METHOD FOR TREATMENT OF CANCER

Abstract

A method of treating cancer is provided wherein the method comprises inhibiting or reducing sialylation of specifically Mesenchymal Stromal Cells (MSCs) to inhibit MSC immunosuppression and restore T cell proliferation in cancer. The method may comprise administering a sialyltransferase inhibitor (e.g. 3Fax-Peracetyl Neu5Ac) or sialidase. Also described is administration of MSCs which have been manipulated prior to administration to remove sialic acids and use of a small molecule or blocking antibody which blocks interactions between MSC-sialic acid and lectins, such as Siglec 7, and/or blocks interactions between MSC-lectins and sialic acid.

Claims

1-18. (canceled)

19. A method of treating cancer in a subject in need thereof, the method comprising inhibiting or reducing sialylation of specifically Mesenchymal Stromal Cells (MSCs) of the subject.

20. The method as claimed in claim 19 wherein inhibiting or reducing sialylation of specifically MSCs comprises administering a sialyltransferase inhibitor to the subject.

21. The method as claimed in claim 20 wherein the sialyltransferase inhibitor inhibits specifically sialyltransferases associated with or specific to MSCs.

22. The method as claimed in claim 20 wherein the inhibitor is targeted for delivery to specifically bone marrow microenvironment.

23. The method as claimed in claim 20 wherein the inhibitor is targeted for delivery to specifically MSCs.

24. The method as claimed in claim 20 wherein the inhibitor is conjugated to a monoclonal antibody that targets an antigen specific to the bone marrow microenvironment or MSCs.

25. The method as claimed in claim 24 wherein the inhibitor is a sialyltransferase inhibitor selected from the group consisting of (i) sialic acid analogs, (ii) CMP-sialic acid analogs, (iii) cytidine analogs, (iv) oligosaccharide derivatives, (v) aromatic compounds, (vi) flavonoids and (vii) lithocholic acid analogs.

26. The method as claimed in claim 25 wherein the inhibitor is a sialic acid analog.

27. The method as claimed in claim 26 wherein the sialyltransferase inhibitor is 3Fax-Peracetyl Neu5Ac.

28. The method as claimed in claim 19 wherein inhibiting or reducing sialylation of specifically MSCs comprises administering a sialidase to the subject wherein the sialidase is targeted for delivery to specifically bone marrow microenvironment or MSCs.

29. The method as claimed in claim 28 wherein the sialidase is targeted for delivery to specifically bone marrow microenvironment.

30. The method as claimed in claim 28 wherein the sialidase is targeted for delivery to specifically MSCs.

31. The method as claimed in any claim 28 wherein the sialidase is conjugated to a monoclonal antibody that targets an antigen specific to the bone marrow microenvironment or MSCs.

32. The method as claimed in claim 19 wherein inhibiting or reducing sialylation of specifically MSCs comprises administering MSCs to the subject wherein the MSCs have been manipulated to remove sialic acids prior to administration to the subject.

33. The method as claimed in claim 32 wherein manipulation of the MSCs to remove sialic acids comprises removing alpha 2,6 linked sialic acids.

34. A method of treating cancer in a subject in need thereof, the method comprising administering a small molecule or blocking antibody that blocks interaction between Mesenchymal Stromal Cell (MSC)-sialic acids and lectins and/or between MSC-lectins and sialic acid.

35. The method as claimed in claim 34 wherein the small molecule or blocking antibody blocks interactions between MSC-sialic acids and Siglec 7 and/or between MSC Siglec 7 and sialic acid.

36. The method as claimed in claim 34 wherein the small molecule comprises a sialic acid mimetic.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0028] The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the following figures in which:

[0029] FIGS. 1(a)-1(c) show sialic acid is upregulated in (a) a pro-inflammatory environment, (b) Multiple Myeloma and (c) colon cancer. CM=Multiple myeloma conditioned media. MWS=multiple myeloma conditioned media without fetal bovine serum. TCM=Colon cancer conditioned media. Statistics: Unpaired T test and one-way ANOVA where applicable P<0.05*<0.005**<0.0005***.

[0030] FIGS. 2(a)-2(f) show pre-treated MSCs have heightened T-cell immunosuppression. FIG. 2(a) shows CD4+ Lymphocytes, FIG. 2(b) shows total CD4+ proliferation, FIG. 2(c) shows CD8+ Lymphocytes, FIG. 2(d) shows total CD8+ proliferation, FIG. 2(e) shows levels of NaNO.sub.2, and FIG. 2(f) shows PGE2 levels.

[0031] FIGS. 3(a)-3(c) show inhibition of glycosylation impairs MSCs immunomodulation.

[0032] FIG. 3(a) shows CD4.sup.+ Lymphocytes, FIG. 3(b) shows total CD4.sup.+ proliferation,

[0033] FIG. 3(c) shows CD8.sup.+ Lymphocytes and FIG. 3(d) shows total CD8.sup.+ proliferation.

[0034] FIGS. 4(a)-4(c) show inhibition of glycosylation impairs MSCs immunomodulation, but that this is NO (FIG. 4(a)), IL-10 FIG. 4(b)) and PGE2 FIG. 4(c)) independent.

[0035] FIGS. 5(a)-5(d) show hMSCs express both Siglec 7 (FIG. 5(a)) and Siglec 9 (FIG. 5(b)) receptors. In a pro-inflammatory environment, Siglec 7 is increased (TNF-α+IL-1β) compared to unstimulated MSCs (FIG. 5(a)). The opposite is seen for Siglec 9 (FIG. 5(b)).

[0036] FIGS. 6(a)-6(b) show multiple myeloma Mesenchymal Stromal Cells (MM MSCs) have increased sialylation profiles compared to healthy MSC controls. The sialic acid profiles of both MM MSCs and healthy MSCs were analysed by flow cytometry.

[0037] FIGS. 7(a)-7(c) show MM MSCs have an increased ability to supress activated T lymphocytes. Both MM MSCs and healthy MSCs were placed into T lymphocyte co-cultures for 96 hours. FIG. 7(a) shows % CD4 proliferation, FIG. 7(b) shows CD4 Counts, FIG. 7(c) shows % CD8 proliferation, and FIG. 7(d) shows CD8 Counts.

[0038] FIGS. 8(a)-8(b) show MM MSCs have an increased ability to supress activated Macrophages. Both MM MSCs and healthy MSCs were placed into macrophage co-cultures for 72 hours in the presence of IFN-γ and LPS. MM MSCs had an increased ability to supress the activated phenotype of IFN-γ stimulated macrophages.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present inventors have identified that inhibition of sialylation of Mesenchymal Stromal Cells (MSCs) reduces the immunosuppressive properties of MSCs. The immunosuppressive component of MSCs is a common feature in cancer and the present invention therefore has broad applicability to cancer in general, effectively acting as a new checkpoint inhibitor.

[0040] Previous attempts focusing on sialylation in cancer were aimed at inhibiting sialylation of specifically tumour cells using, for example, labelled antibodies against tumour antigen. These attempts focused specifically on sialylation of tumour cells as off target toxicity was considered a problem. The present inventors have identified a link between sialylation in specifically MSCs and immunosuppression in cancer, resulting in treatments which target specifically MSCs. Selective delivery to the bone marrow or MSCs reduces the risk of off-target toxicity, in particular nephrotoxicity.

[0041] Inhibition of sialylation of MSCs is shown by the inventors to restore proliferation of T cells, thus overcoming an important immunosuppressive component of the tumour microenvironment in cancer. Without wishing to be bound by theory, a reduction in sialylation of MSCs may also polarize macrophages, which could have beneficial effects independent of T cell proliferation, such as on Natural Killer (NK) cells. For example, if macrophages express less PDL-1, then there may be less of an inhibitory effect on NK cells, which can express PD-1.

[0042] The inventors have also shown an upregulation of Siglec-7 on MSCs in an inflammation setting. Siglec-7 is known to play an important role in negative regulation of T cells (Ikehara Y, Ikehara S K, Paulson J C. Negative regulation of T cell receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9. J Biol Chem. 2004 Oct. 8; 279(41):43117-25. Epub 2004 Aug. 3. PubMed PMID: 15292262). This could be an important link between MSCs and T cells in inflammation.

[0043] Also, without wishing to be bound by theory, siglec ligands on MSCs could directly interact with siglecs (e.g. Siglec 7) on NK cells, causing inhibition of NK cells. Siglec-7 is also known to play an important role in inhibiting NK cells.

[0044] Methods for inhibiting sialylation are described in International Patent Publication No. WO 2008/087256 A1. This document describes specific sialylated structures present on human stem cells and cell populations derived thereof.

[0045] A subject in need thereof may be a subject who is suffering from cancer or a cancer patient.

[0046] Treatment (e.g. sialyltransferase inhibitor, sialidase, modified MSCs, small molecule or blocking antibody) may be combined with one or more standard cancer treatments. The treatment may be administered alone or may be administered as a pharmaceutical composition which will generally comprise a suitable pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutically acceptable excipient, diluent or carrier may be selected depending on the intended route of administration. Examples of suitable pharmaceutical carriers include water, glycerol and ethanol.

[0047] The treatment may be administered to a patient in need of treatment via any suitable route. The treatment may be administered parenterally by injection or infusion. Examples of preferred routes for parenteral administration include, but are not limited to, intravenous, intracardial, intraarterial, intraperitoneal, intramuscular, intracavity, subcutaneous, transmucosal, inhalation and transdermal. Routes of administration may further include topical and enteral, for example, mucosal (including pulmonary), oral, nasal and rectal. The treatment may also be administered via nanoparticles, microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.

[0048] The treatment is typically administered to a subject in a “therapeutically effective amount”, this being an amount sufficient to show benefit to the subject to whom the treatment is administered. The actual dose administered, and rate and time-course of administration, will depend on, and can be determined with due reference to, the nature and severity of the condition which is being treated, as well as factors such as the age, sex and weight of the subject being treated, as well as the route of administration. Further due consideration should be given to the properties of the treatment, for example, its in-vivo plasma life and concentration in the formulation, as well as the route, site and rate of delivery. Prescription of treatment, e.g. decisions on dosage, etc., is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

[0049] Dosage regimens can include a single administration, or multiple administrative doses. The treatment can further be administered simultaneously, sequentially or separately with other therapeutics and medicaments which are used for the treatment of the cancer for which the treatment is being administered.

Definitions

[0050] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

[0051] As used herein, the term “cancer” is understood to refer to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The cancer may, for example, be selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, stomach cancer, bowel cancer, breast cancer, oral cancer, pancreatic cancer and cervical cancer.

[0052] Typically the terms “subject” and “patient” are used interchangeably herein. The subject is typically a mammal, more typically a human.

[0053] The terms “inhibit” and “inhibiting” are used herein to refer to both partial inhibition (i.e. a reduction) and complete inhibition.

[0054] The term “inhibitor of sialylation” is used herein to refer to any compound or drug that inhibits or reduces sialylation by, for example, inhibiting or reducing addition of sialic acids (e.g. a sialyltransferase inhibitor) and/or promoting removal of sialic acids (e.g. sialidase).

[0055] References to “specifically Mesenchymal Stromal Cells (MSCs)”, “targeting specifically Mesenchymal Stromal Cells (MSCs)” or “targeting inhibition of sialylation of specifically MSCs” or similar are used herein to refer to the ability of the treatment (e.g. sialidase or sialyltransferase inhibitors) to inhibit or reduce sialylation of MSCs at a higher rate than sialylation of other cell types, including tumour cells, i.e. the treatment is aimed at reducing or inhibiting sialylation of primarily MSCs rather than sialylation of other cell types. In certain embodiments, there is also an effect on sialylation of other cell types, but this is lower than the effect on sialylation of MSCs, preferably significantly so. In alternative embodiments, the effect on sialylation is restricted to sialylation of MSCs. Similarly, references to “inhibiting specifically sialyltransferases associated with or specific to MSCs” or similar are used herein to refer to the ability of the treatment (e.g. sialyltransferase inhibitor) to inhibit sialyltransferases of MSCs at a higher rate than sialyltransferases of other cell types, including tumour cells, i.e. the treatment is aimed at reducing or inhibiting sialylation of primarily MSCs rather than sialylation of other cell types. In certain embodiments, there is also an effect on sialyltransferases of other cell types, but this is lower than the effect on sialyltransferases of MSCs, preferably significantly so. In alternative embodiments, the effect on sialyltransferases is restricted to sialyltransferases of MSCs.

[0056] The term “treatment” as used herein and associated terms such as “treat” and “treating” means the reduction of the progression, severity and/or duration of cancer or at least one symptom thereof. The term ‘treatment’ therefore refers to any regimen that can benefit a subject. Treatment may include curative, alleviative or prophylactic effects.

[0057] The term “bone marrow microenvironment” as used herein includes all the cellular and structural components of the bone marrow, including, but not limited to, fat cells, hematopoietic stem cells, progenitor cells and precursor cells.

[0058] The term “tumour microenvironment” as used herein refers to the cellular environment in which the tumour/cancerous cells exist.

[0059] The words “comprises/comprising” and the words “having/including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0060] As used herein, terms such as “a”, “an” and “the” include singular and plural referents unless the context clearly demands otherwise.

[0061] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Example—Inhibition of sialylation impairs MSCs immunosuppression

[0062] Materials and Methods Four female BALB/c at approximately 5 to 7 weeks were euthanized and the femur and tibia was dissected and placed in a 50 ml tube with media or PBS. Using a forceps and scissors the bones were cleaned to remove muscles and snipped on both ends to plunge out the marrow into a Petri dish containing MSC medium. A 30G needle fitted on a 1 ml syringe was used to plunge out the marrow and repeated several times till the bones appeared white. The marrow was passed through a 40 μm sieve attached to a 50 ml tube and the sieve was washed twice with medium. Medium was changed, and a cell count was performed. 10 μl cells was mixed with 40 μl PBS and 10 μl 4% acetic acid and cell count was performed. Based on the count approximately 35-50 million cells were seeded per T175 cm flask or 1 animal per flask. After isolation and culturing the MSCs were extensively characterized in vitro before and after treatment with TNF-α+IL-1β.

[0063] Human MSCs (hMSC) were isolated from the bone marrow of three human donors. Cells were seeded in T175 flasks at 5×10.sup.4 mononuclear cells/cm.sup.2. Cells were extensively characterised in vitro before use in experiments.

[0064] Sialic acid content was assayed using lectin coupled flow cytometry. Biotin labelled SNA-I and MAL-II were used to detect a 2-6 sialic acid and a 2-3 sialic acid respectively. Sialyltransferase inhibition was carried out using 100 μM of 3Fax-Peracetyl Neu5Ac. Cells were cultured in the presence of the inhibitor for two successive passages to ensure inhibition.

[0065] To determine if TNF-α+IL-1βMSC displayed enhanced immunoregulatory ability, they were co-cultured in mixed lymphocyte reactions (MLRs). MSC and TNF-α+IL-1βMSC were co-cultured at different MSC: T-cell ratios for 96 hrs. T-cell proliferation, activation, death and differentiation were determined by flow cytometry.

[0066] Both ELISA and the Griess assay were used to determine quantity of immunomodulatory molecules in the supernatant of mixed lymphocyte reaction experiments.

[0067] Results and Discussion

[0068] MSCs have increased T-cell immunosuppressive capacity when they are exposed to a pro-inflammatory microenvironment (FIGS. 1(a)-1(c)). This increased immunosuppressive potential is due to increases in secreted molecules such as nitric oxide (NO), prostaglandin E2 (PGE2), transforming growth factor beta 1 (TGF-β), interleukin 10 (IL-10) and cell surface programed death ligand 1 (PD-L1). In this study, the inventors investigated what role sialic acid plays in the immunosuppressive attributes of MSCs. To study what role sialic acid plays in MSC immunomodulation, the inventors used the sialyltransferase inhibitor 3Fax-Peracetyl Neu5Ac. This is a cell-permeable sialylic acid analog that is converted to a CMP-Neu5Ac and inhibits sialyltransferase. To stimulate the MSCs to be more immunosuppressive, the inventors mimicked the pro-inflammatory microenvironment by pre-treating the MSCs with the pro-inflammatory cytokines IL-1 and TNF-α, conditioning them with supernatants from multiple myeloma cell lines or conditioning them with supernatants from colon cancer cell lines. The inventors observed that sialic acid is increased under these conditions (FIGS. 1(a)-1(c)). The inventors also observed increased lymphocyte suppression from MSCs conditioned in these environments (FIGS. 2(a)-2(f)). The inventors tested the immunomodulatory capacity of MSCs treated with and without the inhibitor (FIGS. 3(a)-3(c)). TNF-α+IL-1β treated MSCs after being exposed to the sialyltransferase inhibitor were not as effective at suppressing activated T-cells (FIGS. 3(a)-3(c)). The inventors then looked to see if the restoration in proliferation was due to inhibition of one of the key proteins involved in MSC modulation (FIGS. 4(a)-4(c)). While the sialyltransferase inhibitor restored proliferation of T-cells, it seems to be independent of nitric oxide (NO), prostaglandin E2 (PGE2), and IL-10.

[0069] The Siglecs are a family of sialic-acid-binding immunoglobulin-like lectins that are thought to promote cell-cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. The inventors have shown for the first time that human MSCs (hMSCs) express both Siglec 7 and Siglec 9. Three human donors showed an increase in Siglec 7 after pro-inflammatory stimulus while Siglec 9 decreases (FIGS. 5(a)-5(d)). This reinforces the importance of sialic acid content in immunomodulation of immune cells by MSCs.

[0070] FIGS. 6(a)-6(b) show that MSCs isolated from myeloma bearing mice had increased sialic acid profiles compared to healthy controls, further supporting that sialic acid is a potential target for cancer treatment. FIGS. 7(a)-7(c) show that MM MSCs have an increased ability to supress activated T lymphocytes compared to healthy controls and FIGS. 8(a)-8(b) show that MM MSCs have an increased ability to supress activated Macrophages compared to healthy controls.