COMBINATION THERAPY COMPRISING A RADIOPHARMACEUTICAL AND A DNA-REPAIR INHIBITOR

Abstract

The present invention provides a method of combination therapy comprising administration of a tissue-targeting radio-pharmaceutical and a DNA-repair inhibitor. The method may be used in the treatment of hyperplastic or neoplastic disease, such as a carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer.

Claims

1. A method of combination therapy, comprising administering a) a tissue-targeting radiopharmaceutical, and b) a DNA-repair inhibitor.

2. The method of claim 1, wherein the tissue-targeting radiopharmaceutical comprises an alpha-emitter.

3. The method of claim 1, wherein the tissue-targeting radiopharmaceutical is a complex comprising the 4+ ion of an alpha-emitting thorium radionuclide such as Thorium-227.

4. The method of claim 1, wherein the tissue-targeting radiopharmaceutical is a targeted thorium conjugate (TTC).

5. The method of claim 1, wherein the tissue-targeting radiopharmaceutical comprises a tissue-targeting moiety selected from a monoclonal or polyclonal antibody, an antibody fragment (such as Fab, F(ab)2, Fab or scFv), a construct of such antibodies and/or fragments, a protein, a peptide or a peptidomimetic.

6. The method of claim 1, wherein the tissue-targeting radiopharmaceutical comprises a tissue-targeting moiety which has binding affinity for the CD22 receptor, FGFR2, Mesothelin, HER-2, PSMA or CD33.

7. The method of claim 1, wherein the DNA-repair inhibitor is an inhibitor of a protein selected from the group consisting of PARP1, ATR, ATM and DNA-PK.

8. The method of claim 1, wherein the DNA-repair inhibitor is selected from the group consisting of BAY1895344, olaparib, AZD0156 and VX984.

9. The method of claim 1, wherein the DNA-repair inhibitor is selected from a PI3k inhibitor, an EGFR inhibitor and/or antibody, an AKT inhibitor, an mTOR inhibitor, an MEK inhibitor, a WEE1 inhibitor, a Chk1 and/or Chk2 inhibitor, or a RAD51 inhibitor.

10. claim for the treatment of hyperplastic or neoplastic disease, The method of claim 1, for treatment of a hyperplastic or neoplastic disease in an animal in need thereof, comprising administering to the animal effective amounts of the tissue-targeting radiopharmaceutical and the DNA-repair inhibitor.

11. The method of claim 1, wherein the tissue-targeting radiopharmaceutical is administered at a dose level below the level required for a monotherapy response.

12. The method of claim 1, wherein the tissue-targeting radiopharmaceutical and the DNA-repair inhibitor are administered sequentially in either order.

13. The method of claim 1, wherein the tissue-targeting radiopharmaceutical is administered before the DNA-repair inhibitor.

14. The method of claim 1, wherein the DNA-repair inhibitor is administered at least 2 days after administration of the tissue-targeting radiopharmaceutical.

15. The method of claim 1, wherein the tissue-targeting radiopharmaceutical is administered at a dose of 20-200 kBq/kg.

16. The method of claim 1, wherein the tissue-targeting radiopharmaceutical comprises a peptide or protein tissue targeting moiety at a level of 0.02-1 mg/kg.

17. The method of claim 1, wherein the DNA-repair inhibitor is administered at a dose of 10-100 mg/kg.

18. The method of claim 1, wherein the DNA-repair inhibitor is administered over the course of at least 3 days.

19. The method of claim 10, comprising administering a) the tissue-targeting radiopharmaceutical, and b) the DNA-repair inhibitor, simultaneously or sequentially in either order.

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. A kit containing a tissue-targeting radiopharmaceutical and a DNA-repair inhibitor for simultaneous, separate or sequential use in the treatment of a hyperplastic or neoplastic disease.

25. (canceled)

26. (Canceled)

27. A kit comprising: a) a tissue-targeting radiopharmaceutical, and b) a DNA-repair inhibitor.

28. The method of claim 6, wherein the tissue-targeting radiopharmaceutical comprises a tissue-targeting moiety which has binding affinity for Mesothelin, FGFR2, HER-2 or CD33.

29. The method of claim 7, wherein the DNA-repair inhibitor is an inhibitor of ATR.

30. The method of claim 9, wherein the DNA-repair inhibitor is a PI3k inhibitor or an EGFR inhibitor and/or antibody.

31. The method of claim 10, wherein the hyperplastic or neoplastic disease is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, or mixed type cancer.

32. The method of claim 31, wherein the hyperplastic or neoplastic disease is Non-Hodgkin's Lymphoma, B-cell neoplasms, breast cancer, colorectal cancer, endometrial cancer, gastric cancer, acute myeloid leukemia, prostate cancer, brain cancer, mesothelioma, ovarian cancer, lung cancer or pancreatic cancer.

33. The kit according to claim 24, wherein the hyperplastic or neoplastic disease is a carcinoma, sarcoma, myeloma, leukemia, lymphoma, or mixed type cancer.

34. The kit according to claim 33, wherein the hyperplastic or neoplastic disease is Non-Hodgkin's Lymphoma, B-cell neoplasms, breast cancer, colorectal cancer, endometrial cancer, gastric cancer, acute myeloid leukemia, prostate cancer, brain cancer, mesothelioma, ovarian cancer, lung cancer or pancreatic cancer.

Description

DESCRIPTION OF FIGURES

[0112] FIG. 1 shows pathways for different DNA repair mechanisms.

[0113] FIG. 2 shows an illustration of an Isobologram.

[0114] FIG. 3 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+ATMi in Ovarian cancer cell line Ovcar-3.

[0115] FIG. 4 shows in vitro combination cytotoxicity assay results showing the synergistic effect of FGFR2-TTC+ATRi combination on KATO-III cancer cell line (Gastric cancer).

[0116] FIG. 5 shows in vitro combination cytotoxicity assay results showing the synergistic effect of FGFR2-TTC+ATRi combination on MFM-223 cancer cell line (Breast cancer).

[0117] FIG. 6 shows in vitro combination cytotoxicity assay results showing the synergistic effect of FGFR2-TTC+ATRi combination on SUM52PE cancer cell line (Breast cancer).

[0118] FIG. 7 shows in vitro combination cytotoxicity assay results showing the synergistic effect of Her2-TTC+ATRi combination on SK-OV-3 cancer cell line (Ovarian cancer).

[0119] FIG. 8 shows in vitro combination cytotoxicity assay results showing the synergistic effect of Her2-TTC+ATRi combination on BT-474 cancer cell line (Breast cancer).

[0120] FIG. 9 shows in vitro combination cytotoxicity assay results showing the synergistic effect of Her2-TTC+ATRi combination on KPL-4 cancer cell line (Breast cancer).

[0121] FIG. 10 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+ATRi in Ovarian cancer cell line Ovcar-3.

[0122] FIG. 11 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+ATRi in lung cancer cell line NCI-H226.

[0123] FIG. 12 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+ATRi in colorectal cancer cell line HT29-Meso.

[0124] FIG. 13 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+DNA-PKi in Ovarian cancer cell line Ovcar-3.

[0125] FIG. 14 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+DNA-PKi in lung cancer cell line NCI-H226.

[0126] FIG. 15 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+DNA-PKi in colorectal cancer cell lines HT29-Meso.

[0127] FIG. 16 shows in vitro combination cytotoxicity assay results showing the synergistic effect of MSLN-TTC+PARPi (Olaparib) in Ovarian cancer cell line Ovcar-3.

[0128] FIG. 17 shows in vitro combination cytotoxicity assay results showing the synergistic effect of CD33-TTC+PARPi (Olaparib) in AML cell line HL-60.

[0129] FIG. 18 shows a schematic representation of the mode of action of DNA damage sensors.

[0130] FIG. 19 shows the suppression of TTC-induced ATR kinase signalling, seen by a reduction in phosphorylated Chk1.

[0131] FIG. 20 shows a DNA histogram of cell cycle analysis showing suppression of TTC-inducedG2/M arrest by ATRi.

[0132] FIG. 21 shows the measurement of double strand DNA breaks (y-H2AX).

[0133] FIG. 22 shows the in vivo efficacy study results showing the synergistic effect of MSLN-TTC+ATRi combination on Ovcar-3 xenograft (ovarian cancer).

[0134] FIG. 23 shows the in vivo efficacy study results showing the synergistic effect of FGFR2-TTC+ATRi combination on MFM-223 (TNBC) xenograft (breast cancer).

[0135] FIG. 24 shows a histogram showing the synergistic increase in cell death by MSLN-TTC+ATRi

[0136] FIG. 25 shows cells stained for cleaved Caspase (Green fluorescence, y-axis) and y-H2AX (Red fluorescence, x-axis)

[0137] FIG. 26 shows In vitro combination cytotoxicity assay results showing the synergistic effect of PSMA-TTC+ATRi (BAY1895344) in prostate cancer cell lines LNCaP-Luc.

[0138] FIG. 27 shows in vitro combination cytotoxicity assay results showing the synergistic effect of PSMA-TTC+PARPi (Olaparib) in prostate cancer cell lines C4-2.

EXAMPLES

Example 1-Combination Cytotoxicity

[0139] Methods

[0140] The in vitro combination studies were performed with either of the two experimental methods explained:

[0141] I. Combination setup in 96 well plates: [0142] 5-20 nM inhibitor was added to cells in 96 well plate [0143] Addition of TTC after 1 hour (titrated from 77 pM .sup.227Th; 20 kBq/ml) [0144] Incubated for 5-7 days [0145] Viability determined by CellTiter-Glo (ATP); luminescence based assay [0146] The data is plotted as % viability based on untreated control [0147] A significant decrease in viability by the combination compared to the TTC monotreatment is defined as synergy

[0148] II. Combination setup in 384 well plates/Isobologram setup

[0149] The assay evaluates the effect of the combination treatment by determining the shift in IC50 from curves established from different combination fractions [1] (see table 1). [0150] TTC and inhibitor was added to the cells in 384 well plate [0151] Incubated for 5-7 days [0152] Viability determined by CellTiter-Glo (ATP); luminescence based assay [0153] The data is plotted as % viability based on untreated control and IC50 values for the 11 curves are calculated. [0154] The IC50 values are plotted in an isobologram, with monotreatments along the y-axis and x-axis and the IC50 values from the combinations in between these two points (see FIG. 2). If the effect is additive a straight curve will be generated between the two monotreatment-IC50 values, if the effect is synergistic the line is below the straight line and antagonistic effect gives a curve over the straight line.

[0155] III. Combination setup in 6 well plates [0156] 5 nM inhibitor was added to cells in 6 well plate [0157] Addition of TTC after 2 hour (5-20 kBq/ml) [0158] Incubated for 5-7 days [0159] Viability determined by CellTiter-Glo (ATP); luminescence based assay [0160] The data is plotted as % viability based on untreated control [0161] A significant decrease in viability by the combination compared to the TTC monotreatment is defined as synergy

[0162] Results

[0163] A range of inhibitors have been tested in combination with TTCs in in vitro cytotoxicity assays (see table 2). The data indicates that the combination treatment results in a synergistic interaction covering a range of TTCs, inhibitor targets and cancer cell lines.

TABLE-US-00001 TABLE 2 Combination cytotoxicity assays Small molecule Cancer cell Combination TTC inhibitor lines Effect FIG.(S) MSLN-TTC ATM inhibitor OVCAR-3 Synergistic 3 FGFR2-TTC ATR inhibitor KATO-III, Synergistic 4, 5, (BAY1895344) MFM-223, 6 SUM52-PE Her2-TTC ATR inhibitor SK-OV-3, Synergistic 7, 8, 9 (BAY1895344) BT-474, KPL-4 MSLN-TTC ATR inhibitor OVCAR3, Synergistic 10, 11, (BAY1895344) NCI-H226, 12 HT29-Meso MSLN-TTC DNA-PK OVCAR3, Synergistic 13, 14, inhibitor NCI-H226, 15 HT29-Meso MSLN-TTC PARP inhibitor OVCAR3 Synergistic 16 CD33-TTC PARP inhibitor HL-60 Synergistic 17 PSMA-TTC ATR inhibitor LNCaP-Luc Synergistic 26 (BAY1895344) PSMA-TTC PARP inhibitor C4-2 Synergistic 27

Example 2-Cellular Mechanistic Assays

[0164] Methods

[0165] Cellular Mechanistic Assays

[0166] p-Chk1 (FIG. 19) and y-H2AX (FIG. 21): [0167] Seeded cells in 6 well plates and incubated with TTC+ATRi (BAY1895344) for 3 days [0168] Detached cells and washed two times with PBS [0169] Cells were fixed and permeabilized cells using 70% ice cold ethanol and incubated 1 hour at 4 C. [0170] Washed with PBS+1% FBS (flow buffer) and transfer to 96 well plate [0171] The cells were spun down and supernatant removed [0172] The cells were resuspended in 100 l anti-yH2AX-A647 antibody (1:50 in flow buffer) and anti-p-Chk1 antibody (1:100 in flow buffer) and incubated for 1 hour in the dark [0173] For cells stained with anti-p-Chk1 antibody: stained with secondary PE-antibody: 100 l per well with Anti-rabbit IgG PE (1:100 in flow buffer) and incubated in dark for 1 hour at 4 C. [0174] Washed two times with flow buffer and removed the supernatant [0175] Resuspended the cells in 200 l flow buffer and transferred to a u-shaped 96 well plate [0176] The plate was analysed by columns on the EasyCyte 8HT (log scale, medium flow rate).

[0177] Cell cycle analysis (DNA histogramFIG. 20): [0178] Seeded cells in 6 well plates and incubated with TTC+ATRi (BAY1895344) for 3 days [0179] Detached cells and washed two times with PBS [0180] Fixed and permeabilized cells using 70% ice cold ethanol and incubate 1 hour at 4 C. [0181] Washed cells with PBS+1% FBS and transfer to 96 well plate [0182] The cells were spun down and supernatant removed [0183] Resuspend the cells in 100 l PI/RNase and incubated for 30 minutes in the dark at 4 C. [0184] Analyse the plate by columns on the EasyCyte 8HT (linear scale, low flow rate).

[0185] Results

[0186] A schematic representation of the mode of action of DNA damage sensors is shown in FIG. 18. The mechanism of action for the combination of TTC and ATRi (BAY1895344) was explored by performing different experiments, including measurement of phosphorylated Chk1 (FIG. 18), cell cycle analysis (FIG. 19) and measurements of double strand DNA breaks (y-H2AX, FIG. 20). In short the data indicates that the combination with ATR inhibitor: [0187] Suppress TTC-induced ATR kinase signaling, seen by a reduction in phosphorylated Chk1 [0188] Suppress TTC induced G2-cell cycle arrest, seen by a shift in cell cycle distribution [0189] Suppress repair of double strand DNA break, seen by a higher degree of double strand DNA breaks compared to TTC monotreatment

[0190] Ultimately this leads to increased cell death by the combination treatment compared to the monotreatment. This can be explained by accumulation of DNA damage leading to mitotic catastrophe.

Example 3-In vivo, Efficacy Studies

[0191] The combination of TTC and ATRi (BAY1895344) was also evaluated in in vivo efficacy studies. Two different xenograft models were evaluated: [0192] Ovcar-3 xenograft in nude mice (FIG. 22)MSLN-positive ovarian cancer cell line, treated with MSLN-TTC in combination with ATRi (BAY1895344) [0193] MFM-223 xenograft in nude mice (FIG. 23)FGFR2-positive breast cancer cell line, treated with FGFR2-TTC in combination with ATRi (BAY1895344)

[0194] Methods

[0195] Ovcar-3 xenograft model (FIG. 22): [0196] At study day 0, animals received a subcutaneous inoculation of 510.sup.6 humane ovarian Ovcar-3 cells/mouse on the right flank. [0197] Upon reaching a palpable tumor size (20-25 mm.sup.2), test item MSLN-TTC (BAY2287411) was injected into the tail vein of the animals at 100 kBq/kg with a protein dose of 0.14 mg/kg. [0198] After initial dosing of MSLN-TTC the ATRi (BAY 1895344) was dosed orally in a cycle of 20 mg/kg twice per day in a row of three days, followed by 4 days off. The first treatment started 7 days after MSLN-TTC had been given and in total 4 cycles of ATRi were given. [0199] The tumor growth and the body weights were measured every other or third day. Upon reaching the humane endpoint, tumor volume >1500 mm.sup.3 or largest diameter of 15 mm, animals will euthanized upon cervical dislocation. Animals will be assessed for any major toxicological signs during necropsy. Major organs (including liver, lung, kidney, spleen and bone marrow) as well as organs with any observed abnormalities will be harvested, fixed and processed to histopathology to assess for histopathological changes due to treatment.

[0200] MFM-223 xenograft model (FIG. 23): [0201] At study day 0, animals received an orthotopic inoculation of 2.510.sup.6 MFM-223 cells/mouse into the upper right mammary fat pad. [0202] Upon reaching a palpable tumor size (30-35 mm.sup.2), test item FGFR2-TTC (BAY2304058) was injected into the tail vein of the animals at 100 kBq/kg with a protein dose of 0.14 mg/kg. [0203] After initial dosing of FGFR2-TTC the ATRi (BAY 1895344) was dosed orally in a cycle of 40 mg/kg twice per day in a row of three days, followed by 4 days off. The first treatment started 7 days after FGFR2-TTC had been given and in total 4 cycles of ATRi (BAY1895344) were given. [0204] The tumor growth and the body weights were measured every other day, on Monday, Wednesday and Friday. Upon reaching the humane endpoint, tumor volume >1500 mm.sup.3 or largest diameter of 15 mm, animals were euthanized upon cervical dislocation. Animals were assessed for any major toxicological signs during necropsy. Major organs (including liver, lung, kidney, spleen and bone marrow) as well as organs with any observed abnormalities will be harvested, fixed and processed to histopathology to assess for histopathological changes due to treatment.

[0205] Results

[0206] Both studies indicated that there was a synergistic effect by the combination of TTC and ATRi (BAY1895344). While no effect was shown for 100 kBq/kg dose level alone, when combined with ATR inhibitor, a significant tumor growth inhibition was observed.

REFERENCES

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