invention, the liposome further comprises: - a second and/or third anticancer agent as defined in any one of the aspects and/or embodiments of a method for producing an auristatin-loaded liposome of the invention; and/or - a first and/or second immunomodulatory agent as defined in any one of the aspects and/or embodiments of a method for producing an auristatin-loaded liposome of the invention.

In another aspect, the invention provides an auristatin-loaded liposome obtainable by a method for producing an auristatin-loaded liposome of the invention, preferably wherein said auristatin-loaded liposome obtainable by a method for producing an auristatin-loaded liposome of the invention is an auristatin-loaded liposome of the invention as disclosed herein.

In a preferred embodiment of an auristatin-loaded liposome obtainable by a method for producing an auristatin-loaded liposome of the invention, said auristatin is loaded as an unconjugated auristatin; preferably wherein said liposome comprises one or more (vesicle-forming) lipids as defined in any one of the aspects and/or embodiments of a method for producing an auristatin-loaded liposome of the invention.

In a preferred embodiment of an auristatin-loaded liposome of the invention, said liposome is a multilamellar vesicle (MLV), large unilamellar vesicle (LUV), small unilamellar vesicle (SUV), oligolamellar vesicle (OLV), paucilamellar vesicle (PLV) or reverse phase evaporation vesicle (REV).

In a preferred embodiment of a method for producing an auristatin-loaded liposome of the invention, said method comprises a step of purifying or isolating (an) auristatin-loaded liposome(s), for instance by removing non-encapsulated drugs, such as auristatins, further anti-cancer agents, and/or immunomodulatory agents. Further, said method for producing an auristatin-loaded liposome of the invention may include, preferably after purification, a step of pH adjustment (of the composition or medium comprising said auristatin-loaded liposomes) in order to allow for parenteral administration of auristatin-loaded liposomes to a subject.

In another aspect, the invention provides an auristatin-loaded liposome of the invention for use as a medicament.

In a preferred embodiment of an auristatin-loaded liposome for use as a medicament, said auristatin-loaded liposome is for use in the treatment of a subject having a tumor, preferably a cancer such as colorectal cancer or hematological cancer.

In a preferred embodiment of an auristatin-loaded liposome for use in the treatment of a subject having a tumor, the auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C, such as 41-45 °C); and wherein said treatment comprises a step of applying heat to said tumor in order to provide for at least said hyperthermic temperature in said tumor so as to allow for release of said auristatin.

The invention also provides a pharmaceutical composition comprising an auristatin-loaded liposome of the invention, preferably said composition further comprising a pharmaceutically acceptable carrier or excipient. Preferably, said composition is pH-adjusted in order to allow for parenteral administration to a subject.

The invention also provides a method for treating a subject suffering, or suspected of suffering, from a tumor such as a colorectal cancer or hematological cancer, comprising the step of: - administering a therapeutically effective amount of an auristatin-loaded liposome of the invention to said subject. In the same manner, the invention also provides a method for treating a subject suffering, or suspected of suffering, from a tumor, preferably a cancer such as colorectal cancer, said method comprising the step of: - administering a therapeutically effective amount of an auristatin-loaded liposome of the invention to said subject.

In a preferred embodiment of said method for treating, said auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C); and wherein said treatment comprises a step of applying heat to said tumor in order to provide for at least said hyperthermic temperature in said tumor so as to allow for release of said auristatin.

In the same manner, the invention provides a use of an auristatin-loaded liposome of the invention in the manufacture of a medicament for the treatment of a subject having a tumor, preferably a cancer such as colorectal cancer or hematological cancer. In a preferred embodiment of said use, said auristatin-loaded liposome is a temperature- sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C); and wherein said treatment comprises a step of applying heat to said tumor in order to provide for at least said hyperthermic temperature in said tumor so as to allow for release of said auristatin.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows a TEM image of PEGylated liposomes containing high MMAE/lipid ratio (HMPL) illustrating vesicles with spherical shape indicating loading of MMAE and not causing morphological changes on liposomes even at high drug to lipid ratios. The dark liposome interior is an indication of a dense medium inside the liposomes resulting from a high concentration of MMAE inside the liposome.

Figure 2 shows chromatographs of the leakage stability study of two different MMAE-loaded liposomes; (A) with low MMAE/lipid ratio (LMPL), and (B) with high drug lipid ratio (HMPL). Thirty days after liposome preparation, aliquots of liposomes (0.5 mb) were transferred into dialysis bags and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 for 24 h at 4°C (i). Then the dialysis was continued further for 72 h at 37 °C (ii). MMAE was quantified in liposomal suspension before dialysis (dash-dot line), in dialysis medium after 24 h incubation at 4°C (solid line, blue), and in dialysis medium after 72 h incubation at 37 °C (dot line, red), using HPLC. For LMPL, the percentage MMAE released under (i) was 0.95% and under (ii) was 3.10%. For HMPL, the percentage MMAE released under (i) was 0.85% and under (ii) was 11.14%.

Figure 3 shows a cytotoxicity analysis of HMPL (A) LMPL (B) and Caelyx (commercial liposomal doxorubicin (DXR)) (C). C26 colon carcinoma cells were exposed to different liposomal preparations in serial dilutions based on phospholipid concentration and incubated for 3 h in culturing condition, washed 3 times and allowed to proliferate for 72 h, cell viability was then assayed using XTT. Each drawn line is based on an average of 3 wells. Figure 4 shows the results of an in vivo study of safety of MMAE-liposomes in mice. Increasing doses of MMAE either as encapsulated in LMPL or HMPL or as free MMAE were injected into BALB/c mice (5/group) and monitored for 3 weeks. Occurrence of animal death was reported as percentage of death in each group of mice.

Figure 5 shows an HPLC analysis of MMAE-loaded temperature-sensitive liposomes. Non-encapsulated MMAE was separated from liposomes by filter centrifugation (filled peak) and compared against total MMAE content of liposome before separation (black line, fine of upper peak).

Figure 6 shows loading efficiencies of MMAE and doxorubicin (DXR) coloaded into liposomes encapsulating AS250 in two different MMAE/DXR ratios. Liposomes encapsulating AS250 were incubated with DXR and MMAE in combination ratios of Img MMAE+2 mg DXR (Combo-PLl) and 2mg MMAE+1 mg DXR (Combo-PL2) for 1 h at 65°C, purified and the content of both drugs in liposomes were quantified by HPLC.

Figure 7 shows an HPLC analysis of MMAE and DXR content of dually loaded liposomes using triethylammonium sulfate gradient. DXR and MMAE were incubated with liposomes encapsulating TEAS 250 mM for 1 h at 65°C, purified and the content of both drugs in liposomes were quantified by HPLC.

Figure 8 shows an HPLC analysis of MMAE content of liposomes remotely loaded with MMAE using triethylammonium sulfate gradient. MMAE were incubated with liposomes encapsulating TEAS 250 mM for 1 h at 65°C, purified and the content of MMAE in liposomal suspension before (blank peak) and after (filled peak) purification were quantified by HPLC.

Figure 9 shows MMAE loading efficiency at a varying drug/lipid ratios but at constant pH of around 4-4.5. Different amounts of MMAE were added to constant amount of liposomes encapsulating ammonium sulfate 250 mM (12.5 jimol phospholipid) and incubated for 1 h at 65°C and nonencapsulated drug was separated with dialysis. Encapsulation efficiencies were measured based on drug/lipid ratios before and after dialysis.

Figure 10 shows MMAE loading efficiency at different pH but at constant drug to lipid ratio of Img MMAE/12.5 jimol liposomal phospholipid. It clearly shows a beneficial effect on loading efficiency when the pH of the external medium (containing MMAE) is between 3-7, especially when the pH of the external medium pH is about 4.5.

Figure 11 shows remote loading efficiency of MMAF into liposomes encapsulating ammonium sulfate at different pH. Two mg MMAF was added to liposomes (6 jimol liposomal phospholipid) with different external pH, incubated for 1 h at 65°C and the non-encapsulated drug was separated with dialysis. Encapsulation efficiencies were measured based on drug/lipid ratios before and after dialysis.

Figure 12 shows in panel A the molecular structure of MMAE with the different acidic and basic residues of the molecules and their corresponding calculated pKa values. Panel B shows the predicted distribution of all microspecies of the MMAE molecule that exist at different pH values calculated by in silico modeling using Chemicalize. Panels C represents the molecular structure of a positively charged species of MMAE (corresponds to red line in panel B, as indicated by arrow), and panel D represents the molecular structure of an uncharged species of MMAE (corresponds to blue line in panel B, as indicated by arrow). Calculations were made using Chemicalize. Panel B shows that at pH values between 2-6 a positively charged species with a distribution of 99.99% is predicted to be the dominant species (i.e. the microspecies indicated in panel C), whereas at pH values above 7 the distribution of the uncharged species (i.e. the species indicated in panel D) starts to increase, reaching 50% at a pH of about 8.9, which is the basic pKa of MMAE. In silico modelling thus shows that the predicted optimum loading pH should be around a pH of 8.9, since only uncharged microspecies (i.e. the microspecies of panel D) can pass through the liposomal membrane during loading. This contrasts with the experimentally observed optimum loading pH for MMAE, which unexpectedly was significantly lower.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “auristatin”, as used herein, includes reference to a group of antimitotic agents comprising monomethyl auristatin E (“MMAE”), monomethyl auristatin F (“MMAF”), also known as desmethyl-auristatin F, and auristatin PE, also known as soblidotin. Preferably, the auristatin is unconjugated, i.e. in free form, when loaded into a liposome. For instance, the auristatin is preferably not in the form of a conjugate such as an antibody-drug conjugate (ADC) when loaded into a liposome. Preferably, the auristatin is an MMAE or MMAF.

The term “MMAE”, as used herein, includes reference to monomethyl auristatin E. MMAE is an antimitotic agent which inhibits cell division by blocking the polymerization of tubulin. The IUPAC name of MMAE is (S)-N-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l- phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrroli din- 1-yl)- 3-methoxy-5-methyl-l-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamido)butanamide. Preferably, the MMAE is unconjugated, i.e. in free form, when loaded into a liposome. For instance, the MMAE is preferably not in the form of a conjugate such as an antibodydrug conjugate (ADC). Derivatives of MMAE are also included in said definition.

The term “MMAF”, as used herein, includes reference to a compound with the IUPAC chemical name of (S)-2-((2R,3R)-3-((S)-l- ((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamido)butanamido)-3-methoxy-5- methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3- phenylpropanoic acid. Derivatives of MMAF are also included in said definition.

The term “auristatin PE”, as used herein, includes reference to a compound with the IUPAC chemical name of 2-[[(2S)-2-(dimethylamino)-3- methylbutanoyl]amino]-N-[(3R,4S,5S)-3-methoxy-l-[(2S)-2-[(lR,2R)-l- methoxy-2-methyl-3-oxo-3-(2-phenylethylamino)propyl]pyrrolidin-l-yl]-5- methyl-l-oxoheptan-4-yl]-N,3-dimethylbutanamide. Derivatives of auristatin PE are also included in said definition.

The term “liposome”, as used herein, includes reference to a lipidic vesicle having one or more bilayer membranes comprising vesicle-forming amphipathic lipid molecules such as phospholipids. Liposomes may entrap an aqueous internal medium (core) and are preferably capable of encapsulating a drug. The liposomes as disclosed herein may comprise one or more (vesicle-forming) lipids such as phospholipids, diglycerides, dialiphatic glycolipids, sphingolipids, sphingomyelin and glycosphingolipid, cholesterol, and their derivates. The term also includes reference to modified liposomes such as PEG-modified liposomes. One example of a liposome is a liposome that is composed of POPC and DSPG. The term “liposome”, as used herein, includes reference to a liposome that is in suspension, i.e. a liposomal suspension.

The term “vesicle-forming lipids” as used herein, includes reference to lipophilic or amphiphilic molecules that can either form a liposomal bilayer structures such as phospholipids or cannot individually form a liposome bilayer but could be incorporated into a liposome bilayer to modify liposome membrane properties. Examples of vesicle-forming lipids include but are not limited to sterols, PEGylated lipid conjugates, acyl chains and lysolipids. The term “phospholipid”, as used herein, includes reference to amphiphilic agents having an hydrophobic group formed of longchain alkyl chains, and a hydrophilic group containing a phosphate moiety. The group of phospholipids includes amongst others phosphatidic acid, phosphatidyl glycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, and mixtures thereof. Preferably, the phospholipids are chosen from l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), dimyristoyl-phosphatidylcholine (DMPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (SPC), dimyristoylphosphatidylglycerol (DMPG), disrearoylphosphatidylglycerol (DSPG), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPO), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)distearoyl phosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EYPC) or hydrogenated egg yolk phosphatidylcholine (HEPC), sterol modified lipids (SML), cationic lipids and inverse zwitterlipids.

Liposomes can be modified by for instance PEGylation. Preferably, a liposome of the invention is a PEGylated liposome. As is generally known, polyethylene glycol (PEG)-lipid conjugates are used extensively to improve circulation times for liposome-encapsulated active ingredients in order to avoid or reduce uptake of the injected liposomes by the reticuloendothelial system of patient. PEGylation refers to the process of both covalent and non- covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures such as vesicle-forming lipids. The liposomes as disclosed herein are preferably PEGylated liposomes. PEGylation can be performed by incubating a reactive derivative of PEG with a functionalized vesicle-forming lipid or functionalized cholesterol or with a liposome containing said functionalized vesicle-forming lipid. Suitable PEGylated lipids that can be used in a method for producing an auristatin -loaded liposome as disclosed herein, include for instance conjugates of DSPE-PEG, optionally functionalized, in which the molecular weight of PEG is 50 - 5000 g/mol, preferably 2000 g/mol. The said PEGylated liposome may comprise about 3-20 mol % of PEG-lipid conjugates. Preferably, the PEGylated DSPE is an mPEG 2000-DSPE (e.g l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). The skilled person understands that, in order to provide a PEGylated liposome, numerous (vesicle-forming) lipids can be employed that provide an anchor for PEGylation. DSPE is therefore not an essential lipid in the present invention. For instance, instead of DSPE-PEG, cholesterol-PEG or another PEGylated lipid can be included in a liposome of the invention.

Liposomes as disclosed herein may comprise surface ligands for targeting unhealthy tissue such as cancer cells or may comprise an environment-sensitive trigger for increasing the bioavailability of the drug specifically at the site of disease, such as cancer.

The liposomes as disclosed herein can be unilamellar vesicles such as small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs), and multilamellar vesicles (MLV), for instance with a size of 10-300 nm, preferably 30 - 200 nm. The liposomal membrane, as referred to herein, indicates the bilayer of amphipathic molecules that separates an internal aqueous medium (also referred to as a first aqueous medium) from an external medium, such as an external aqueous medium (also referred to as a second aqueous medium herein). In methods as disclosed herein, typically the liposome that encapsulates a first aqueous medium is itself comprised in an external aqueous medium. The external aqueous medium can be a first aqueous medium as disclosed herein or a second aqueous medium as disclosed herein, depending on the method step. The liposome and said external aqueous medium can be contained in any appropriate reaction vessel. The term “remote loading agent”, as used herein, can be used interchangeably with the term “trapping agent” or “active loading agent”. The term includes reference to agents such as ions that mediate remote loading of anti-cancer agents into liposomes. Preferred remote loading agents include ammonium salts such as ammonium sulfate (AS) or triethylammonium sulfate (TEAS). For instance, ammonium sulfate can be employed at a concentration range of 200-500 mM and can be prepared by dissolving ammonium sulfate in deionized water. For instance, triethylammonium sulfate (e.g. CAS number 27039-85-6) can be employed in a concentration range of 200-500 mM and can be prepared by titrating 1 M sulfuric acid with triethylamine to a final pH of 7.3 and sulfate concentration of 500 mM. Other suitable remote loading agents include triethylammonium dextran sulfate, ammonium chloride and EDTA. A further group of remote loading agents that can be employed are metal salts of a member selected from a carboxylate, sulfate, phosphonate, phosphate or an acetate. Further examples of remote loading agents are sulfur-containing remote loading agents such as sulfate, 1,5-naphthalenedisulfonate, dextran sulfate, sulfobutlyether beta cyclodextrin, sucrose octasulfate benzene sulfonate or poly(4-styrenesulfonate) trans resveratrol-trisulfate. It is also noted that cation acetate-gradients can be used to actively load amphiphilic weak acids. Exemplary amines that can be used as remote loading agents include, monoamines, polyamines, tributyl ammonium, trimethylammonium, diethylmethylammonium, triethylammonium, triisopropylammonium, diisopropylethyl ammonium, N-ethylmorpholinium, Nmethylmorpholinium, N-hydroxyethylpiperidinium, N,N- dimethylpiperazinium, N-methylpyrrolidinium, diisopropylammonium, dicychohexylammonium, isopropylethylammonium, isopropylmethylammonium, tert-butylethylammonium, protonized forms of morpholine, pyridine, piperidine, pyrrolidine, imidazole, 2-amino-2- methylpropanol, piperazine, tert-butylamine, 2-amino-2-methylpropandiol, tris- (hydroxymethyl)-aminomethane tetramethylammonium, tris- (hydroxyethyl)-aminomethane, diethyl-(2-hydroxyethyl)amine, tetraethylammonium, tetrabutylammonium and Nmethylglucamine, polyamidoamine dendrimers and polyethyleneimine.

As an alternative, or in addition, to using a remote loading agent to mediate loading of an auristatin into a liposome, a transmembrane pH gradient can be employed to actively load an auristatin into a liposome. In such an embodiment, the pH of the liquid medium (preferably a water-based medium such as a (first) aqueous medium) inside the liposome (first pH) is lower or higher, preferably higher, than the pH in the liquid medium (preferably a water-based medium such as a (second) aqueous medium) outside the liposome (second pH). This transmembrane pH gradient mediates loading of an auristatin into said liposome. If remote loading of an auristatin using a transmembrane pH gradient is employed, the transmembrane pH gradient can be provided by adding an aqueous medium with a second pH, wherein said second pH is higher or lower, preferably lower, than the pH of the liquid inside the liposome (first pH).

A combination of a remote loading agent concentration gradient across the liposomal membrane and a specified second pH, or a pH gradient across the liposomal membrane optionally in combination with said remote loading agent concentration gradient, are foreseen in driving loading of an auristatin. Thus, in one aspect, the invention provides a method for producing an auristatin-loaded liposome, comprising the steps of: - providing a liposome that encapsulates a first aqueous medium having a first pH; - mixing said liposome with a second aqueous medium comprising an auristatin; wherein said second aqueous medium has a second pH that is acidic, preferably an acidic pH between 2-7, more preferably a pH of 3.5-7 or 3.5-6, even more preferably a pH of 4.0-4.5 such as about 4.5; and wherein said second pH is different from (such as higher or lower than) the first pH; - loading said auristatin into said liposome; wherein said loading is driven by said pH gradient. In preferred embodiments of a method for producing an MMAE-loaded liposome of the invention, the second pH is lower (e.g. a pH of 3.5-7, preferably a pH of 4.0-4.5, more preferably a pH of about 4.5) than the first pH (e.g. a pH of about 6-7, such as a physiological pH, or a pH of 7-9). In embodiments, said pH gradient loading is in combination with loading driven by a remote loading agent concentration gradient as disclosed herein.

The term “remote loading” can be used interchangeably with the term “active loading”. In methods from producing auristatin-loaded liposomes as disclosed herein, the auristatin is loaded by remote loading, which may also be referred to as active loading.

The term “mixing”, as used herein, includes reference to combining or bringing together a liposome that encapsulates a remote loading agent and an aqueous medium comprising an auristatin. The mixing is such that a mixture is formed which can for instance be a dispersion or suspension. Suitable mixing processes are known in the art. Non-limiting examples of suitable mixing processes include vortex mixing processes and static mixing processes. The mixing is such that it allows for remote loading of an auristatin into a liposome.

The term “transmembrane gradient”, as used herein, includes reference to a difference in remote loading agent concentration or pH between the inside (interior) and outside (exterior) of a liposome as separated by the liposomal membrane. The remote loading agent concentration is higher in the interior of the liposome than in the exterior of the liposome. The term includes reference to a discontinuous increase of the concentration of the remote loading agent across the liposomal membrane from outside (exterior) aqueous medium to inside the liposome (internal aqueous medium). The transmembrane gradient in remote loading agent concentration or pH is set at a gradient that allows for active or remote loading of an auristatin such as MMAE into the liposome. For instance the difference between remote loading agent concentration inside (e.g. first aqueous medium, interior) and outside (first aqueous medium, exterior; or second aqueous medium, exterior) the liposome can be at least 5%, 10%, 30%, 40%, 50% or at least 100%. Preferably, the remote loading agent concentration is zero or about zero in the aqueous medium on the outside (exterior) of the liposomal membrane. To create (generate or establish) the concentration gradient, the liposome is typically formed in a first aqueous medium, followed by replacing or diluting said first aqueous medium that is on the outside of said liposome with a second aqueous medium. The diluted or new external aqueous medium has a different concentration of the remote loading agent, thereby establishing the ion-gradient. The replacement of the external aqueous medium can be performed by multiple techniques, for instance, by passing the prepared liposome through a gel filtration column, e.g., a Sephadex or Sepharose column, which has been equilibrated with a second aqueous medium, or by centrifugation, dialysis, or other common procedures.

Preferably, non-encapsulated remote loading agent is removed from the exterior of said liposome, said exterior typically being an exterior aqueous medium which can be a first aqueous medium as disclosed herein. Non-encapsulated remote loading agent can for instance be removed by dialyzing a liposome that encapsulates a first aqueous medium (said liposome being comprised in an exterior aqueous medium that is a first aqueous medium as disclosed herein) against a buffer solution. Dialysis is one way to generate a concentration gradient of said remote loading agent. Another way to generate a concentration gradient as disclosed herein is by replacing or diluting an exterior aqueous medium that is a first aqueous medium with a second aqueous medium as disclosed herein that does not contain remote loading agent or lower concentrations thereof. Further, in a method for producing of the invention, after said steps of providing a liposome and generating a concentration gradient are performed, said liposome can be provided in an aqueous medium of which the pH is adjusted to (i) a pH of 3.5-7, preferably a pH of 4.0-4.5, more preferably a pH of about 4.5 (in case of MMAE); or (ii) a pH of 2.5-4, preferably a pH of about 3 (in case of MMAF), prior to said step of mixing said liposome with an aqueous medium comprising an auristatin.

In embodiments, where a method for producing an auristatin- loaded liposome as disclosed herein refers to a step of mixing a liposome with an aqueous medium comprising an auristatin, and to generating a concentration gradient, the generated concentration gradient may be the direct result of the mixing of said liposome with said aqueous medium comprising an auristatin, which automatically or spontaneously generates said concentration gradient. In other words, the step of mixing and the step of generating can be interpreted as a single step, the latter being the consequence of the first step. This is explicitly covered in a method for producing an auristatin-loaded liposome as disclosed herein when it refers to a step of generating a concentration gradient and a step of mixing said liposome with an aqueous medium comprising an auristatin. In an embodiment, the liposome that encapsulates the remote loading agent is a dried or dehydrated liposome containing the remote loading agent. The dried or dehydrated liposome containing the remote loading agent can subsequently be mixed with an aqueous medium comprising an auristatin to generate or establish a concentration gradient of said remote loading agent and to (actively) load said auristatin into said liposome.

A liposome as disclosed herein which is comprised in an exterior aqueous medium (which is preferably the case) can also be referred to as a liposomal suspension. In embodiments, the pH of such a liposomal suspension is adjusted as disclosed herein in order to beneficially load MMAE or MMAF into said liposome. Preferably, prior to remote loading of said auristatin into said liposome, the remote loading agent concentration in the exterior aqueous medium is as low as possible, preferably the remote loading agent is essentially absent in the exterior aqueous medium. The term “aqueous medium”, as used herein, includes reference to a water-based medium, for instance an aqueous phase comprising water and optionally further comprising water-soluble components such as an organic solvent such as DMSO; ethanol; propylene glycol; glycerol; and/or conventional water-soluble components. An aqueous medium can be an aqueous solution or aqueous suspension. In aqueous media, vesicle-forming lipids form liposomes. The skilled person is well aware which conditions allow for formation of liposomes from vesicle-forming lipids in aqueous media. An aqueous medium as described herein can be an aqueous solution. An example of a first aqueous medium as described herein is an ammonium sulphate or triethyl ammonium sulphate (TEAS) solution. An example of a second aqueous medium as described herein is an aqueous solution comprising an organic solvent such as DMSO.

The term “drug” as used herein refers to a therapeutic agent or any substance used in the prevention, diagnosis, alleviation, treatment, or cure of disease. Preferably, the drug is an auristatin.

The term “immunomodulatory”, as used herein, includes reference to an agent that is capable of stimulating or suppressing an immune response for instance by affecting the expression of chemokines, cytokines and/or other mediators of immune responses. The term “immunomodulatory agent”, as used herein, refers to an agent that stimulates or suppresses the immune system, preferably stimulates the immune system. The term “immunomodulatory agent”, can also be referred to as a biological response modifier.

The term “tumor”, as used herein, includes reference to abnormal cellular growth that can be benign, pre-cancerous, malignant, or metastatic. The tumor can be a solid tumor such as a carcinoma or a blood (liquid) tumor such as a lymphoma or leukemia. Preferably, the tumor is a solid tumor. Preferably, the tumor is a cancer. The term “cancer”, as used herein, is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer and primary carcinomas are included. The cancer can be selected from the group formed by adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, lip and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer, lymphomas, urethral cancer, vaginal cancer, and/or vulvar cancer. The term colorectal cancer, as used herein, includes colon cancer.

The term “optionally PEGylated”, as used in relation to “optionally PEGylated lipids” such as “optionally pegylated DSPE”, refers to the lipid such as DSPE being either PEGylated or non-PEGylated. Hence, the optionality refers to the PEGylation, not to the presence of the lipid.

The term “therapeutically effective amount”, as used herein, includes reference to an amount of an auristatin-loaded liposome that, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated, e.g. at a reasonable benefit/risk ratio applicable to any medical treatment. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and can be determined by the skilled person in a routine manner.

The term “subject”, as used herein, includes reference to a recipient of an auristatin-loaded liposome as disclosed herein, for instance a subject that is suffering, or suspected of suffering, from a tumor. Preferably, the subject is a mammal, more preferably a human. The terms “patient” and “subject” may be used interchangeable.

The terms “treating” and “treatment”, as used herein, include reference to reversing, reducing, and/or arresting the symptoms, clinical signs, and/or underlying pathology of a condition with the goal to improve or stabilize a subject's condition.

Methods for producing auristatin-loaded liposomes

The present inventors have now for the first time loaded an auristatin, more specifically MMAE and MMAF, into a liposome with a beneficial loading (encapsulation) efficiency, a beneficial drug to lipid ratio, and with high leakage stability that allows for liposome storage over longer periods of time and allows for low MMAE exposure to normal tissue upon administration.

In the present invention, it was established that an auristatin such as MMAE or MMAF could be loaded into two different liposomes with different lipid composition. It was shown that an auristatin could be loaded into liposomes made of rigid and impermeable membranes (stable, long- circulating liposomes). It was also shown that an auristatin could be loaded into liposomes composed of less rigid lipid composition that undergoes transition behavior at around 42°C (temperature-sensitive liposomes). Liposomes composed of HSPC:Cholestrol:mPEG-DSPE are stable and long circulating liposomes due to rigidity of membrane and presence of PEG. HSPC exhibits transition behavior at temperatures around 55 °C and therefore liposomes composed of solely HSPC have high leakage stability at physiological temperature. Cholesterol enhances liposomes stability and impermeability by diminishing phase behaviors of phospholipids. Therefore, liposomes of HSPC and chol (60:40 mol%) do not undergo phase transition and remains stable and impermeable even at temperatures above 70 °C. The two different liposomes with different lipid composition successfully tested in the Examples have opposite membrane characteristics and are together a good representative of the spectrum of different liposome compositions.

In the method for producing an auristatin-loaded liposome as disclosed herein, a remote loading method (also referred to as active loading method) is used to load an auristatin such as MMAE, optionally together with one or more further anti-cancer agent (for instance a second anticancer agent and optionally a third anti-cancer agent) such as an anthracycline (for instance doxorubicin), into a liposome.

Preferably, in a method for producing an auristatin-loaded liposome as disclosed herein, the liposome that encapsulates a remote loading agent is obtainable by a method comprising the steps of: - providing a solvent, preferably an organic solvent such as chloroform, comprising one or more (vesicle-forming) lipids; - drying said one or more (vesicle-forming) lipids to thereby provide a dried lipid film; - hydrating said dried lipid film with a first aqueous medium under conditions that allow for the formation of a liposome encapsulating said first aqueous medium, wherein said first aqueous medium comprises a remote loading agent; - optionally, extruding said liposome through a membrane with pores in order to downsize said liposome; and -optionally, drying or dehydrating said liposome encapsulating said first aqueous medium. In the same manner, in a method for producing an auristatin- loaded liposome as disclosed herein, the step of providing a liposome that encapsulates a remote loading agent can be replaced by the steps of: - providing an organic solvent comprising one or more (vesicle-forming) lipids; - drying said one or more (vesicle-forming) lipids to thereby provide a dried lipid film; - hydrating said dried lipid film with a first aqueous medium under conditions that allow for the formation of a liposome encapsulating said first aqueous medium, wherein said first aqueous medium comprises a remote loading agent; - optionally, extruding said liposome through a membrane with pores in order to downsize said liposome; and -optionally, drying or dehydrating said liposome encapsulating said first aqueous medium, to thereby provide a liposome that encapsulates said remote loading agent.

Alternatively, the liposome that encapsulates a remote loading agent can be obtained through solvent injection, which may also be referred to as solvent removal or solvent displacement. In such a method, (vesicleforming) lipids are first dissolved in an organic phase that is miscible with water (for instance ethanol, methanol, ether, tert-butanol, etc.) which is then injected into an aqueous medium (such as an aqueous solution) comprising a remote loading agent such as ammonium sulfate 250mM. By the dissipation of organic solvent in said aqueous medium, a liposome that encapsulates a remote loading agent is formed. Subsequently, optionally said liposome can be extruded through a membrane with pores in order to downsize said liposome, and/or optionally, dried or dehydrated.

Exemplary vesicle-forming lipids that may find application in the present invention are selected from the group formed by, or consisting of, sphingomyelin; glycosphingolipid; a dialiphatic chain lipid, such as a phospholipid, a dialiphatic glycolipid, and a diglyceride; cholesterol and derivates thereof; and combinations thereof. Typical phospholipids are a phosphatidyl glycerol, a phosphatidic acid, a phosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, and a phosphatidylinositol, and mixtures thereof.

Preferably, the phospholipid is selected from the group formed by, or consisting of, hydrogenated soy phosphatidylcholine (HSPC), optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), distearoyl phosphatidylcholine (DSPC), hydrogenated egg yolk phosphatidylcholine (HEPC), dimyristoyl-phosphatidylcholine (DMPC), soy phosphatidylcholine (SPC), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPO), sterol modified lipids (SML), egg yolk phosphatidylcholine (EYPC), inverse zwitterlipids and cationic lipids, and combinations thereof.

Preferably, the liposome that encapsulates a remote loading agent comprises a first (vesicle-forming) phospholipid that is a phosphatidylcholine and a second (vesicle-forming) phospholipid that is phosphatidylethanolamine.

Examples of suitable phosphatidylcholines are hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC) and distearoyl phosphatidylcholine (DSPC). An example of a suitable phosphatidylethanolamine is a PEGylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), preferably a methoxyPEGylated(mPEG) DSPE, more preferably a mPEG2000-DSPE.

The liposome may include further lipids or proteins, such as sterols such as cholesterol, ergosterol, campesterol and stigmasterol. Especially if stable, long-circulating liposomes are envisioned, cholesterol is a suitable lipid for inclusion into the liposome.

Preferably, in a method for producing an auristatin -loaded liposome as disclosed herein, (i) said first (vesicle-forming) phospholipid that is a phosphatidylcholine is a hydrogenated soy phosphatidylcholine (HSPC), and said (vesicle-forming) phospholipid that is a phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine (PEG-DSPE); and wherein said hposome further comprises a sterol such as cholesterol; or (ii) said first (vesicle-forming) phospholipid that is a phosphatidylcholine is a dipalmitoylphosphatidylcholine (DPPC), and said second (vesicle-forming) phospholipid that is phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), and wherein said liposome further comprises a second phosphatidylcholine that is a distearoyl phosphatidylcholine (DSPC). It is envisaged that further (vesicleforming) lipids such as phospholipids and/or proteins may be included in said liposome. Preferably, the PEGylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) is a methoxyPEGylated(mPEG) DSPE, more preferably a mPEG2000-DSPE. The liposome components indicated under (i) above provide for a long-circulation liposome, wherein the liposome components indicated under (ii) above provide a temperature-sensitive (and fast release) liposome.

In a method for producing an auristatin-loaded liposome of as disclosed herein, the HSPC can be present in a mole percent of 40-100% such as 40-80%, a sterol such as cholesterol in a mole percent of 0-50% such as 20-50%, and optionally PEGylated DSPE in a mole percent of 0.5-20% such as 0.5-10%, calculated over the total mole content of HSPC, sterol such as cholesterol, and optionally PEGylated DSPE in said liposome, totaling a mole percent of 100%. In a method for producing an MMAE-loaded liposome as disclosed herein, the DPPC can be present in a mole percent of 55-100% such as 55-95%, DSPC in a mole percent of 0-50% such as 5-30% and optionally PEGylated DSPE in a mole percent of 1-10%, calculated over the total mole content of DPPC, DSPC and optionally PEGylated DSPE in said liposome, totaling a mole percent of 100%. Preferably, the mole percent in a method for producing an auristatin-loaded hposome that is a long circulating liposome composed of HSPC/cholesterol/mPEG2000-DSPE is 56.5/38.2/5.3 mole%, respectively. Preferably, the mole percent in a method for producing an auristatin-loaded liposome that is a temperature sensitive liposomes composed of DPPC/DSPC/mPEG2000-DSPE is 80/15/5 mol%, respectively.

In one embodiment, the auristatin-loaded liposome is a long- circulating liposome and in another embodiment the auristatin-loaded liposome is a temperature-sensitive liposome.

In embodiments of a method for producing an auristatin-loaded liposome as disclosed herein, the step of drying said one or more (vesicleforming) lipids can be performed by common laboratory techniques such as drying by an evaporator such as an rotary evaporator, optionally connected by an (overnight) freeze drying step.

Embodiments are envisioned wherein the liposome that encapsulates a remote loading agent, after preparation, is dehydrated or dried, for instance for storage purposes. In such embodiments, the method for producing an auristatin-loaded liposome as disclosed herein may involve addition of the dried or dehydrated liposomes directly to a (second) aqueous medium comprising an auristatin as disclosed herein, to thereby hydrate said dehydrated or dried liposome and to thereby establish or generate said concentration gradient of said remote loading agent. Another option is to hydrate a liposome containing remote loading agent in a further external medium first, as will be readily apparent by a person skilled in the art. Optionally, freeze-drying means can be employed in order to provide a reduced pressure when dehydrating a liposome. In embodiments, a liposome encapsulating a remote loading agent, and its surrounding medium, is frozen in liquid nitrogen before being dehydrated and placed under reduced pressure. The skilled person is well aware of methods and means to keep the lipid vesicle membranes intact as the water in the system is removed, such as the inclusion of one or more protective sugars that interact with the lipid vesicle membranes. This allows for survival of liposomes during the dehydration process without losing a substantial portion of their internal contents. Exemplary sugars are selected from the group formed by maltose, trehalose, sucrose, glucose, dextran and lactose. In practice, disaccharide sugars are more suitable for this purpose than monosaccharide sugars, especially the disaccharide sugars trehalose and sucrose.

A method for producing an auristatin-loaded liposome as disclosed herein may further comprise a step of hydrating said dried lipid film with a first aqueous medium under conditions that allow for the formation of a liposome encapsulating said first aqueous medium, wherein said first aqueous medium comprises a remote loading agent. The remote loading agent of interest can for instance be an ammonium-based remote loading agent, preferably an ammonium salt such as ammonium sulphate or triethyl ammonium sulphate (TEAS). When in an aqueous medium, said ammonium-based remote loading agent is dissolved. The concentration of the remote loading agent can vary such as from lOOmM to lOOOmM, preferably from 200mM to 600 mM including 250mM and 500mM.

In principle, liposomes encapsulating the remote loading agent can be prepared by any liposome preparation methods such as solvent removal methods, detergent solubilization and microfluidization which are readily available to the skilled person. One suitable example is adding a volume of remote loading agent dissolved in an aqueous solution to said dried lipid film, followed by gentle shaking, and optionally sonication, at a temperature range of for instance 60-65 °C. It is noted that the skilled person is well aware of the conditions under which liposomes form in an aqueous solution.

In embodiments of a method for producing as disclosed herein, or a liposome as disclosed herein, the liposome is a multilamellar vesicle (MLV) liposome or a large unilamellar vesicle (LUV) liposome. One conventional technique for preparing multilamellar lipid vesicle (MLVs) liposomes is by depositing on the inside walls of a suitable vessel one or more lipids, by dissolving the lipids in chloroform and subsequently evaporating the chloroform, after which an aqueous medium that is to be encapsulated is added to the vessel, allowing the aqueous medium to hydrate the lipid, and finally swirling or vortexing the resulting lipid suspension. Conventional techniques for preparing large unilamellar lipid vesicles (LUVs) are for instance infusion procedures, detergent dilution, reverse-phase evaporation. These, and other conventional techniques for producing liposomes, are described in numerous text books including Gregoriadis, Liposome Technology, Volume I (1984), which is incorporated herein by reference.

In embodiments of a method for producing as disclosed herein, or a liposome as disclosed herein, a moiety that specifically targets the liposome to a particular cell type, tissue and/or organ is incorporated into the membrane of a liposome as disclosed herein. Targeting mechanisms typically require that said targeting moiety is positioned on the surface of the liposome such that said targeting moiety is available for interaction with the target, for instance a cancer cell, e.g. a cell surface receptor of a cancer cell. The skilled person has routine techniques at his disposal for including a targeting moiety, for instance a targeting moiety that specifically targets a tumor (cell), in the liposomal membrane. Suitable targeting moieties are generally known in the art.

Examples of targeting moieties include plasminogen activator inhibitor (PAI -I), anti-ErbB family antibodies and antibody fragments, hyaluronic acid, tissue- and urokinase-type plasminogen activator (tPA/uPA), receptor associated protein (RAP), lipoprotein lipase (LPL), desmoteplase, lactoferrin, [a]2-macroglobulin ([a]2M), melanotransferrin (or P97), thrombospondin 1 and 2, tPA/uPA:PAI-l complexes, factor Vila/tissue- factor pathway inhibitor (TFPI), hepatic lipase, pseudomonas exotoxin A, factor Villa, A[p]l-40, amyloid -[P] precursor protein (APP), factor IXa, CI inhibitor, complement C3, HIV-I Tat protein, matrix metalloproteinase 9 (MMP-9), apolipoproteinE (apoE), CRM66, MMP-13 (collagenase-3), pregnancy zone protein, spingolipid activator protein (SAP), heparin cofactor II, antithrombin III, [a]l-antitrypsin, platelet-derived growth factor (PDGF), heat shock protein 96 (HSP-96), apolipoproteinJ (apoJ, or clusterin), very-low-density lipoprotein (VLDL), A[p] bound to apoJ and apoE, aprotinin, angiopep-2 (TFFYGGSRGKRNNFKTEEY), leptin, transferrin, insulin, epidermal growth factors, an insulin-like growth factor, peptidomimetic and/or humanized monoclonal antibodies, lectins, hemoglobin, dingle chain antibodies or peptides specific for said receptors (e.g., sequences HAIYPRH and THRPPMWSPVWP that bind to the human transferrin receptor, or anti-human transferrin receptor (TfR) monoclonal antibody A24), all or a portion of a non-toxic mutant of diphtheria toxin CRM197, non-toxic portion of a diphtheria toxin polypeptide chain, all or a portion of the diphtheria toxin B chain, apolipoprotein E, apolipoprotein B, vitamin A/retinol- binding protein, vitamin D-binding protein, vitamin B12/cobalamin plasma carrier protein, transcobalamin-B and glutathione.

Folate and transferrin receptors are overexpressed on cancer cells and have been used to make liposomes tumor cell specific. Therefore, a suitable targeting moiety is a folate, transferrin, or anti-human transferrin or folate receptor monoclonal antibody. Other targeting moieties that can suitably be used in targeting cancer cells are anti-ErbB family antibodies and antibody (binding) fragments thereof, anti- matrix metalloproteases (MMPs) antibodies or binding fragments thereof, anti-E- and P-selectins, anti-VCAM-1 and anti-ICAM antibodies or binding fragments thereof, anti- aB-integrin antibodies antibodies or binding fragments thereof, and all or a portion of a non-toxic mutant of diphtheria toxin CRM197.

The prepared liposomes encapsulating remote loading agent and/or encapsulating a solution with a first pH, can optionally be subjected to a step of liposome (down)sizing to create a homogeneous populations of liposomes. Such a step may for instance involve extruding the hydrated liposomes through a membrane, preferably a polycarbonate membrane that has a pore size (diameter) in the nanometer range such as 200 nm, 100 nm or 50 nm or using high-pressure homogenization technique such as microfluidization. This step can be performed using standard laboratory equipment such as a LIPEX® extruder (Evonik, Germany). Preferably, the hydrated liposomes are sequentially extruded through membranes with a decreasing pore size, such as extruding through membranes with a pore size of 200 nm, 100 nm or 50 nm, sequentially. Typically, the step of downsizing occurs at a temperatures above the transition temperature of phospholipid mixture such as in HSPC liposomes it is about 65°C.

The optional sizing of the prepared liposomes serves the purpose of achieving a desired liposome size range and relatively narrow distribution of liposome sizes. When the size range of the prepared liposomes is approximately 20-200 nanometers, the liposomal suspension can be sterilized by filtration through a conventional filter, for instance a 0.22 or 0.4 micron filter. If the liposomes have been sized down to about 20- 200 nm, the filter sterilization method can be carried out on a high through-put basis. Multiple techniques are available for sizing (down) liposomes to a desired size. For instance, sonicating a liposome suspension provides for a size reduction down to small unilamellar vesicles less than about 50 nanometer in size. Liposome sonication can be performed by for instance bath or probe sonication. A further method is homogenization. This technique provides for shearing energy to fragment larger liposomes into smaller liposomes. A preferred method of sizing (down) of liposomes is extrusion of liposomes through a (polycarbonate) membrane with pores in the nm range. Another technique for sizing (down) liposomes is the use of an asymmetric ceramic membrane. In such a method, the liposomal suspension is cycled through the membrane one or more times until a desired liposome size distribution is achieved. A liposome as disclosed herein may also be extruded through successively smaller-pore membranes. This provides for a gradual reduction in liposome size as indicated above.

In embodiments, the process of active loading of an auristatin as described herein, involves the use of a transmembrane concentration gradient of remote loading agent or a pH gradient. The principle of active loading, in general, has been described in the art. During active loading, the auristatin molecules transfer from an external aqueous medium (also referred to as a second aqueous medium herein) across the liposomal membrane to the internal aqueous medium and accumulate inside liposomes, which process is driven by said transmembrane remote loading agent concentration gradient or transmembrane pH gradient, which results in entrapment or capture of said auristatin inside the liposomes.

To create (generate or establish) the concentration gradient, the liposome is typically formed in a first aqueous medium, followed by replacing or diluting said first aqueous medium that is on the outside of said liposome. The diluted or new external aqueous medium has a different concentration of the remote loading agent, thereby establishing the remote loading agent concentration gradient. The replacement of the external aqueous medium can be performed by multiple techniques, for instance, by passing the prepared liposome through a gel filtration column, e.g., a Sephadex or Sepharose column, which has been equilibrated with a second aqueous medium, or by dialysis, centrifugation, or other common procedures.

Preferably, a liposome that encapsulates a remote loading agent as described herein (i.e. a liposome that is pre-formed and yet unloaded with an auristatin), contains an active- or remote-loading buffer which contains water (also referred to as a first aqueous medium herein) or is a dried or dehydrated liposome, and contains a remote loading agent as described herein. In an exemplary embodiment, the concentration of salts in the internal (first) aqueous medium of a yet unloaded liposomes is between 1 and 1200, preferably 1 and 1000 mM.

The encapsulation or loading efficiency, for instance defined as encapsulated amount of auristatin (e.g., as measured in grams of auristatin I moles of phospholipid (e.g. combined total mole content of the phosphatidylcholine and phosphatidylethanolamine phospholipids) or gram of auristatin/gram total lipid (e.g. combined total grams of the phosphatidylcholine and phosphatidylethanolamine phospholipids and sterol such as cholesterol)) in the internal aqueous medium (also referred to as the first aqueous medium) divided by the initial amount in the external aqueous medium (also referred to as the second aqueous medium) multiplied by 100%, is preferably at least 10% or at least 20% or at least 30% (such as at least 31, 32, 33, 34, 35, 36, 37, 38 or at least 39%), more preferably at least 40% (such as at least 41, 42, 43, 44, 45, 46, 47, 48 or at least 49%), or at least 50% (such as at least 51, 52, 53, 54, 55, 56, 57, 58 or at least 59%) or at least 60% (such as at least 61, 62, 63, 64, 65, 66, 67, 68 or at least 69%), and even more preferably at least 70%, 80%, 81%, 82%, 83%, 84%, or at least 85%, or at least 90% such as 100%.

Preferably, the drug (auristatin) to lipid ratio in an auristatin- loaded liposome as disclosed herein is in a range of 0.1-300 jig auristatin/ 1 jimol liposomal phospholipid, preferably a ratio of least 8 or at least 10, more preferably at least 40 or at least 50 or at least 100, even more preferably at least 150 or at least 250. Examples of suitable drug (auristatin) to lipid ratio ranges are 8-240, 40-160 or 50-300. Preferably, the drug to lipid ratio is calculated based on the concentration of auristatin (pg/ml) divided by the concentration of liposomal phospholipid (pmol/ml) in the final (purified) liposomal suspension. Preferably, the auristatin is MMAE or MMAF. The term “liposomal phospholipid”, as used herein in relation to drug to lipid ratios, includes reference to the total (molar) content of phospholipids in said liposomal suspension (e.g. expressed in pmol/ml), either during production of the liposome or in the final (purified) liposomal suspension, and thus comprises the total combined molar content of inter alia phosphatidylcholines and phosphatidylethanolamines, and other phospholipids if present.

In embodiments, the auristatin that is loaded into the liposome is not covalently attached to a component of the liposome, and/or is not covalently attached to any component used to generate, create or establish the remote loading agent concentration gradient.

Preferably, in a method for producing an auristatin -loaded liposome as disclosed herein, said aqueous medium comprising an auristatin (also referred to as the exterior or second aqueous medium herein) is at a pH of 2-8 or at a pH of 2-6, preferably at a pH of 3-7, 3.5-7 or 4-7, more preferably at a pH of 4-5 or 4-4.5, and most preferably at a pH of about 4.5. It was unexpectedly established that at these pH values high MMAE loading efficiencies could be achieved. When an MMAE is loaded, said aqueous medium comprising an MMAE (also referred to as the exterior or second aqueous medium herein) is at a pH of 2-8, preferably at a pH of 2.5- 5, more preferably 2.5-4, even more preferably about 3. In other words, in a method for producing an auristatin-loaded liposome as disclosed herein, preferably (i) said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAE; and wherein said loading is performed at a pH of 3.5-7, preferably at a pH of 4.0-4.5, more preferably at a pH of about 4.5; or (ii) wherein said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAE; and wherein said loading is performed at a pH of 2.5-4, preferably at a pH of about 3.

In a method for producing an auristatin-loaded liposome as disclosed herein, a step of loading said auristatin into said liposome, wherein said loading is driven by said concentration gradient, is included. Preferably, the auristatin that is loaded into said liposome is an unconjugated auristatin, i.e. an auristatin in free form not conjugated to an binding molecule such as an antibody.

The auristatin is preferably dissolved in an aqueous medium comprising DMSO when mixed with said liposome. A typical amount of auristatin mixed with, or added to, said liposome is about 1 mg, such as in a concentration of about 72 mg/mL auristatin. The remote loading of auristatin can be performed under different conditions that the skilled person is aware of.

Preferably, in said aqueous medium comprising an auristatin, the auristatin is dissolved. In other words, in said aqueous medium comprising an auristatin, said auristatin is preferably not in the form of a precipitate.

Remote loading of auristatin can be performed at temperatures between 0-100 °C, such as room temperature, 50-55 °C or 60-65 °C, the exact temperature depending on the liposomal phase transition temperature of the liposome employed, which can be routinely established by a skilled person. The phase transition temperature is also referred to as the temperature that is needed to induce a change in the physical state of the lipids that make up the liposome, from the gel-like phase (ordered), where the hydrocarbon chains are fully extended and closely packed, to the liquidlike phase (disordered), where the hydrocarbon chains are randomly oriented and packed less closely. The permeability of the liposomal membrane increases above the phase transition temperature of the liposome. A liposomal phase transition temperature of -20°C to 105°C, for instance 0°C to 65°C is an exemplary liposomal phase transition temperature range. Preferably, loading of an auristatin occurs at a temperature above the liposomal phase transition temperature of the liposome, although this is not a requirement for effective loading. Thus, loading of an auristatin can occur at a temperature of -20°C to 105°C, for instance 3°C - 65°C which may be dependent on the liposomal phase transition temperature of the liposome. The skilled person is well aware of the fact that a liposomal phase transition temperature is influenced by the choice of vesicle-forming lipid(s) and by the presence in the membrane of steroids such as cholesterol. For instance, less cholesterol in the liposomal membrane provides for less stable liposomes. When stable, long-circulating liposomes are envisaged, an appropriate mol% of steroid, preferably, cholesterol, is 5-75 mol%, preferably 10-50 mol%.

In certain embodiments, a second anti-cancer agent and/or a first immunomodulatory agent is co-loaded with said auristatin into said liposome. Said second anti-cancer agent is preferably not an auristatin. In a further embodiment, a third anti-cancer agent and/or a second immunomodulatory agent is co-loaded with said auristatin into said liposome, wherein said third anti-cancer agent is different from said second anti-cancer agent and/or said second immunomodulatory agent is different from said first immunomodulatory agent.

A typical second and/or third anti-cancer agent is selected from the group formed by: - a proteasome inhibitor such as carfilzomib, oprozomib, bortezomib and/or ixazomib; - a tyrosine kinase inhibitor such as imatinib, lapatinib, acalabrutinib, afatinib, alectinib, avapritinib, axitinib, bosutinib, cabozantinib, crizotinib, dacomitinib, dasatinib, entrectinib, erlotinib, gilteritinib, ibrutinib, midostaurin, neratinib, nilotinib, pacritinib, pazopanib, pexidartinib, ponatinib, quizartinib, regorafenib, midostaurine, sorafenib, dasatinib, sunitinib, vandetanib, aflibercept, zanubrutinib and/or ziv-aflibercept; - an anthracycline such as a doxorubicin, daunorubicin, idarubicin, mitoxantrone, valrubicin, epirubicin, pirarubicin, rubidomycin, carcinomycin and/or N-acetyl adriamycin; - an alkylating agent such as busulfan, cyclophosphamide, bendamustine, carboplatin, chlorambucil, cyclophosphamide, cisplatin, temozolomide, melphalan, bendamustine, carmustine, lomustine, lomustine, dacarbazine, oxaliplatin, melphalan, lomustine, ifosfamide, mechlorethamine, thiotepa, trabectedin and/or streptozocin; - an camptothecin such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan and/or rubitecan;

- an anti-metabolite such as gemcitabine; - a taxane such as paclitaxel and/or docetaxel; - a targeted anti-cancer agent such as an antibody including for instance herceptin, nivolumab and/or bevacizumab; - an anticancer agent selected from the group formed by mitomycin C; a plant- derived alkaloid such as vincristine, vinblastine, vinorelbine, vinflunine, vinpocetine, vindesine, ellipticine or 6-3-aminopropyl-ellipticine; 2- diethylaminoethyl-ellipticinium; datelliptium; or orretelliptine.

A typical first and/or second immunomodulatory agent is selected from the group formed by: a thalidomide, lenalidomide, pomalidomide and/or imiquimod.

One preferred example of a second or third active ingredient is a proteasome inhibitor such as carfilzomib or bortezomib. Another preferred example of a second or third active ingredient is an anthracycline such as a doxorubicin.

The encapsulation or loading efficiency, which can be defined as encapsulated amount (e.g., as measured in grams of auristatin I moles of phospholipid or gram of auristatin/moles of total lipid) of the auristatin in the internal aqueous medium (also referred to as the first aqueous medium) divided by the initial amount in the external aqueous medium (also referred to as the second aqueous medium) multiplied by 100%, is preferably at least 20%, more preferably at least 50% or at least 60% or at least 70%, and even more preferably at least 80%, 81%, 82%, 83%, 84%, 85%, or at least 90 % such as 100%. Total lipid refers to phospholipids, sterols and any other lipid that is not a phospholipid. Preferably, loading efficiency or encapsulation efficiency is calculated using the formula: /auristatin) . ratio of ( — — I after dialisis encapsulation efficiency (%) = 100 * - auristatin - ratio of ( — — ) before dialisis

preferably wherein lipid is phospholipid, and wherein lipid (preferably phospholipid) and auristatin content (i.e. amount, for instance grams of auristatin I moles of phospholipid, or grams of auristatin/grams total lipid)) are measured using Bartlett assay and HPLC, respectively. The Bartlett assay is inter alia described in Bartlett, J Biol Chem.234:466-8 (1959), the contents of which are incorporated herein by reference. The skilled person can routinely establish encapsulation efficiency percentages of loaded liposomes.

Preferably, encapsulation efficiency or loading efficiency is calculated using the Bartlett assay.

A method for producing an auristatin-loaded liposome as disclosed herein may optionally comprise a step of purifying or isolating (an) auristatin-loaded liposome(s) by removing non-encapsulated drugs, such as an auristatin, a further anti-cancer agent and/or an immunomodulatory agent. Further, said method, preferably after said step of purifying, may comprise a step of pH adjustment in order to allow for parenteral administration to a subject. The skilled person is well aware how to remove non-encapsulated drugs, such as auristatin and additional drugs. It is also routine to adjust the pH of the liposomes in order to allow for parenteral administration of said liposomes. Preferably, the pH is adjusted to isotonic conditions. Typical techniques that can be used for the removal of nonencapsulated drugs and pH adjustment include dialysis, gel filtration, centrifugation and tangential flow filtration. Dialysis is preferably performed against an isotonic solution. Typical columns that can be used for gel filtration include Sephadex columns and separose columns.

Auristatin-loaded liposomes

The invention also provides an auristatin-loaded liposome, wherein the auristatin is loaded as an unconjugated auristatin or auristatin unconjugated to an antibody or antibody fragment. Preferably, the auristatin is an MMAE or MMAF.

An auristatin-loaded liposome as disclosed herein may comprises one or more (vesicle-forming) lipids selected from the group formed by phospholipids, sphingolipids, diglycerides, dialiphatic glycolipids, cholesterol and derivates thereof, and combinations thereof.

Preferably, an auristatin-loaded liposome as disclosed herein comprises a first (vesicle-forming) phospholipid that is a phosphatidylcholine and a second (vesicle-forming) phospholipid that is a phosphatidylethanolamine. More preferably, in a liposome as disclosed herein, (i) said first (vesicle-forming) phospholipid that is a phosphatidylcholine is a hydrogenated soy phosphatidylcholine (HSPC), and said (vesicle-forming) phospholipid that is a phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); and wherein said liposome further comprises a sterol such as cholesterol; or (ii) said first (vesicle-forming) phospholipid that is a phosphatidylcholine is a dipahnitoylphosphatidylcholine (DPPC), and said second (vesicle-forming) phospholipid that is phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and wherein said liposome further comprises a second phosphatidylcholine that is a distearoyl phosphatidylcholine (DSPC).

Preferably, the optionally PEGylated l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) is a methoxyPEGylated(mPEG) DSPE, more preferably a mPEG2000-DSPE.

Typically, in a liposome as disclosed herein, the HSPC is present in a mole percent of 40-100% such as 40-80%, cholesterol in a mole percent of 0-50% such as 10-50% and optionally PEGylated DSPE (such as mPEG DSPE) in a mole percent of 0-15% such as 0.5-10%, calculated over the total mole content of HSPC, cholesterol and optionally PEGylated DSPE in said liposome, totaling a mole percent of 100%. In embodiments of a liposome as disclosed herein, the DPPC is present in a mole percent of 55-100% such as 55-95%, DSPC in a mole percent of 0-50% such as 5-30% and optionally PEGylated DSPE in a mole percent of 1-10% calculated over the total mole content of DPPC, DSPC and optionally PEGylated DSPE in said liposome, totaling a mole percent of 100%.

In certain embodiments, a liposome as disclosed herein may further comprise a second and/or third anti-cancer agent as disclosed herein in relation to a method for producing an auristatin-loaded liposome, and/or may further comprise- a first and/or second immunomodulatory agent as disclosed herein in relation to a method for producing an auristatin-loaded liposome. The auristatin that is loaded into a liposome is to be regarded as a first anti-cancer agent.

The invention also provides an auristatin-loaded liposome obtainable by a method for producing an auristatin-loaded liposome according to the invention. Preferably, such a liposome is an auristatin- loaded liposome as disclosed herein above.

Embodiments of the liposome that are described in relation to a method for producing an auristatin-loaded liposome as disclosed herein, also apply in relation to an auristatin-loaded liposome as disclosed herein.

Methods of treatment using liposomes as disclosed herein

The auristatin-loaded liposomes as disclosed herein find application in the treatment of a subject suffering, or suspected of suffering, from a tumor.

Therefore, the invention provides an auristatin-loaded liposome as disclosed herein for use as a medicament, preferably for use in the treatment of a tumor, preferably a cancer such as colorectal cancer.

In the same manner, the invention provides a method for treating a subject suffering, or suspected of suffering, from a tumor, preferably a cancer such as colorectal cancer, comprising the steps of: - administering a therapeutically effective amount of an auristatin-loaded liposome as disclosed herein to said subject. In the same manner, the invention provides a use of an auristatin-loaded liposome as disclosed herein in the manufacture of a medicament for the treatment of a tumor, preferably a cancer such as a colorectal cancer.

Preferably, an auristatin-loaded liposome as described herein is formulated in a pharmaceutical composition. The invention therefore also provides a pharmaceutical composition comprising an auristatin-loaded liposome as disclosed herein, optionally said pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients.

A pharmaceutical composition as disclosed herein preferably comprises one or more pharmaceutically-acceptable carriers, adjuvants, excipients, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. The specific characteristics of the pharmaceutical composition depend on the route of administration, as is generally known by the skilled person.

The term “pharmaceutically acceptable”, as used herein, refers to a nontoxic material that is compatible with the physical and chemical characteristics of an auristatin-loaded liposome as disclosed herein and does not interfere with the active principle.

Preferably, a pharmaceutical composition as disclosed herein is adapted for parenteral administration, wherein the composition is for intravenous, intra-arterial, subcutaneous, and/or intramuscular administration. Parenteral administration involves the injection or infusion of a pharmaceutical composition as disclosed herein into a body tissue or body fluid, whereby preferably a syringe, needle, or catheter is used. As an alternative, needle-less high-pressure administration may be used as means for parenteral administration.

A suitable pharmaceutical formulation for parenteral administration includes an (isotonic or somewhat hypertonic) aqueous solution or suspension which can be prepared or formulated prior to administration, for example from preparations which contain the active ingredient alone or together with a pharmaceutically acceptable carrier. The pharmaceutical preparations may be sterilized and/or contain adjuncts, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical compositions disclosed herein can be produced according to manners routinely known in the art, for example by means of conventional dissolving processes.

A pharmaceutical composition as disclosed herein is formulated in a therapeutically effective amount.

It is within the metes and bounds of the skilled person to devise appropriate dosing regimens that provide for therapeutic efficacy. A suitable dose can be selected from the dose range of 0.01 mg to 100 gram of auristatin-loaded liposome. Such a dose can be administered once a day, daily, weekly, biweekly (such as every other week or every 14 days), monthly or periodically as deemed necessary or beneficial. Such administration can be a parenteral administration, such as an intravenous administration, for instance in a dose of 5 mg to 10 gram. Another example of a suitable dose range is 0.1- 1.2 mg/kg.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the disclosure includes embodiments having combinations of all or some of the features described.

The content of the documents referred to herein is incorporated by reference. EXAMPLES

Example 1. Encapsulation of MMAE into stable long circulating liposomes.

Materials and methods

Preparation of stable long circulating liposomal MMAE Lipids for preparation of long circulating liposome composed of hydrogenated soy phosphatidylcholine (HSPC, Lipoid, Germany)/cholesterol (Choi, sigma, Germany)/ l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (poly ethylene glycol)-2000] (mPEG2000-DSPE) (Lipoid, Ludwigshafen, Germany) (56.5 jimol Z38.2 jimol Z5.3 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by a rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 5 mL of ammonium sulfate 250 mM (AS250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C.

As a negative control a dried lipid film was hydrated with 5 mL of HEPES 10 mM buffered saline pH 7.4. These primary liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes with pore sizes of 200, 100, and 50 nm, sequentially and in that order, using a LIPEX extruder (Evonik Transferra Nanosciences, BO, Canada) at 65°C.

To establish the gradient for remote loading, liposomes were dialyzed against HEPES 10 mM buffered sucrose 10% (w/v), pH 7.4, at 4 °C. The phospholipid content of the dialyzed liposomal suspensions were then quantified by Bartlett inorganic phosphate assay (Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234:466-8.). For remote loading of MMAE (Advanced Chemblocks Inc. Burlingame, CA, USA) into liposomes, the pH of liposomal suspensions was first adjusted to pH =5 and then 1 or 3.5 mg of MMAE dissolved in DMSO (71.45 mg/mL) were added to the liposomes (7.5 jimol phospholipid) encapsulating AS250, vortexed and incubated for 60 min at 65 °C under argon atmosphere. The aim was to produce two types of liposomal MMAE: PEGylated liposomes with low MMAE content (LMPL) and PEGylated liposome with high MMAE content (HMPL).

The final concentration of DMSO in liposomal suspension was kept identically at 5% (v/v) by addition of excessive amount of DMSO to LMPL. After this, non-encapsulated free MMAE was separated from liposomal MMAE by dialyzing liposomes against HEPES 10 mM buffered sucrose 10% (w/v), pH 7.4. As controls to validate the separation procedure free MMAE treated with free trapping agents was also placed in a dialysis bag and treated the same as liposomal samples.

Liposome characterization

Samples of liposomes before and after the dialysis were withdrawn and characterized respect to colloidal properties including size and poly dispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAE contents were also quantified using Bartlett assay and HPLC, respectively. From which the encapsulation efficiency was measured as follow:

MMAE) .. .. . ratio of . , after dialisis

lipid / encapsulation efficiency (%) = 100 * ratio o before dialisis

Liposomes were finally sterilized by passing through sterile syringe filter with 0.2 gm pore size and kept under argon atmosphere at 4 °C. Morphology of liposomes with high MMAE content (HMPL) was also evaluated by transmittance electron microscopy (TEM).

Leakage stability analysis of MMAE -liposome Leakage stability of HMPL was assessed using dialysis. Briefly, a month after liposome preparation 500 pL of liposomal suspension was transferred into a dialysis bag and dialyzed against 80 mL HEPES buffered sucrose pH 7.4 for 24 h at 4 °C under gentle stirring. Sample from dialysis medium was withdrawn and the dialysis bags were incubated further for 72 h at 37 °C. The amount of MMAE in dialysis medium at each time point was quantified by HPLC and compared against the initial content of MMAE inside the dialysis bags.

In vitro cytotoxicity

C26 colon carcinoma cells were seeded at the density of 2500 cells/well in 96- well microplates. After overnight incubation at 37 °C, in RPMI 1640 (Sigma- Aldrich, Germany) supplemented with 10% fetal calf serum (FCS, Gibco Invitrogen) the medium was replaced with serum free RPMI 1640 containing serial dilutions of different liposomal MMAE and commercial liposomal doxorubicin Caelyx® (Janssen Pharmaceutica NV, Belgium). Dilutions were made based on phospholipid concentration in liposomal preparations. After 3 h of liposome -cells exposure at 37 °C, cells were washed three times with pre-warmed complete medium and were allowed to proliferate for 72 h at the culturing condition. The cytotoxic impact of liposomes on C26 cells was finally assessed using XTT assay. Briefly, at the end of proliferation period culture medium was replaced with 100 pL of 2,3- Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT , Img/mL)/ Phenazine methosulfate (PMS, 7.66 pg/mL) solution freshly prepared in phenol red and serum free culture medium. After 1 h incubation at 37 °C in a CCL-incubator the absorbance at 490 nm was recorded and the relative cell death (R) was calculated as follows: R = l-[ (Atest - Abiank)/( Acontroi - Abiank)] , where A_test and Acontroi were the absorbances of the cells treated with the test solutions and the culture medium (negative control), respectively. Abiank was the absorbance of XTT/PMS solution added in cell free wells. IC50 were then calculated using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA).

In vivo evaluation of systemic toxicity of liposomal MMAE

Increasing doses of MMAE either as free or encapsulated in LMPL or HMPL prepared as described before were injected to into healthy BALB/c mice (5/group) and monitored for 3 weeks. Occurrence of animal death was reported as percentage of death in each group of mice.

Results

Liposome characteristics

Loading of MMAE into liposome did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical. The particle size of LMPL and HMPL liposomes was 76.23 ± 0.30 nm, and 77.73 ± 0.55 nm and the PDI was 0.04 ± 0.015 and 0.057 ± 0.013, respectively. The MMAE content in LMPL and HMPL at identical phospholipid content was 0,6067 and 1,7029 mg/ml, respectively. Indicating drug to lipid ratios of 109.8 and 274.7 pg MMAE/pmol phospholipid. Encapsulation efficiency for LMPL and HMPL was about 62% and 48 % respectively. Control liposomes encapsulating HBS revealed 0% encapsulation, indicating the MMAE is actively loaded into liposomes by means of ammonium sulfate gradient.

The TEM image (Figure 1) illustrates vesicles with spherical shape indicating loading of MMAE does not cause morphological changes on liposomes even at high drug to lipid ratio. In addition, the dark liposome interior is an indication of a dense medium inside the liposomes resulted from high concentration of MMAE inside liposomes.

Leakage stability of MMAE-liposome HPLC analysis of free MMAE inside the dialysis medium indicated almost no release from both LMPL and HMPL during a month of incubation at 4 °C. Liposomes were also well stable within a subsequent 72 h storage at 37 °C with 3.1% and 11.14 % release of encapsulated MMAE from LMPL and HMPL, respectively (Figure 2).

In vitro cytotoxicity

Based on phospholipid concentration LMPL, HMPL and Caelyx® exhibited IC50 values of 0.329 ± 0.028, 0.036 ± 0.004 and 19.700 ± 0.071 pM against cultured C26 cells. LMPL and HMPL were found 60 and 547 times more toxic than liposomal doxorubicin, respectively (Figure 3).

In vivo evaluation of systemic toxicity of liposomal MMAE

Injection of different doses of free or liposomal MMAE into healthy mice revealed that encapsulation of MMAE into liposomes improved the systemic toxicity of MMAE since mice tolerated higher doses of liposomal MMAE compared to receiving free form of the drug. In dose of 1.2 mg/kg while no death occurred within mice receiving liposomal MMAE (n=10), 80% of mice received free MMAE died within a period of 3 weeks (Figure 4).

Example 2. Encapsulation of MMAE into fast-release temperature sensitive liposomes.

Materials and Methods

Preparation of fast-release temperature sensitive liposomal MMAE Lipids for preparation of fast-release temperature sensitive liposomes composed of dipalmitoylphosphatidylcholine (DPPC)/ distearoyl phosphatidylcholine (DSPC)/mPEG2000-DSPE (80/15/5 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of ammonium sulfate 250 mM (AS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As a negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. This primary liposome suspension were then downsized and homogenized by passing through polycarbonate membranes of 200 and 100 nm, sequentially, using LIPEX extruder at 65°C.

To establish the gradient for remote loading, liposomes were dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C. The phospholipid content of the dialyzed liposomal suspensions were then quantified by Bartlett inorganic phosphate assay.

For remote loading of MMAE into liposomes, the pH of liposomal suspensions were first adjusted at 4.5 and then 1.4 mg of MMAE dissolved in DMSO (71.45 mg/mL) was added to the thermosensitive liposomes (size: 120 nm) encapsulating AS250 (5.5 jimol phospholipid). DMSO content was adjusted at 5%, vortexed and incubated for 60 min at 37 °C under argon atmosphere. After this, non-encapsulated free MMAE was separated from liposomal MMAE by dialyzing liposomes against HEPES 10 mM buffered sucrose 10% pH 7.4. The aim was to check the possibility of loading of MMAE into liposomes which have temperature sensitivity and release payload upon exposure to mild hyperthermia of around 42 °C.

Liposome characterization

Samples of liposomes before and after the dialysis were withdrawn and characterized respect to colloidal properties including size and poly dispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAE contents were also quantified using Bartlett assay and HPLC, respectively.

In order to evaluate the drug loading efficiency, non-encapsulated free MMAE was separated from liposomal MMAE by filter centrifugation using Amicon® Ultra 0.5 (MWCO 100K, Millipore - Merck, Germany) and the concentration of MMAE in liposomal suspension before centrifugation and inside the filtrate passed through the filter upon 10 min centrifugation at 6000g was quantified using HPLC. Loading efficiency was measured as follow: encapsulation efficiency (%)

Concentration of MMAE in filtrate solution

= 100 * -

Concentration of MMAE in liposomal suspention before filtration

Results

Loading of MMAE into temperature-sensitive liposomes did not change the colloidal properties of liposomes and sizes and PDIs before and after loading were identical. The particle size of MMAE loaded temperature sensitive liposomes was 119 ± 1.32 nm and the PDI was 0.051 ± 0.019, respectively. MMAE was successfully loaded into temperature sensitive liposomes by drug loading efficiency of around 40% (Figure 5) and resulted in liposomes containing MMAE with drug to lipid ratio of 95 pg MMAE/pmol phospholipid. Control liposomes encapsulating HBS revealed 0% encapsulation, indicating the MMAE is actively loaded into liposomes by means of the ammonium sulfate gradient.

In Examples 1 and 2 it is established that MMAE could be loaded into two liposomes with different lipid composition. MMAE was loaded into (i) liposomes made of rigid and completely impermeable membranes (Example 1) and (ii) liposomes composed of less rigid lipid composition that undergo liposomal phase transition at around 42°C (Example 2). Liposomes composed of HSPC:Cholestrol:mPEG-DSPE are stable and long circulating liposomes due to rigidity of membrane and presence of PEG. HSPC exhibits liposomal phase transition behavior at temperatures around 55 °C and therefore liposomes composed of solely HSPC provide for high leakage stability at physiological temperature. Cholesterol enhances liposomes stability and impermeability by diminishing phase behaviors of phospholipids. Therefore, liposomes of HSPC and cholesterol (60:40 mol%) do not undergo phase transition and remains stable and impermeable even at temperatures above 70 °C. Because MMAE was successfully loaded into these two liposomes, with two opposite membrane characteristics, it is envisaged that liposomes with any kind of lipid composition can be loaded with MMAE via remote loading methods.

Example 3. Co-encapsulation of MMAE and DXR into liposomes.

Materials and Methods

Preparation of stable long circulating liposomal MMAE+DXR Lipids composed of hydrogenated soy phosphatidylcholine (HSPC)/cholesterol(Chol)/mPEG2000-DSPE (56.5/38.2/5.3 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 5 mL of ammonium sulfate 250 mM (AS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

The pH of liposome suspension were then adjusted at 5 and then MMAE and DXR were added into liposomes encapsulating AS250 (12 jimol phospholipid content) in two different combinations. In one combination (Combo-PLl) 1 mg MMAE and 2 mg DXR were added and in the other one (Combo-PL2) 2 mg MMAE and 1 mg DXR mg were added. Liposomes were then vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 to separate free drugs from encapsulated drugs.

Liposomes were then characterized respect to colloidal properties including size and polydispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and drug contents were also quantified using Bartlett assay and HPLC, respectively.

Results

Co-loading of MMAE into liposomes did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical and remained around 80 nm. DXR and MMAE were successfully coencapsulated into liposomes and resulted into two liposomal preparations containing 28 jig MMAE plus 147 pg DXR, and 91 pg MMAE plus 97 pg DXR per jimol phospholipid. Loading efficiency respect to DXR was about 100% in both preparations while loading of MMAE resulted in loading efficiencies of 41 and 58 % in MMAE/DXR combinations of 1/2 and 2/1, respectively (Figure 6).

Herein it was established that MMAE could be co loaded into liposomes by means of remote loading methods. One may adjust the initial amounts of each compound to be co-loaded into liposomes to achieve a desired ratio of MMAE and the combined compound.

Example 4. Co-encapsulation of MMAE and DXR into liposomes using TEAS gradient.

Lipids composed of hydrogenated soy phosphatidylcholine HSPC/cholesterol (Chol)/mPEG2000-DSPE (56.5/38.2/5.3 jimol) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of triethylammonium sulfate 250 mM (TEAS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

The pH of liposome suspension were then adjusted at 5 and then 0.58 mg MMAE and 0.29 mg doxorubicin (DXR) were added to liposomes encapsulating TEAS250 (3.9 jimol phospholipid content), vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 to separate free drugs from encapsulated drugs.

Results

Liposome characteristics

Co-loading of MMAE into liposome did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical. DXR and MMAE were successfully co-encapsulated into liposomes via the TEAS gradient, where liposome suspension contained 58.7 pg DXR/jimol phospholipid and 59.5 pg MMAE/jimol phospholipid, co-encapsulated with loading efficiencies of 79% and 40%, respectively (Figure 7).

Meanwhile in control liposomes encapsulating HEPES buffer minimal drugs were detected.

Example 5. Encapsulation of MMAE into liposomes using TEAS gradient. Lipids composed of hydrogenated soy phosphatidylcholine (HSPC)Zcholesterol (Chol)/mPEG2000-DSPE (56.5/38.2/5.3 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of triethylammonium sulfate 250 mM (TEAS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

The pH of liposome suspension was then adjusted at 5 and then 0.58 mg MMAE was added to 3.9 jimol liposomes encapsulating TEAS250, vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 to separate free drugs from encapsulated drugs.

Liposomes were then characterized respect to colloidal properties including size and polydispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAE contents were also quantified using Bartlett assay and HPLC, respectively.

Results

Liposome characteristics

Loading of MMAE into liposomes did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical. MMAE was successfully encapsulated into liposomes via the TEAS gradient, where liposome suspension contained 81.1 jig MMAE/jimol phospholipid, encapsulated with a loading efficiency of 59.23% (Figure 8). In control liposomes encapsulating HEPES buffer, minimal drugs were detected.

Example 6. Effect of drug/lipid ratio on encapsulation of MMAE into liposomes encapsulating ammonium sulfate.

Lipids composed of hydrogenated soy phosphatidylcholine (HSPC)/cholesterol (Chol)/mPEG2000-DSPE (113/76.4/10.6 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of ammonium sulfate 250 mM (AS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

Aliquots of liposomes containing 12.5 jimol phospholipid were withdrawn and pH were adjusted at 4.5 and different amounts of 1, 2 and 4 mg MMAE was added to each liposome suspension, vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 to separate free drugs from encapsulated drugs.

Liposomes were then characterized respect to colloidal properties including size and poly dispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAE contents were also quantified using Bartlett assay and HPLC, respectively. Results

Liposome characteristics

Loading of MMAE at different drug/lipid ratios did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were remained identical. Highest loading efficiencies achieved at MMAE/phospholipid ratio around 1 (mg)/12.5 (jimol) and significantly decreases at high drug content of 4 mg at the same lipid content (Figure 9).

Example 7. Effect of pH on encapsulation of MMAE into liposomes encapsulating ammonium sulfate.

Lipids composed of hydrogenated soy phosphatidylcholine (HSPC)Zcholesterol (Chol)/mPEG2000-DSPE (113/76.4/10.6 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of ammonium sulfate 250 mM (AS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

Aliquots of liposomes containing 12.5 jimol phospholipid were withdrawn and pH were adjusted at different values of 3.5, 5, 7, 8 and 7. Then 1 mg MMAE was added to each liposomal suspensions, vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4 to separate free drugs from encapsulated drugs. Liposomes were then characterized respect to colloidal properties including size and poly dispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAE contents were also quantified using Bartlett assay and HPLC, respectively.

Results

Liposome characteristics

Loading of MMAE at different pH did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical. MMAE was successfully encapsulated into liposomes via the remote loading methods at different pH while no MMAE was found in control liposomes containing HEPES saline. Loading of MMAE at different pH of 3.5, 4.5, 7, 8 and 9 resulted in 51, 81, 63, 25 and 0, respectively (Figure 10). The maximum loading rate achieved when external pH of liposome suspension during drug loading was adjusted at 4.5. Increasing the pH above 7 unexpectedly dramatically decreased encapsulation rate of MMAE reaching to 0% at pH around 9. Lowering the pH below 4 also reduces encapsulation of MMAE into MMAE liposomes.

Example 8. Encapsulation of MMAF into liposomes encapsulating ammonium sulfate

Lipids composed of hydrogenated soy phosphatidylcholine (HSPC)Zcholesterol (Chol)/mPEG2000-DSPE (56.5/38.2/5.3 jimol, respectively) were first mixed from stocks of lipids dissolved in chloroform, and then dried by rotary evaporator and overnight connection to freeze dryer. The dried lipid film was then hydrated with 10 mL of ammonium sulfate 250 mM (AS 250) followed by gentle shaking and 10 min bath sonication, all at 60-65 °C. As negative control a dried lipid film was hydrated with HEPES 10 mM buffered saline pH 7.4. Liposome suspensions were then downsized and homogenized by passing through polycarbonate membranes of 200, 100, and 50 nm, sequentially, using LIPEX extruder at 65°C and dialyzed against HEPES 10 mM buffered sucrose 10% pH 7.4, at 4 °C.

Aliquots of liposomes containing 6 jimol phospholipid were withdrawn and pH were adjusted at different values of 3, 4, 5 and 6.5. then 2 mg MMAF was added to each liposomal suspensions, vortexed and incubated for 60 min at 65 °C under argon atmosphere. Liposomes were then dialyzed against HEPES 10 mM buffered sucrose 10% pH 6.5 to separate free drugs from encapsulated drugs.

Liposomes were then characterized respect to colloidal properties including size and poly dispersity index (PDI) using a Dynamic Light Scattering instrument (Nano-ZS; Malvern, UK). Lipid and MMAF contents were also quantified using Bartlett assay and HPLC, respectively.

Results

Liposome characteristics

Loading of MMAF into liposomes did not change the colloidal properties of liposomes and both sizes and PDIs before and after loading were identical. MMAF was successfully encapsulated into liposomes via the remote loading methods at different pH. Loading of MMAF at different pH of 3, 4, 5 and 6.5 resulted in 46, 41, 33 and 9 percent loading efficiency, respectively (Figure 11). The maximum loading rate achieved when external pH of liposome suspension during drug loading was adjusted at 3. Increasing the pH decreases the encapsulation rate of MMAF loading into liposomes containing ammonium sulfate (250 mM). Herein we established that like MMAE it is also possible to load MMAF into liposomes by means of remote loading methods.

Claims

65 Claims

1. A method for producing an auristatin -loaded liposome, comprising the steps of:

- providing a liposome that encapsulates a remote loading agent;

- generating a concentration gradient of said remote loading agent across the membrane of said liposome;

- mixing said liposome with an aqueous medium comprising an auristatin;

- loading said auristatin into said liposome; wherein said loading is driven by said concentration gradient; and

- optionally, purifying an auristatin-loaded liposome.

2. The method according to claim 1, wherein said auristatin is a monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

3. The method according to any one of the preceding claims, wherein

(i) said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAE; and wherein said loading is performed at a pH of 3.5- 7, preferably at a pH of 4.0-4.5, more preferably at a pH of about 4.5; or

(ii) wherein said aqueous medium comprising an auristatin is an aqueous medium comprising an MMAF; and wherein said loading is performed at a pH of 2.5-4, preferably at a pH of about 3.

4. The method according to any one of the preceding claims, wherein said remote loading agent is an ammonium-based remote loading agent, preferably an ammonium salt such as ammonium sulphate or triethyl ammonium sulphate (TEAS).

5. The method according to any one of the preceding claims, wherein said liposome comprises one or more vesicle-forming lipids selected from the 66 group formed by phospholipids, sphingolipids, diglycerides, dialiphatic glycolipids, cholesterol and derivates thereof, and combinations thereof.

6. The method according to any one of the preceding claims, wherein said liposome comprises a first vesicle-forming phospholipid that is a phosphatidylcholine and a second vesicle-forming phospholipid that is an optionally PEGylated phosphatidylethanolamine.

7. The method according to claim 6, wherein

(i) said phosphatidylcholine is a hydrogenated soy phosphatidylcholine (HSPC), said phosphatidylethanolamine is an optionally PEGylated l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and wherein said liposome further comprises a sterol such as cholesterol; or

(ii) said phosphatidylcholine is a dipalmitoylphosphatidylcholine (DPPC), said phosphatidylethanolamine is an optionally PEGylated 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and wherein said liposome further comprises a second phosphatidylcholine that is a distearoyl phosphatidylcholine (DSPC).

8. The method according to any one of the preceding claims, wherein said liposome that encapsulates a remote loading agent is obtainable by a method comprising the steps of:

- providing an organic solvent comprising one or more vesicle-forming lipids as defined in any one of the preceding claims;

- drying said one or more vesicle-forming lipids to thereby provide a dried lipid film;

- hydrating said dried lipid film with a first aqueous medium under conditions that allow for the formation of a liposome encapsulating said first 67 aqueous medium, wherein said first aqueous medium comprises a remote loading agent;

- optionally, extruding said liposome through a membrane with pores in order to downsize said liposome;

-optionally, drying or dehydrating said liposome encapsulating said first aqueous medium.

9. The method according to any one of the preceding claims, wherein said concentration gradient of said remote loading agent is generated by:

- removing non-encapsulated remote loading agent, preferably by dialyzing said liposome against a buffer solution; and/or

- mixing said liposome with said aqueous medium comprising an auristatin.

10. The method according to any one of the preceding claims, wherein the auristatin loading efficiency is at least 40%, more preferably at least 70%, and even more preferably at least 100%.

11. The method according to any one of the preceding claims, wherein the drug (auristatin) to lipid ratio (pg drug/p mol lipid) is in a range of 0.1- 300, preferably 8-240.

12. The method according to any one of the preceding claims, wherein a second anti-cancer agent and/or a first immunomodulatory agent is coloaded with said auristatin into said liposome; optionally wherein a third anti-cancer agent and/or a second immunomodulatory agent is co-loaded into said liposome, wherein said third anti-cancer agent is different from said second anti-cancer agent and/or said second immunomodulatory agent is different from said first immunomodulatory agent. 68

13. The method according to claim 12, wherein said second and/or third anti-cancer agent is selected from the group formed by

- a proteasome inhibitor such as carfilzomib, oprozomib, bortezomib and/or ixazomib;

- a tyrosine kinase inhibitor such as imatinib, lapatinib, acalabrutinib, afatinib, alectinib, avapritinib, axitinib, bosutinib, cabozantinib, crizotinib, dacomitinib, dasatinib, entrectinib, erlotinib, gilteritinib, ibrutinib, midostaurin, neratinib, nilotinib, pacritinib, pazopanib, pexidartinib, ponatinib, quizartinib, regorafenib, midostaurine, sorafenib, dasatinib, sunitinib, vandetanib, aflibercept, zanubrutinib and/or ziv-aflibercept;

- an anthracycline such as a doxorubicin, daunorubicin, idarubicin, mitoxantrone, valrubicin, epirubicin, pirarubicin, rubidomycin, carcinomycin and/or N-acetyl adriamycin;

- an alkylating agent such as busulfan, cyclophosphamide, bendamustine, carboplatin, chlorambucil, cyclophosphamide, cisplatin, temozolomide, melphalan, bendamustine, carmustine, lomustine, lomustine, dacarbazine, oxaliplatin, melphalan, lomustine, ifosfamide, mechlorethamine, thiotepa, trabectedin and/or streptozocin;

- an camptothecin such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan and/or rubitecan;

- an anti-metabolite such as gemcitabine;

- a taxane such as paclitaxel and/or docetaxel;

- a targeted anti-cancer agent such as an antibody including for instance herceptin, nivolumab and/or bevacizumab;

- an anti-cancer agent selected from the group formed by mitomycin C; a plant-derived alkaloid such as vincristine, vinblastine, vinorelbine, vinflunine, vinpocetine, vindesine, ellipticine or 6-3-aminopropyl-elhpticine; 2-diethylaminoethyl-elhpticinium; datelliptium; or orretelliptine; and/or said first and/or second immunomodulatory agent is selected from the group formed by: 69

- an immunomodulatory agent that is a thalidomide, lenalidomide, pomalidomide and/or imiquimod.

14. An auristatin-loaded liposome obtainable by a method according to any one of claims 1-13, wherein said auristatin is loaded as an unconjugated auristatin; preferably wherein the liposome comprises one or more vesicle-forming lipids as defined in any one of claims 5-7.

15. The auristatin-loaded liposome according to claim 14, wherein said auristatin-loaded liposome is not a sustained-release liposome.

16. The auristatin-loaded liposome according to claim 14 or claim 15, wherein said auristatin-loaded liposome is for tumor-directed drug delivery.

17. The auristatin-loaded liposome according to claim 16, wherein said tumor-directed drug delivery is by:

- passive tumor targeting, preferably wherein the auristatin-loaded liposome is a stable, long-circulating liposome;

- active tumor targeting, preferably wherein the auristatin-loaded liposome comprises a tumor-targeting moiety that specifically binds to a tumor cell and/or a tumor-associated cell; and/or

- controlled or triggered release, preferably wherein said auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C).

18. An auristatin-loaded liposome according to any one of claims 14- 17, wherein said liposome is for use as a medicament, preferably for use in the treatment of a subject having a tumor, preferably a cancer such as colorectal cancer. 70

19. An auristatin-loaded liposome for use according to claim 18, wherein the auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C); and wherein said treatment comprises a step of applying heat to said tumor in order to provide for at least said hyperthermic temperature in said tumor so as to allow for release of said auristatin.

20. A method for treating a subject suffering, or suspected of suffering, from a tumor, preferably a cancer such as colorectal cancer, said method comprising the step of:

- administering a therapeutically effective amount of an auristatin-loaded liposome of any one of claims 14-17 to said subject.

21. The method according to claim 20, wherein said auristatin-loaded liposome is a temperature-sensitive liposome that releases auristatin at a hyperthermic temperature (preferably a temperature of at least 40 °C); and wherein said treatment comprises a step of applying heat to said tumor in order to provide for at least said hyperthermic temperature in said tumor so as to allow for release of said auristatin.