CA3009293A1 - Metal complexes to induce immunogenic cell death and uses thereof - Google Patents

Abstract

Provided herein are compositions and methods for the use of copper and at least one agent to induce immunogenic cell death (ICD), in combination or sequentially, to treat or prevent a disease in a patient. A combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGB1 release, and CRT exposure of cells. The copper, the agent, or both, may be formulated in a liposome.

Description

METAL COMPLEXES TO INDUCE IMMUNOGENIC CELL DEATH AND USES THEREOF
TECHNICAL FIELD
Disclosed herein are pharmaceutical compositions and uses thereof to induce immunogenic cell death for the treatment or prevention of cancer or other disease conditions.
Further disclosed are methods for identifying compositions with enhanced ability to induce immunogenic cell death. Additional embodiments are directed to treatment methods to induce immunogenic cell death.
BACKGROUND
Immunogenic cell death (ICD) is a phenomenon whereby cancer cells die in a manner that activates the immune system. Activation of the immune system can in turn elicit an immune response against the cancer. The phenomenon is distinct from regular apoptosis in which cell death does not generally lead to the activation of the immune system.
In particular, during immunogenic cell death, dying tumour cells emit signals known as damage-associated molecular patterns (DAMPs). ICD is often characterized by three distinct DAMPs, which are evidenced by the presence of one or more of the following molecular determinants (referred to in the literature as "markers") of ICD: (1) the pre-apoptotic expression of endoplasmic reticulin (ER) calreticulin (CRT), (2) the secretion of ATP, and (3) the secretion of nuclear high mobility group box 1 (HMGB1). It has been reported that the presence of these DAMPs during tumor cell death leads to the release of pro-inflammatory cytokines, activation of innate immune cells such as dendritic cells (DCs) and macrophages, and ultimately stimulation of the adaptive immune response.
A number of agents used in cancer treatment are capable of inducing immunogenic cell death as determined by one or more assay to detect one or more of the above markers.
Generally, ICD inducers can be categorized as Type I or Type ll inducers. Type 1 inducers induce ICD as an off-target effect, while Type II inducers typically cause primarily ER
stress, which is directly related to ICD induction. However, there is an ongoing need in the art for additional or improved compositions for inducing 1CD for the treatment of cancer and other disease conditions. The present disclosure seeks to address that need or to provide useful alternatives to known approaches.

SUMMARY
According to a one aspect of the disclosure, there is provided an immunostimulatory pharmaceutical composition to treat or prevent cancer by inducing immunogenic cell death comprising: copper and at least one agent, such as an anti-cancer agent, that is capable of complexing with copper to form a metal complex. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone. The increase in the ICD response of the combination as measured by extracellular ATP may be 1.2 to 10,000 times that of the agent alone measured under otherwise identical conditions. The copper, the agent, or both, in certain embodiments are formulated in a liposome.
According to a further aspect of the disclosure, there is provided the use of copper and at least one agent, such as an anti-cancer agent, that induces immunogenic cell death (ICD), in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure of cells. The copper, the agent, or both, in certain embodiments, are formulated in a liposome.
According to another aspect of the disclosure, there is provided a method of inducing immunogenic cell death (ICD) comprising administering copper and at least one agent, such as an anti-cancer agent, in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMG131 release, and CRT exposure. The copper, the agent, or both, may be formulated in a liposome.
In yet a further aspect, there is provided a method of producing an immunostimulatory pharmaceutical composition for inducing immunogenic cell death (ICD), comprising the steps of:
(i) exposing an agent, such as an anti-cancer agent, and copper, alone and in combination, to an assay that measures at least one of ATP secretion, HMGE31 release, and CRT exposure in cell culture;
(ii) determining whether the agent exhibits an increase in ICD response in combination with the copper compared to results in which the ICD of the anti-cancer agent is measured alone; and

2 (iii) wherein, if the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, formulating the agent, the copper, or both in one or more pharmaceutical formulations, such as a liposome, to treat or to prevent cancer, either sequentially or in combination, to a patient in need thereof.
In one embodiment of any of the foregoing aspects, the copper and the agent are capable of inducing the ICD response with a further, second agent.
In yet a further aspect of the disclosure, there is provided a kit comprising copper and an agent, such as an anti-cancer agent, to induce immunogenic cell death (ICD), wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures ATP secretion, HMGE31 release, CRT
exposure or a combination thereof, and wherein the copper, the agent, or both, in non-limiting embodiments, are formulated in a liposome. The kit may comprise instructions to induce immunogenic cell death to a patient in need thereof.
According to any of the foregoing aspects of the disclosure, the copper and the at least one agent are capable of inducing immunogenic cell death with radiation.
According to any of the foregoing aspects of the disclosure, the increase in the ICD response in the presence of copper is measured by ATP secretion.
In a further embodiment of the any of the foregoing aspects, the copper is incorporated in the same or a different liposome as the agent.
According to a further embodiment of any of the above aspects, a further, second agent is utilized.
According to any of the foregoing aspects of the disclosure, a cytotoxic effect is realized, as measured by a cytotoxic assay.
According to any of the foregoing aspects of the disclosure, the liposome is a pre-formed liposome. That is, the liposome is prepared in solution and subsequently the anti-cancer agent is loaded into the liposome by complexation with the metal.
In embodiments of any of the foregoing aspects, the agent is a therapeutic agent that can treat or prevent disease and that can complex with a metal. For example, the agent may be an anti-

3 cancer agent, an anti-viral agent or other therapeutic agent to treat or prevent disease by inducing ICD in the presence of a metal. Moreover, in a further embodiment of any of the foregoing aspects, a nanoparticle or other suitable delivery vehicle is utilized instead of a liposome. Such alternate drug delivery vehicles are known to those of ordinary skill in the art.
BRIEF DESCRIPTION OF FIGURES
FIGURE 1A shows extracellular ATP (nM) of CT26 murine colon cancer cells (CT26 cells) that were untreated, treated with copper (100 pM) and treated with zinc (100 pM).
FIGURE 1B shows extracellular ATP (nM) of CT26 cells that were untreated, treated with PX-478 (25 pM) and treated with PX-478 and copper (1:1 molar ratio).
FIGURE 1C shows extracellular ATP (nM) of CT26 cells that were untreated, treated with Emodin (100 pM) and treated with Emodin and copper (1:1 molar ratio).
FIGURE 1D shows extracellular ATP (nM) of CT26 cells that were untreated, treated with clioquinol (CQ; 25 pM), treated with CQ and copper (2:1 molar ratio) and CQ
and zinc (2:1 molar ratio).
FIGURE lE shows extracellular ATP (nM) of 0T26 cells that were untreated, treated with pyrithione (Pyr; 5 pM), treated with Pyr and copper (2:1 molar ratio) and Pyr and zinc (2:1 molar ratio).
FIGURE IF shows extracellular ATP (nM) of CT26 cells that were untreated, treated with flavopiridol (FLV; 1 pM) and treated with FLV and copper (1:1 molar ratio).
FIGURE 1G shows extracellular ATP (nM) of CT26 cells that were untreated, treated with diethyldithiocarbamate (DDC; 12.5 pM), treated with DDC and copper (2:1 molar ratio) and treated with DDC and zinc (2:1 molar ratio).
FIGURE 1H shows extracellular ATP (nM) for CT26 cells that were untreated, treated with epigallocathecin gallate (EPGG; 100 pM), treated with EPGG and copper (1:1 molar ratio) and treated with EPGG and zinc (1:1 molar ratio).
FIGURE 2A shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.

4 FIGURE 2B shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, cisplatin (CDDP) alone, PX-478 and CDDP, Cu(PX-478) alone, and CDDP and Cu(PX-478) in combination.
FIGURE 2C shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4,4',4"-tri-ter-butyl-2,2':6',2" ¨
terpyridine (TTT) alone, cisplatin (CDDP) alone, TTT and CDDP, Cu(TTT)2 alone, and cisplatin and Cu(TTT)2 in combination.
FIGURE 2D shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mito, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
FIGURE 2E shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: clioquinol (CQ) alone, gemcitabine (Gem) alone, CQ and Gem in combination, Cu(CQ)2 alone, and Gem and Cu(CQ)2 in combination.
FIGURE 2F shows fold increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4' (4-chlorophenyl) ¨ 2,2':6',2"-terpyridine (4CPT) alone, cisplatin (CDDP) alone, 4CPT and CDDP, Cu(4CPT)2 alone, and CDDP and Cu(4CPT)2in combination.
FIGURE 3A shows HMGB, (ng/mL) of CT26 cells for the following: untreated, PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.
FIGURE 3B shows HMGBi (ng/mL) of CT26 cells for the following: untreated, flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mito, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
FIGURE 4 shows CRT expression on CT26 cells at various time points post-treatment with mitoxantrone and HEPEs buffered saline solution (HBSS). The inset shows the relative viability of the CT26 cells at each time point after treatment by mitoxantrone.

DETAILED DESCRIPTION
Induction and measurement of Immunogenic Cell Death (ICD) The compositions and methods described in the present disclosure enhance immunogenic cell death phenotypes, which is also referred to herein as "ICD". In particular, the inventors have examined the ability of various agents to induce ICD by utilizing in vitro assays to measure molecular determinants indicative of ICD and have found that the addition of copper can enhance or induce ICD responses for certain agents. In certain embodiments, after selection of suitable combinations by the foregoing assay, the agents are most advantageously loaded into pre-formed liposomes comprising copper, using, for example, methods described herein.
As noted, during ICD, cells emit signals known as damage-associated molecular patterns (DAMPs). ATP is one such DAMP that is secreted during ICD and, without being limiting, is believed to attract monocytes to the site of tumor cell death. ATP secretion can be measured in vitro by exposing cells in culture to the agents being tested for a pre-determined period of time.
ATP is subsequently measured by a reaction in which the ATP secreted is measured by luminescence.
Prior to measuring ATP secretion, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in multi-well plates, such as 24-well plates.
At 24 hours, cells are treated with the agents. The metal ion is added to the agent preparation under conditions such that the metal complexes are formed optimally prior to their addition to the cells.
Cells are exposed to the tested agents for 24 hours (or left untreated), after which the supernatant of each well is transferred to a new plate. The Promega CellTiter-Glo TM 2.0 Kit is then used, as per the manufacturer's instructions, to determine the extracellular ATP level under each treatment condition. The assay measures ATP by luminescence. Luciferin is converted to oxyluciferin in the presence of ATP, Mg2+ and 02. Oxyluciferin in turn emits light that is quantified by a luminometer and is correlated with the amount of ATP present by a standard curve. The assay for use in the present disclosure is described in Promega Technical Manual, CellTiter-Glo0 2.0 Assay, Instructions for Use of Products, G9241, G9242 and G9243 available on-line at www.promeqa.com and which is incorporated herein by reference.
A further DAMP is high-mobility group box 1, also referred to herein as "HMGE31". Without being limiting in any manner, HMGBi has been reported to be a late apoptotic marker and its release to the extracellular space may facilitate the release and presentation of tumour antigens to dendritic cells. This molecular determinant of ICD can be measured by the assay described below.
Similar to the assay for ATP release, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Where copper complexes are tested, the metal ion is added to the agent at a molar ratio such that the metal complexes are formed at a pre-determined metal-to-ligand ratio prior to addition to the cells for optimal metal complexation. Cells are exposed to the tested agents for 24 hours, after which the supernatant of each well is collected and the amount of HMG131 release is assessed using a commercial EL1SA kit purchased from IBL
lnternationalTM.
The test quantifies HMG131 by an anti-HMGBi antibody that is immobilized.
HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody.
Substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology.
Calreticulin (CRT) is another DAMP that is exposed on the surface of tumor cells during cell death. CRT can be measured by flow cytometric analysis of CRT cell surface expression.
According to this assay, after 24 hours of treatment of the cells, such as CT26 cells, with an agent, the agent is removed and replaced with culture media. Cells are harvested at 0, 24, 48, and 96 hours post-treatment and stained with anti-CRT antibody followed by Alexa-488-conjugated secondary antibody. Propidium iodide (PI) is used as a counterstain to differentiate between viable and dead cells. The relative CRT mean fluorescence intensity (MFI) of P1-negative viable cells is determined by subtracting the MFI of isotype control-stained cells from the MF1 of anti-CRT stained cells, and then normalising to HBSS-treated cells.
An immunofluorescence assay may also be used to visualize cell surface expression of CRT on cells treated with an agent for 4 hours and stained with the anti-CRT antibody (green), as described above. To enhance visualization, the cell membrane may be labelled with wheat germ agglutinin (red) and the nucleus with Hoechst 33342 (blue). Images may be taken using an IN Cell Analyzer 2200TM.
In a further embodiment, ICD induction by copper is evidenced by each of the three assays or any combination of two of any of the three assays or one of any of the three assays. In a further embodiment, the presence of copper increases the levels of ATP secretion caused by an ICD
inducer. In yet a further embodiment, the presence of copper enhances the level of HMGB, released by an ICD inducer. According to yet further embodiments described herein, ICD
induction by copper is evidenced by CRT expression. The ability of cancer to enhance ICD
induction is measured by quantifying ICD induction of an agent in the presence and absence of copper.
In one embodiment, the ICD response of the metal-agent complex as measured by extracellular ATP is 1.2 to 10,000, 1.5 to 10,000, 2 to 10,000, 3 to 10,000, 4 to 10,000, or

5 to 10,000 times that of the agent alone measured under otherwise identical conditions. In another embodiment, the ICD response of the metal-agent complex as measured by extracellular ATP
is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5,6,

6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times that of the agent alone measured under otherwise identical conditions. The upper limit of the ICD response of the metal- agent complex as measured by extracellular ATP may be 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 10,000 times relative to agent alone. Any combination of the foregoing lower and upper limits is included in select embodiments of the disclosure.
As discussed, the ICD of an agent in combination with copper is determined by measuring a phenotype of ICD (ATP secretion, CRT release and/or HMGB1). In addition, the cytotoxicity of a particular pharmaceutical formulation and/or treatment regime can be measured using a cytotoxicity assay. A cytotoxic effect is exhibited if an additive or synergistic effect is observed (non-antagonistic) as determined by the Chou Talelay method, which is known to those of ordinary skill in the art.
Without being limiting, in one embodiment, the cytotoxicity assay can be used to determine whether a cytotoxic effect of the agent of interest in combination with copper is present.
Moreover, in those embodiments that additionally include radiation and/or a second agent, the composition or treatment will be considered to have a cytotoxic effect if any combination selected from at least two of (i) copper, (ii) an agent, (iii) a second agent if two or more are used, and (iii) radiation treatment exhibits a cytotoxic effect in vitro. By way of example, if a pharmaceutical composition comprises copper as well as a first and a second agent in combination, and if the second agent exhibits a cytotoxic effect in combination with copper, but the first agent in combination with copper does not, nor the first agent in combination with the second agent, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. Yet in a further example, if a pharmaceutical composition comprises copper and a first and a second agent in combination, and if the first agent exhibits a cytotoxic effect in combination with the second agent, but the first or second agent in combination with copper does not, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. In another non-limiting, illustrative example, if a treatment comprises an agent in combination with copper and additionally radiation treatment, and if the agent and the radiation treatment exhibit a cytotoxic effect in combination, then the treatment will be considered cytotoxic, even if the copper and agent demonstrate no cytotoxic effect in combination.
Additional examples will be readily envisioned by those of ordinary skill in the art.
As noted, whether a cytotoxic effect is additive or synergistic can be measured using an in vitro assay. Cells are seeded in 384-well plates and treated with various concentrations of copper and agent (0.001 nM to 10 mM) for 72 hours. Cells are then stained with Hoechst 33342 and ethidium homodimer-I for total and non-viable (cells that have lost membrane integrity), respectively. The cells are subsequently imaged using an automated fluorescent microscopic platform such as the IN Cell Analyzer 2200. All images are processed through software such as the IN Cell Developer Toolbox which provides total and dead cell counts. The viable cell counts are used to generate dose responsive curves using curve-fitting programs such as Prism (GraphPad) to determine the potency of each treatment as determined by IC50 values.
For combinations involving radiation, the cells are seeded in 6-well plates and treated with the agent of interest with or without copper for 24 hours, after which the cells are irradiated at different doses (2, 4, 6, 8 10 Gy). The irradiated cells are immediately plated in culture dishes to achieve 100-500 colonies two weeks later. The colonies are stained with aqueous malachite green or crystal violet and dried overnight prior to colony counting. The surviving fraction (SF) is calculated using the equation SF = [no. of colonies formed after treatment /
(no. of cells seeded x plating efficiency)]. The dose response curves from each treatment are then compared to assess treatment efficacy in vitro.
As noted, the metal ion of select embodiments is copper. The metal ion may also be an ion of a transition metal or a Group IIlb metal. The transition metal may be from Group IB, 2B, 3B, 48, 5B, 6B, 7B and 8B (groups 3-12). Examples of transition metals include copper, zinc, manganese, iron, cobalt and nickel. In another embodiment, the metal has d-orbitals. The Group IIlb metal is from the boron family, which includes boron, aluminum, gallium, indium, thallium and nihonium. In one embodiment, the metal is in the 2+ oxidation state.

Agents The agent of select embodiments used in combination with copper typically comprises at least one complexation moiety or ligand to enable complexation with the copper. This includes a chemical group selected from an S-donor, 0-donor, N, 0 donor, a Schiff base, hydrazones, P-donor phosphine, N-donor or a combination thereof. In another embodiment, the moiety is a hard electron donor. Other moieties or ligands known to those of skill in the art suitable for corriplexation with a metal ion are included within the scope of certain embodiments as well. In a further embodiment, the complexation via metal coordination facilitates drug loading as described in co-owned WO 2017/100925 (Bally et al.). This could be measured by incubating an agent of interest with a liposome containing copper, or other metal ion of interest, under optimal conditions to facilitate drug uptake and measuring drug uptake after one hour. In one embodiment, the drug uptake after one hour is at least 30%, 40%, 50%, 60%, 70%
or 80% as determined by measuring the drug:lipid ratio relative to the theoretical drug:lipid ratio that could ideally be obtained. In another embodiment, the drug uptake after one hour is between 60% and 100% as measured above.
The agent may be selected from antineoplastic agents such as cytotoxic agents, including nucleoside analogues, anthracyclines, anti-folates, topoisomerase 1 inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTor inhibitors, retinoids, immunomodulatory agents and histone deacetylase inhibitors.
In one embodiment, an agent suitable for use in certain embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone, 4' (4-chlorophenyl) ¨ 2,2':6',2"-terpyridine (4CPT), 4,4',4"-tri-ter-butyl-2,2':6',2" ¨ terpyridine (TTT), shikonin and wogonin.
Without being limiting, the agent for use in select embodiments may be poorly soluble in solution prior to or after corn plexation with the metal ion. By this it is meant that the poorly soluble agent in free form has a solubility of less than 1 mg/mL in either water or a solution of the metal ion which complexes with the agent. Solubility of the agent in water or in the presence of the metal ion (10 mM to 500 mM) is measured at conditions of physiological pH and temperature after 60 minutes of incubation under these conditions. Measurement of solubility of the agent is conducted as described in co-owned WO 2017/100925.
In yet further embodiments, the agent has a solubility that is greater than 1 mg/mL in either water or a metal ion solution, as determined by the assay described above (see WO
2017/100925).
Preparation of pharmaceutical formulations As discussed, the agent may be formulated in a liposome. A liposome is a vesicle comprising a bilayer having amphipathic lipids enclosing an internal solution. The liposome may be a large unilamellar vesicle (LUV), which can be prepared as described below using extrusion. In one embodiment, the average diameter of the liposome may be between 60 nm and 2,000 nm, 70 and 1,000 nm, 70 and 500 nm, 70 and 200 nm or 70 and 180 nm. The liposome may comprise lipids including phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pahnitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also encompassed by certain embodiments. The phospholipids may comprise two acyl chains from 6 to 24 carbon atoms selected independently of one another and with varying degrees of unsaturation.
Additionally, the amphipathic lipids described above may be mixed with other lipids including triacylglycerols and sterols. As would be appreciated by those of skill in the art, lipids that interfere with liposome formation in the presence of a metal should typically be avoided.
Whether or not a given lipid is suitable for liposome formation in the presence of a metal ion can be determined by those of skill in the art. In one embodiment, the liposome comprises the lipids 1,2-distearoyl-sn-glycero-3- phosophocholine (DSPC) and cholesterol. The precise ratios of the lipids may vary as required. A non-limiting example of a suitable ratio of DSPC/Cholesterol is 55:45 mol:mol.
The liposomes may also comprise a hydrophilic polymer-lipid conjugate. The hydrophilic polymer may be a polyalkylether, such as polyethylene glycol. The hydrophilic polymer-lipid conjugate is generally prepared from a lipid that has a functional group at the polar head moiety that is chemically conjugated to the hydrophilic polymer. An example of such a lipid is phosphatidylethanolamine. The inclusion of such hydrophilic polymer-lipid conjugates in a liposome can increase its circulation longevity in the bloodstream after administration. The hydrophilic polymer is biocompatible and has a solubility in water that permits the polymer to extend away from the outer surface of the liposome. The polymer is generally flexible and may provide uniform surface coverage of the liposome outer surface. In addition, it has been found herein that the inclusion of such a hydrophilic polymer-lipid conjugate can increase the amount of the transition metal encapsulated in the liposome. This can be used as a methodology to increase the amount of the agent encapsulated in the liposome. In one embodiment, the liposome may include a hydrophilic polymer, such as polyethylene glycol (PEG) at between 1 and 20 mol% or between 2 and 10 mol%. A non-limiting example of a formulation comprising PEG is DSPC/CHOL/PEG (50:45:5, mole ratio) or DSPC /PEG (95:5, mole ratio).
The specific ratios of the lipids, however, may vary according to embodiments that would be apparent to those of ordinary skill in the art.
Liposomes can be prepared by any of a variety of suitable techniques known to those of skill in the art. An example of one suitable method involves cycles of freeze-thaw and subsequent extrusion of lipid preparations. According to one such method, lipids selected for inclusion in a liposome may be dessicated and dissolved in a solvent, such as an organic solvent, at a desired ratio. After removal of the solvent, the resultant lipids are hydrated in an aqueous solution. The solution in which the lipids are hydrated forms the internal solution of the liposomes.
Subsequently the hydrated lipids may be subjected to cycles of freezing and thawing. The hydrated lipids are passed through an extrusion apparatus to obtain liposomes of a defined size.
The size of the resulting liposomes may be determined using quasi-electric light scattering (e.g., using a NanoBrook ZetaPALSTM Potential Analyzer).
Without being limiting, the liposomes may be prepared so that they comprise an internal solution comprising the metal ion. For example, when preparing liposomes by freeze-thaw and subsequent extrusion as described above, the lipids are hydrated in a solution comprising a metal ion. Generally, the liposomes so formed will comprise the metal ion not only in the internal solution of the liposomes, but also in the external solution.
Unencapsulated metal ion may be removed from the external solution of the liposome prior to loading of the agent. For example, the external copper solution may be exchanged with a solution containing substantially no copper ions by passage through a column equilibrated with a buffer. Other techniques may be employed such as centrifugation, dialysis, the addition of a chelating agent, such as EDTA (to chelate the metal) or related technologies. Typically the solution that exchanges with the metal-containing solution is a buffer, although other solutions may be used as desired. The liposomes may be subsequently concentrated to a desired lipid concentration by any suitable concentration method, such as by using tangential flow dialysis. In one embodiment, the solution external to the liposome contains substantially no metal ions that complex with the poorly soluble agent. By this it is meant that the concentration of metal ions in the external solution is less than that of the metal ion concentration in the liposome, for example less than one fifth of the concentration of metal ion in the liposome.
Alternatively, or in addition, the external solution may comprise a chelating agent that chelates with the metal ions. As noted, the metal ion may be encapsulated in the liposome as a metal salt.
Examples include copper sulfate, copper chloride or copper gluconate.
The pre-formed liposomes comprising the metal ion may be incubated with the agent to facilitate uptake via metal complexation. The agent may be added in any suitable form, including' as a powder or as a solution. If the agent is insoluble in water, it can be added as a powder. The amount of free agent in solution can subsequently be increased by increasing the temperature.
Incubation of the pre-formed liposomes with the one or more agents is performed under conditions sufficient to allow the agent to move across the phospholipid bilayer of the liposome into the internal solution thereof. Such a method is referred to by those of skill in the art as "loading". Movement of the agent across the phospholipid bilayer of the pre-formed liposome during loading may occur independently of any pH gradient across the bilayer.
The loading may, however, be dependent on other factors. As will be appreciated, the loading conditions can be readily selected by those of skill in the art to achieve a desired rate of loading. For example, the diffusion of the agent across the bilayer may be dependent on the temperature and/or lipid composition of the liposome.
Without being bound by theory, the formation of the drug-metal complex incorporated in the pre-formed liposomes may be characterized as an inorganic synthesis reaction. In certain embodiments, the uptake of drug during the loading reaction is visualized as a colour change as many metal complexed agents have different spectral characteristics that can be detected by eye. For example, a colour change to purple, brown, green or yellow can be observed during loading with copper.
By formulating complexes through such an inorganic synthesis reaction occurring within the pre-formed liposome, a high drug-to-lipid ratio may be attained according to certain embodiments.

For example, the drug-to-lipid ratio may be at least 0.3:1 0.4:1, 0.5:1, at least 0.6:1, at least 0.7:1, or at least 0.8:1. In further embodiments, the drug-to-lipid ratio is about 0.1:1 to about 0.6:1 (mol:mol), about 0.15:1 to about 0.5:1 (mol:mol) or about 0.2:1 to about 0.4:1 (mol:mol).
Such a high drug-to-lipid ratio may be dependent on the number of metal ions inside the liposome and/or the nature of the complex formed. Formation of a transition metal complex with a given agent may be rapid (e.g., Cu(DDC)2), occurring in minutes, or more gradual. The complexation reaction rate may be temperature dependent. The rate of metal-agent complex formation may also be dependent on the rate at which the externally added agent crosses the lipid bilayer of the liposome. As will be appreciated by those of skill in the art, these variables can be adjusted as desired to achieve a desired reaction rate for the complexation reaction.
Although metal-agent complexes incorporated within a liposome are described, other embodiments encompass formulations for ICD induction in which copper is administered to a patient separately from the liposomal formulation comprising the agent.
According to such embodiments, the copper is typically administered separately or together with the liposomal formulation. In one embodiment, the copper is administered as a copper chelate incorporated in a liposome. Typically, a copper chelate is administered by injection. Examples of copper chelates include Cu(ATSM) and Cu(GTSM). In another embodiment, the copper is formulated as a salt. For example, copper gluconate may be administered orally. The agent may be administered as a liposomal formulation before, during or after administration of the free copper.
In such embodiments, the agent may be loaded into the liposome using active loading methods, such as pH gradient loading, or passive loading methods as known to those of ordinary skill in the art. After administration, the free copper may complex with the agent.
In a further embodiment, a copper-containing liposome is administered separately or together with one or more agents in free form. Likewise, after administration, the copper may complex with the agent in free form.
Combinations of anticancer agents and metal ions for inducing ICD
According to certain features of select embodiments, one or more additional agents may be used to induce ICP as described herein. The combinations of two or more drugs may exhibit increased ATP secretion, HMBG1 release and/or CRT expression relative to treatment with one agent alone and/or complexed with metal. The one or more additional agent may also complex with the metal or may be present in free form. These additional agents may be selected from antineoplastic agents such as cytotoxic agents, including nucleoside analogues, anthracyclines, anti-folates, topoisomerase I inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTor inhibitors, retinoids, immunomodulatory agents and histone deacetylase inhibitors. In one embodiment, a second agent suitable for use in certain embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone, 4' (4-chlorophenyl) ¨ 2,2':6',2"-terpyridine (4CPT), ¨ terpyridine (UT), shikonin and wogonin.
In a further embodiment, one or more agents complexed with a metal may be used to treat cancer in combination with an additional therapy such as radiation.
Advantageously, the methods described in select embodiments herein can be used to load multiple agents, either simultaneously or sequentially into a liposome. Each of the agents incorporated into the liposome can be loaded by the complexation method described herein.
Moreover, the liposomes into which the agent is loaded may themselves be prepared so that the internal solution comprises not only the metal ion but also an additional agent. Loading of an agent in this manner is often referred to as passive loading. The subsequent loading of a second agent which complexes with the metal in the pre-formed liposome (as described above) will result in incorporation of two agents in the liposome, one of which is loaded passively and the other actively via complexation. Since the passively loaded agent need not complex with a metal ion to effect loading, this approach provides greater flexibility in preparing liposome-encapsulated drug combinations for use to treat or prevent a disease of interest. Moreover, in certain embodiments, the two or more agents may be loaded at a predetermined ratio that exhibits synergistic or additive effects as elucidated by the Chou-Talalay determination, which is a known methodology for measuring such effects.
A formulation of liposomes may also comprise two or more liposome populations, which incorporate the same or different agents, comprise different lipid formulations, or comprise liposomes of different vesicle sizes. Moreover, one or more agents may be in free form, while one or more agents may be incorporated in a liposome. For example, the copper may be in free form and the agent or agents incorporated in a liposome. In another embodiment, the copper may be incorporated in a liposome and the agent or agents may be in free form in the pharmaceutical composition. The combinations of agents selected for the pharmaceutical formulation may achieve greater therapeutic efficacy, safety, prolonged drug release or targeting after administration.
According to one embodiment, a second agent in free form is included in a treatment regime such that the drug becomes active in the presence of the metal ion. Examples of such drug combinations include co-encapsulation of metal-CQ and free DSF, the precursor of DDC. The DSF is metabolized to form DDC and DDC and is subsequently activated in the presence of a metal ion, such as copper, at the tumour site.
Administration The pharmaceutical composition is generally administered to treat and/or prevent cancer, although other diseases are contemplated in which ICD induction by metal complexation provides a prophylactic or therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage. In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly.
In other embodiments, the pharmaceutical compositions described herein may be administered topically. In still further alternative embodiments, the pharmaceutical compositions described herein may be administered orally. In yet a further embodiment, the pharmaceutical compositions are for pulmonary administration by aerosol or powder dispersion.
The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.
The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.
EXAMPLES
Example 1: Extracellular ATP secretion of anti-cancer agents in the presence of copper This example shows that ATP secretion, which is a marker of immunogenic cell death, is enhanced when metal-binding compounds of interest are complexed with copper.
The results are shown in Figure 1.
CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, the cells were treated with the indicated agents, which included PX-478, Emodin, clioquinol (CQ), pyrithione (Pyr), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG). Where copper or zinc complexes were tested, the metal ion was added to the ligand at the indicated molar ratio such that the metal complexes were formed prior to addition to the cells. The cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was transferred to a new plate. The Promega CellTiter-Glo 2.0 Kit was then used immediately, as per the manufacturer's instructions, to determine the extracellular ATP level under each treatment condition.
The results of ATP secretion from CT26 cells for each anti-cancer agent in the presence and absence of copper sulphate or zinc sulphate are shown in Figures 1A-1H. Copper and zinc were mixed with the ligand at the indicated molar ratio (ligand:metal ion). As shown in Figure 1A, CuSO4 or ZnSO4 treatments alone at the highest tested concentration (100 pM) resulted in extracellular ATP levels comparable to the untreated control. The data in Figures 1B to 1H
demonstrate that treatment with ligands that can coordinate with metal ions yields increased ATP release when these ligands were complexed with Cu2+, but not with Zn2+. In all cases, the increase in extracellular ATP of the drug in the presence of Cu2+ was increased by a statistically significant amount relative to treatment of cells with drug alone.
Example 2: Combinations of anti-cancer agents with copper for increased ATP
secretion This example examines the effect of copper-binding anti-cancer agents (alone or complexed with copper) in combination with existing anti-cancer agents to enhance ATP
secretion, a marker of immunogenic cell death. The results are shown in Figure 2.
C126 cells were treated with anti-cancer agents of interest comprising a ligand or their corresponding copper complex as single agents or in combination with the clinically approved anti-cancer agents mitoxantrone (Mito; 1 pM), gemcitabine (Gem; 0.25 pM), or cisplatin (CDDP;
25 pM). The level of ATP secretion was determined by analyzing the supernatant at 24 hours following treatment. The anti-cancer agents with copper-binding ligands include PX-478 (25 pM), flavopiridol (FLV; 1pM), clioquinol (CQ; 25pM), and analogues of terpyridine 4,4'4"-Tri-ter-Buty1-2,2':6',2"-terpyridine (TTT; 25 pM) and 4'-(4-chloropheny1)-2,2':6',2"-terpyridine (CPT; 25 pM).
As shown in Figures 2A-F, the combination of the anti-cancer agent complexed with copper (PX-478, TTT, FLV, CQ and CPT) and the clinically approved agent (Mito, Gem and CDDP) yielded greater ATP release than the approved anti-cancer alone. The use of the copper complex instead of the anti-cancer agent alone, in some cases, leads to additional enhancement in ATP release.
Example 3: HMG131 release of anti-cancer combinations The effect of combining copper-binding agents with existing anti-cancer agents to increase HMGBi secretion from cells was next examined and the results are presented in Figure 3.
C126 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, cells were treated with the indicated anti-cancer agents. The release of HMGB, was assessed for the combination of PX-478 or Cu(PX-478) and gemcitabine (Gem) and the combination of flavopiridol (FLV) or Cu(FLV) with mitoxantrone (Mito). Where copper or zinc complexes were tested, the metal ion was added to the ligand at the indicated molar ratio such that the metal complexes were formed prior to addition to the cells. Cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL
International Tm.
Figure 3B shows that the use of flavopidirol, either in free form or as a copper complex, leads to greater HMGB, secretion compared to mitoxantrone alone. As seen in Figure 3A, combining PX-478 or Cu(PX-478) with gemcitabine led to a modest to comparable increase in HMGE31 release relative to gemcitabine alone.
Example 4: Methods for measuring CRT expression This example outlines a methodology for measuring CRT expression on the surface of cells using flow cytometric analysis. In this example, the cells were treated with mitoxantrone (Mito).
Following 24 hour treatment of CT26 cells with Mito (1 pM), the drug was removed and replaced with culture media. The cells were harvested at 0, 24, 48, and 96 hours post-treatment and stained with anti-CRT antibody followed by Alexa-488-conjugated secondary antibody.
Propidium iodide (PI) was used as a counterstain to differentiate between viable and dead cells.
The relative CRT mean fluorescence intensity (MFI) of P1-negative viable cells was determined by subtracting the MFI of isotype control-stained cells from the MFI of anti-CRT stained cells, and then normalising to HBSS - treated cells. The results are shown in Figure 4. As indicated, the CRT fluorescent intensity of cells treated with Mito increased with time post-treatment, whereas cells treated with HBSS did not exhibit any effect.

The insert of Figure 4 shows decreasing viability of Mito-treated cells at the indicated time points post-treatment.
In addition, an immunofluorescence assay was used to visualize cell surface expression of CRT
on CT26 cells treated with Mito (1pM) for 4 h and stained with anti-CRT
antibody that fluoresces as a green colour when visualized with an IN Cell Analyzer 2200TM (results not shown). Green stain was visible around the cell surface for Mito-treated cells whereas with HBSS treatment, no green stain was visible in the image.
The foregoing description should not be construed as limiting and includes embodiments and equivalents thereof that would be known to those of ordinary skill in the art.

Claims (19)

1. An immunostimulatory pharmaceutical composition to treat or prevent a disease by inducing immunogenic cell death comprising:
(i) copper and at least one agent that is capable of complexing with copper to form a metal complex; and (ii) wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, and wherein the increase in the ICD response of the combination as measured by extracellular ATP is 1.2 to 10,000 times that of the agent alone measured under otherwise identical conditions.

2. The pharmaceutical composition of claim 1, wherein the copper, the agent, or both, are formulated in a liposome.

3. The pharmaceutical composition of claim 1, wherein the copper is incorporated in the same or a different pre-formed liposome in the pharmaceutical composition as the agent.

4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a further, second agent.

5. The pharmaceutical composition of claim 1, 2 or 3, wherein the pharmaceutical composition provides a cytotoxic effect, or wherein the at least one agent or copper is cytotoxic in combination with radiation as measured by a cytotoxic assay.

6. Use of copper and at least one agent to induce immunogenic cell death (ICD), in combination or sequentially, to a patient in need thereof, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGB1 release, and CRT exposure of cells.

7. The use of claim 6, wherein the copper, the agent, or both, are formulated in a liposome.

8. The use of claim 6, wherein the copper and the agent are capable of inducing the ICD
response with a further, second agent.

9. The use of claim 6 or 7, wherein the copper and the at least one agent are capable of inducing immunogenic cell death with radiation.

10. The use of claim 6 or 7, wherein the increase in the ICD response in the presence of copper is measured by ATP secretion.

11. A method of inducing immunogenic cell death (ICD) comprising administering copper and at least one agent, in combination or sequentially, to a patient in need thereof, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGB1 release, and CRT exposure.

12. The method of claim 11, wherein the copper, the agent, or both, are formulated in a liposome.

13. The method of claim 11, further comprising a step of radiation treatment.

14. The method of claim 11 or 12, wherein the immunogenic cell death is measured by ATP
secretion.

15. A method of producing an immunostimulatory pharmaceutical composition for inducing immunogenic cell death (ICD), comprising the steps of:
(iv) exposing an agent and copper, alone and in combination, to an assay that measures at least one of ATP secretion, HMGB, release, and CRT exposure in cell culture;
(v) determining whether the agent exhibits an increase in ICD response in combination with the copper compared to results in which the ICD of the agent is measured alone; and (vi) wherein, if the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, formulating the agent, the copper, or both in one or more pharmaceutical formulations to treat or to prevent cancer, either sequentially or in combination, to a patient in need thereof.

16. The method of claim 15, wherein the pharmaceutical formulation comprises copper.

17. A kit comprising copper and an agent, to induce immunogenic cell death (ICD), wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures ATP secretion, HMGB, release, CRT exposure or a combination thereof.

18. The kit of claim 17, wherein the copper, the agent, or both, are formulated in a liposome.

19. The kit of claim 17, further comprising instructions for use thereof to induce immunogenic cell death to a patient in need thereof.