Composition and uses thereof

Abstract

The present invention relates, inter alia, to methods for the prevention or treatment of cancer, and to compositions, combinations and kits for the treatment or prevention of cancer. In one embodiment, the invention provides a method of treating or preventing cancer, comprising administering to a patient in need thereof an immune checkpoint modulator together with a compound of Formula (I): ##STR00001##
wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; and wherein at least 70% of the X groups is SO.sub.3M.

Claims

1. A method of treating or preventing cancer, comprising administering to a patient in need thereof an immune checkpoint inhibitor together with a compound of Formula (I): ##STR00018## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; wherein at least 70% of the X groups is SO.sub.3M; and wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

2. The method of claim 1, wherein at least 90% of the X groups is SO.sub.3M.

3. The method of claim 1, wherein the compound of Formula (I) is a compound of Formula (VI): ##STR00019##

4. The method of claim 1, wherein the immune checkpoint inhibitor targets one or more of a T cell, a Natural Killer (NK) cell, a Natural Killer T cell (NKT), a Gamma Delta T cell, an invariant T cell, or an invariant Natural Killer T cell (NKT).

5. The method of claim 1, wherein the immune checkpoint inhibitor is a T cell immune checkpoint inhibitor.

6. A method of treating or preventing cancer, comprising administering to a patient in need thereof Nivolumab together with a compound of Formula (I): ##STR00020## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; and wherein at least 70% of the X groups is SO.sub.3M.

7. The method of claim 1, wherein the cancer is a solid tumour.

8. The method of claim 3, wherein the method is a method of treating cancer.

9. A pharmaceutical composition or combination comprising a compound of Formula (I) and an immune checkpoint inhibitor, wherein the compound of Formula (I) is: ##STR00021## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; wherein at least 70% of the X groups is SO.sub.3M; and wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

10. A pharmaceutical composition or combination, comprising a compound of Formula (I) and Nivolumab, wherein the compound of Formula (I) is: ##STR00022## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; and wherein at least 70% of the X groups is SO.sub.3M.

11. A kit comprising a compound of Formula (I) and an immune checkpoint inhibitor, wherein the compound of Formula (I) is: ##STR00023## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; wherein at least 70% of the X groups is SO.sub.3M; and wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

12. The pharmaceutical composition or combination of claim 9, wherein at least 90% of the X groups is SO.sub.3M.

13. The pharmaceutical composition or combination of claim 9, wherein the compound of Formula (I) is a compound of Formula (VI): ##STR00024##

14. The kit of claim 11, wherein at least 90% of the X groups is SO.sub.3M.

15. The kit of claim 11, wherein the compound of Formula (I) is a compound of Formula (VI): ##STR00025##

16. A kit, comprising a compound of Formula (I) and Nivolumab, wherein the compound of Formula (I) is: ##STR00026## wherein X is SO.sub.3M or H, wherein M is any pharmaceutically acceptable cation; and wherein at least 70% of the X groups is SO.sub.3M.

17. The method of claim 1, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

18. The pharmaceutical composition or combination of claim 9, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

19. The kit of claim 11, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) Examples of the invention will now be described by way of example with reference to the accompanying Figures, in which:

(2) FIG. 1 shows the effect of treatments on 4T1.2 tumour bearing mouse body weight. PG545 was administered intraperitoneally on days 1, 8 and 15 (vertical lines) and antibodies on days 1, 4, 8, 11 and 15;

(3) FIG. 2 shows the anti-tumour activity of PD-1 antibody and PG545 alone and in combination against 4T1.2 tumours in vivo;

(4) FIG. 3 shows the mean tumour volume and mean weight change of the mice over the course of the study of Example 2;

(5) FIG. 4 shows a flow cytometric analysis of the bulk tumour cultures from each treatment group;

(6) FIG. 5 shows therapy-induced changes in 4T1.2 tumour and spleen associated CD8 T cell frequency;

(7) FIGS. 6A and 6B shows the gating strategy for analysing the CD8 T responses to therapy in the 4T1.2 mammary tumours;

(8) FIG. 7 shows histograms demonstrating the granzyme B status for both the tumour-associated effector and central memory CD8 T cell populations in each treatment group;

(9) FIG. 8 shows therapy-induced changes in 4T1.2 tumour and spleen associated CD4 T cell frequency;

(10) FIG. 9 shows flow cytometric analysis demonstrating the tumour-associated changes in the CD4 T cell compartment within each treatment group (concatenated results shown for each treatment group (4 tumours/group));

(11) FIG. 10 shows therapy-induced changes in 4T1.2 tumour and spleen associated NK cell frequency;

(12) FIGS. 11A and 11B shows the gating strategy for analysing the NK cell responses to therapy in the 4T1.2 mammary tumours;

(13) FIG. 12 shows therapy-induced changes in 4T1.2 tumour myeloid cell frequency;

(14) FIG. 13A shows the gating strategy for analysing the myeloid cell responses to therapy (Group 1—Vehicle+Control antibody) in the 4T1.2 mammary tumours;

(15) FIG. 13B shows the gating strategy for analysing the myeloid cell responses to therapy (Group 2—PG545+Control antibody) in the 4T1.2 mammary tumours;

(16) FIG. 13C shows the gating strategy for analysing the myeloid cell responses to therapy (Group 3—Vehicle+Anti-PD-1) in the 4T1.2 mammary tumours;

(17) FIG. 13D shows the gating strategy for analysing the myeloid cell responses to therapy (Group 4—PG545+Anti-PD-1) in the 4T1.2 mammary tumours;

(18) FIG. 14 shows therapy-induced changes in PD-L1 expression on 4T1.2 tumour associated myeloid cells;

(19) FIG. 15 shows results from administration of PG545 and nivolumab in a first human patient with microsatellite stable colorectal cancer; and

(20) FIG. 16 shows results from administration of PG545 and nivolumab in a second human patient with microsatellite stable colorectal cancer.

(21) Preferred features, embodiments and variations of the invention may be discerned from the following Examples which provides sufficient information for those skilled in the art to perform the invention. The following Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.

EXAMPLES

(22) Examples of the present invention will now be described with reference to FIGS. 1 to 14.

(23) Compounds Used

(24) PG545 (or 3β,5α-Cholestanyl 2,3,4,6-tetra-O-sodium sulfonato-α-D-glucopyranosyl-(1.fwdarw.4)-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl-(1.fwdarw.4)-2,3,6-tri-O-sodium sulfonato-α-D-glucopyranosyl-(1.fwdarw.4)-2,3,6-tri-O-sodium sulfonato-β-D-glucopyranoside), has the structure below. PG545 has been allocated a proposed International Nonproprietary Name (INN) by the World Health Organization of pixatimod (for the acid). This compound is described in WO2009/049370. PG545 may be synthesised as provided in Ferro et al 2012 or in WO2009/049370.

(25) ##STR00017##

(26) Anti-PD-1 antibody (RMP1-14) was purchased from Bio-X-Cell (NH, USA).

(27) Isotype Control antibody (2A3) was purchased from Bio-X-Cell (NH, USA).

Example 1—Study in the 4T1.2 Tumour Model

(28) Female Balb/c mice (Age 8 weeks; Walter and Eliza Hall Institute of Medical Research (WEHI), Victoria Australia) were inoculated in the intramammary fatpad with 1×10.sup.5 4T1.2 cells in phosphate buffered saline. 4T1.2 is a mouse mammary tumour model.

(29) Mice were weighed and tumours measured 2-3 times weekly using electronic callipers. Tumour volume (mm.sup.3) was calculated as length (mm)/2×width (mm).sup.2. One week following implantation mice with similar sized tumours (mean tumour volume 56 mm.sup.3) were randomised into 4 groups of 6 animals (Day 1). Treatment groups were saline+control antibody, PG545+control antibody, saline+anti-PD-1 and PG545+anti-PD-1.

(30) PG545 was given at 15 mg/kg weekly (0.1 ml/10 g body weight) for 3 weeks and anti-PD-1 or control antibody (200 μg; 100 μL of a 2 mg/mL solution) was given intraperitoneally on days 1, 4, 8, 11 and 15. Mice received a small dish containing a food supplement (Ensure mixed with food dust) daily. A dosage of 15 mg/kg PG545 was selected in view of the body weight issues observed at 20 mg/kg PG545 in these mice (i.e. issues with maximum tolerated dose at 20 mg/kg PG545. The maximum tolerated drug dose (MTD) is defined as that causing 10% body weight loss and from which the animals recovered weight to baseline levels within 7-10 days).

(31) The experiment was ended on day 18 or earlier if an ethical endpoint was met. Blood was collected via cardiac bleed and serum was prepared and frozen at −80° C.

(32) The mean body weight changes following the treatments are shown in FIG. 1. In FIG. 1, the data represent the mean percent weight change from day 1 for each group; bars are standard error of the mean (SEM). At day 15 one mouse in the saline+control antibody group was found dead. At day 18: (i) in the PG545+anti-PD-1 group one mouse was found dead, and the remaining mice showed signs of distress (ruffled fur); and (ii) in the PG545+control antibody group four of the six mice were bloated and showed signs of distress (ruffled fur). It was decided to harvest the experiment on day 18.

(33) The percentage tumour growth inhibition was determined according to the following formula: 100×(1−ΔT/ΔC) where ΔC and ΔT were calculated by subtracting the mean tumour volume in each group on day 1 of treatment from the mean tumour volume on the day of analysis. Statistical analysis of the in vivo data was performed using Graph Pad Prism Version 6.0 (Graph Pad La Jolla, Calif.). An ANOVA analysis was performed followed by Dunnett's post hoc test to compare the tumour growth in the treated groups to the vehicle control.

(34) FIG. 2 summarises the effect of the treatment on 4T1.2 tumour growth. Tumour growth in the PG545+control antibody, saline (or vehicle)+anti-PD-1 and PG545+anti-PD-1 groups was inhibited by 68%, 44% and 84%, respectively on day 18. Tumour growth in the PG545+control antibody and PG545+anti-PD-1 groups was significantly inhibited compared to the saline (or vehicle)+control antibody group (P<0.01 for both). No significant differences were seen between any other groups. In FIG. 2, tumour volumes are expressed as mean tumour volume (±SEM); **=P<0.01 compared to vehicle+control antibody.

Example 2—Study to Evaluate Tumour Infiltrating Leukocytes

(35) Sixteen mice from the cohort implanted in Example 1 above were randomised 1 week after inoculation into 4 groups of 4 mice as for Example 1 (mean tumour volume=77 mm.sup.3). PG545 was given intraperitoneally at 15 mg/kg (0.1 ml/10 g body weight) on days 1 and 8 with anti-PD-1 or control antibody (100 μl) given intraperitoneally on days 1, 4 and 8.

(36) On day 11, mice were euthanized and tumours harvested. The tumours were mechanically disaggregated and then digested in collagenase IV and DNase 1 before being filtered through a cell strainer to yield a single cell suspension. Spleens were also removed and used as background staining controls. Where appropriate, the cells were fixed and permeabilised [Ebioscience or BD Pharmingen Fixation/Permeabilisation Kits; also see the procedure provided in the technical data sheet for BD Cytofix/Cytoperm™ available from www.bdbiosciences.com]. The cells were then incubated with the antibody cocktails summarised in Table 2. Flow cytometry analysis was performed on the LSR II analyser (BD Biosciences).

(37) TABLE-US-00002 TABLE 2 Immune Staining Cocktails Stain 1 Stain 2 Stain 3 Stain 4 CD45.2- CD45.2-APC CD45.2-PEcy7 CD45.2- eFluor450 TCRb-PE CD49b-APC eFluor450 TCRb-APC-cy7 CD4- CD27-PE Ly6C-PEcy7 CD8b-PEcy7 eFluor450 CD11b-APCcy7 CD11b-APCcy7 CD44-FITC FoxP3-FITC CD69-FITC Ly6G-BV711 CD62L-BV510 CD8-PEcy7 CD335- CD11c-FITC CD69-PE NO DAPI eFluor450 MHCII-APC Granzyme B DAPI PD-L1-PE NO DAPI DAPI

(38) Staining Set Summary: Stain 1 (fixed group due to Granzyme B intracellular stain). Stopping gate for collection was set on CD45.2 for all groups to remove any bias associated with tumour size. Stain 2. Did not produce a clear data set and was excluded from further analysis. Stains 3 and 4. Stopping gate for collection was set on DAPI negative cells (viable cell collection date) for all groups to remove any bias associated with tumour size.

(39) In this study statistical analysis was performed using the Mann Whitney U Test.

(40) The mean tumour volume and body weight change of the mice in this study are provided in FIG. 3.

(41) A significant increase in lymphocyte frequency was observed within the PG545+anti-PD-1 treated tumours relative to the single agent and control treated tumours (FIG. 4). This increase in 4T1.2 tumour-associated lymphocyte frequency correlated with a reduction in CD45.2 negative cells in the PG545+anti-PD-1 treated tumours; highlighting the impact of the combination therapy on tumour and/or stromal cell viability. In FIG. 4, data shows the concatenated (merged) results from 4 mice in each group. Group 1=Vehicle (saline)+control antibody (CIg); Group 2=PG545+Control antibody; Group 3=Vehicle (saline)+anti-PD-1; Group 4=PG545+anti-PD-1. Row 1 shows the concatenated fluorescence-activated cell sorting (FACs) blots for CD45.2 versus FSC. Row 2 shows the concatenated FACs blots for SSC versus FSC (morphology plot) on the CD45+leukocyte gate.

(42) The increase in tumour-associated lymphocytes in response to PG545+anti-PD-1 treatment correlated with a significant increase in both CD8 (TCRb+CD8+) and CD4 (TCRb+CD8−) T cells (FIG. 5; FACS data collected for Stain 2 was deemed uninterruptible, so the treatment-associated effects on CD4+T effector cells and T regulatory cells was not accurately quantitated). The gating strategy used to analyse Stain 1 is shown in FIG. 6 (in which concatenated results are shown for each treatment group (4 tumours/group)). Notably these therapy-induced changes in T cell frequency were not observed in the spleen (FIG. 5); highlighting the tumour-specific nature of these effects. In FIG. 5, each symbol represents an individual tumour. The horizontal line shown in each treatment group represents the average frequency of tumour-associated CD8 T cells for the group of 4 mice. Spleens from each treatment group were combined for analysis. Each horizontal bar represents the average splenic associated CD8 T cell frequency for each treatment group.

(43) Within the tumour-associated CD8+ T cell compartment, a significant increase in the frequency of Central Memory CD8.sup.− T cells (CD62L+CD44+ cells) was observed (FIG. 5). Within the PG545+anti-PD-1 treated tumours the frequency of Effector Memory CD8+ T cells was elevated relative to the control and anti-PD-1 treated groups (FIG. 5). However, the frequency of CD69+Granzyme B+Effector CD8+ T cells appeared comparable across the four treatment groups (FIGS. 6 and 7). In FIG. 7, concatenated results are shown for each treatment group (four tumours/group).

(44) The frequency of tumour-associated CD4.sup.+CD44.sup.+CD62L.sup.+ and CD4.sup.+CD44.sup.+CD62L.sup.− T cells was elevated in the PG545+anti-PD-1 group compared to the single agent and control groups (FIG. 8, FIG. 9). These changes in the CD4 T cell compartment of the PG545+anti-PD-1 treated tumours were not evident within the spleens of these mice (FIG. 8), again highlighting the tumour-specific nature of these effects. In FIG. 8, each symbol represents an individual tumour. The horizontal line shown in each treatment group represents the average frequency of tumour-associated CD4 T cells for the group of four mice. Spleens from each treatment group were combined for analysis. Each horizontal bar represents the average splenic associated CD4 T cell frequency for each treatment group.

(45) NK cell frequency is low in the 4T1.2 tumours. Co-treatment with PG545+anti-PD-1 significantly increased the frequency of tumour-associated CD69.sup.+ NK cells compared to the single agent and control treatments (FIG. 10, FIGS. 11A-11B). Notably this effect of the PG545+anti-PD-1 treatment was not observed within the spleens of these mice. Therapy-associated changes in NK cell expression of CD335 expression were not detected (FIG. 10, FIGS. 11A-11B). In FIG. 10, each symbol represents an individual tumour. The horizontal line shown in each treatment group represents the average frequency of tumour-associated NK cells for the group of four mice. Spleens from each treatment group were combined for analysis. Each horizontal bar represents the average splenic associated NK cell frequency for each treatment group. In FIGS. 11A-11B, concatenated results are shown for each treatment group (four tumours/group).

(46) Drug-associated changes within the 4T1.2 tumour-associated myeloid populations were only observed in the neutrophil cell subset (Ly6C.sup.intCD11b.sup.+CD11.sup.−Ly6G.sup.+ cells) (FIG. 12; FIGS. 13A-13D (in which concatenated results are shown for each treatment group, four tumours/group)). Anti-PD-1 treatment was the primary mediator of the observed reduction in tumour-associated neutrophils (FIG. 12). In FIG. 12, each symbol represents an individual tumour and the horizontal line shown in each treatment group represents the average frequency of tumour-associated myeloid cells for the group of four mice.

(47) Drug-associated changes in PD-L1 expression within the 4T1.2 tumour-associated myeloid compartment were not detected (FIG. 14; FIGS. 13A-13D). In FIG. 14, each symbol represents the mean fluorescence intensity (MFI) for PD-L1 expression within an individual tumour. The horizontal line shown in each treatment group represents the average MFI for PD-L1 expression.

CONCLUSIONS

(48) The % TGI for the PG545 and anti-PD-1 group (84%) was almost two-fold higher compared with the vehicle+anti-PD1 antibody group (44%) and higher than the PG545+control antibody group (68%).

(49) Immune analysis revealed significant synergy of PG545 and anti-PD-1 in the resected 4T1.2 mammary tumours. Significant increases in tumour-associated lymphocytes were observed, with the accumulation of Effector Memory CD8+ T cells, Central Memory CD8+ T cells, and CD4+ T cells in the 4T1.2 tumours.

(50) Synergistic effects of PG545 and anti-PD-1 were also evident on the 4T1.2 tumour-associated NK cells. Significant increases in tumour associated NK cell frequency and CD69 expression within the NK cell compartment were observed in the PG545+anti-PD-1 treated 4T1.2 tumours.

(51) The PG545 and anti-PD1 antibody combination enhanced the TGI almost two-fold compared with anti-PD1 alone. Moreover, synergistic increases in tumour-associated CD8+T, CD4+T and NK cells were identified in the 4T1.2 tumours treated with both PG545 and anti-PD1 therapy.

Example 3—Human Colorectal Cancer Trial

(52) Five human patients with advanced solid tumours (microsatellite stable colorectal cancer (MSS CRC)) were treated with a combination of PG545 and nivolumab. These patients received a dose of 25 mg PG545 via a 1-hour intravenous infusion administered weekly, and 240 mg nivolumab via an intravenous infusion administered once every two weeks.

(53) The biological activity of the combination was assessed using RECIST v1.1 criteria and by analysing relevant biomarkers of immunomodulation in the peripheral blood. Subjects continued to be treated until they exhibited disease progression (increase of tumour size>20%), were discontinued for reasons of intolerability, or the study was terminated.

(54) The results from two of the patients are provided in FIGS. 15 and 16. These subjects responded to treatment with the combination of PG545 and nivolumab. Moreover, increases in the numbers of effector memory T cells (CD4 or CD8 T cells) were reported in the peripheral blood of these subjects which is consistent with the expansion of T cells reported in tumour-bearing mice (Hammond et al, 2018).

(55) In contrast with these results, a Phase Ia study in patients with advanced solid tumours identified the maximum tolerated dose of PG545 when administered as a once weekly IV infusion, namely 100 mg. However, in that study PG545 monotherapy did not lead to reductions in tumour size (Dredge et al, 2018). Furthermore, despite responses to PD-1 inhibition in microsatellite instability (MSI) high metastatic colorectal cancer (mCRC), no objective response (i.e. tumour shrinkage) has been observed in MSI-low or microsatellite stable (MSS) mCRC subject with nivolumab (Topalian et al. NEJM, 2012) or another PD-1 inhibitor called pembrolizumb (Le et al. Annals of Oncol, 2016). Therefore, the combination of PG545 and the PD-1 inhibitor nivolumab clearly has a synergistic effect on these tumours that could not have been expected based on the activity of either of PG545 or nivolumab alone.

(56) In the present specification and claims (if any), the word ‘comprising’ and its derivatives including ‘comprises’ and ‘comprise’ include each of the stated integers but does not exclude the inclusion of one or more further integers.

(57) Reference throughout this specification to ‘one embodiment’ or ‘an embodiment’ means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.

(58) In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims (if any) appropriately interpreted by those skilled in the art.

CITATION LIST

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