Method of treating metastatic triple negative breast cancer with radiotherapy combined with phosphatidyl inositol-3 kinase delta/gamma inhibitors and anti-PD-1 antibodies

Abstract

The present invention provides a pharmaceutical composition and a treatment method to be combined with radiotherapy, for treatment of triple negative breast cancer. More specifically, the pharmaceutical composition comprises a PD-1 blockade and a PI3Kγ δ inhibitor, and it has excellent effects of inhibiting tumor and enhancing immunity by combining the PD-1 blockade and PI3Kγ δ inhibitor with radiotherapy, compared to single therapy of each therapeutic agent.

Claims

1. A method of treating triple negative breast cancer, comprising administering a therapeutically effective amount of 1) a PI3K inhibitor against one or more selected from the group consisting of PI3K delta and PI3K gamma, and 2) a PD-1 blockade; and performing radiotherapy, wherein the PI3K inhibitor is one or more selected from the group consisting of duvelisib, tenalisib and idelalisib, wherein the PD-1 blockade is one or more selected from the group consisting of pembrolizumab and nivolumab, wherein the triple negative breast cancer is one selected from the group consisting of (i) metastatic tumor, (ii) metastatic tumor and primary tumor, (iii) metastatic tumor and recurrent tumor, and (iv) metastatic tumor, primary tumor and recurrent tumor.

2. The method according to claim 1, wherein the step of administering the PI3K inhibitor and the PD-1 blockade is performed by mixing the PI3K inhibitor and the PD-1 blockade and administering them together, or by administering the PI3K inhibitor and the PD-1 blockade simultaneously or sequentially.

3. The method according to claim 1, wherein the radiotherapy is performed at a dosage of 8 to 12 Gy in 1 to 5 times fractions (Fx), at an irradiation interval of 1 to 3 days.

4. The method according to claim 1, wherein the method reduces the tumor volume of a subject to 0.25 times or less of the tumor volume in a no-treatment control group.

5. The method according to claim 1, wherein the method increases the ratio of CD8 positive cells in tumor tissue to 2 times to 100 times compared to a no-treatment control group.

6. The method according to claim 1, wherein the method of treatment reduces the ratio of regulatory T cells (T.sub.reg) in tumor tissue to 60% or less than a no-treatment control group, and the ratio of myeloid-derived suppressor cells (MDSC) to 90% or less than a no-treatment control group.

7. The method according to claim 1, wherein the method of treatment reduces the ratio of M2 tumor-associated macrophages (M2 TAM) in tumor tissue to 50% or less than a no-treatment control group, and increases the ratio of M1 tumor-associated macrophages (M1 TAM) to 1.1 to 3 times compared to a no-treatment control group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1a is a graph showing the volume change of tumor according to each treatment method in the triple negative breast cancer allograft mouse model.

(2) FIG. 1b is a graph showing the survival rate according to each treatment method in the triple negative breast cancer allograft mouse model.

(3) FIG. 2a to FIG. 2c are graphs showing the FACS analysis result for spleen tissue, and the ratios of the regulatory T cells (FIG. 2a); polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC, FIG. 2b); and CD8 positive T cells (FIG. 2c), analyzed from the FACS result.

(4) FIG. 3a to FIG. 3c are graphs showing the FACS analysis results for tumor draining lymph nodes, and the ratios of the regulatory T cells (FIG. 3a), myeloid-derived suppressor cells (PMN-MDSC, FIG. 3b); and CD8 positive T cells (FIG. 3c), analyzed from the FACS results.

(5) FIG. 4a to FIG. 4c are graphs showing the FACS analysis results for tumor microenvironment (tumor tissue), and the ratios of the regulatory T cells (FIG. 4a); myeloid-derived suppressor cells (PMN-MDSC and M-MDSC, FIG. 4b); and CD8 positive T cells (FIG. 4c), analyzed from the FACS results.

(6) FIG. 5 is the FACS analysis results for tumor microenvironment (tumor tissue) and the result of performing M1 or M2 TAMs therefrom.

(7) FIG. 6a to FIG. 6c are showing the imaging result (FIG. 6a); a luminescence graph for primary tumor (FIG. 6b); and a luminescence graph for secondary tumor (FIG. 6c).

(8) FIG. 7a to FIG. 7b are showing the macroscopic images of primary and secondary tumor of mouse model (FIG. 7a) and nude mouse model (FIG. 7b).

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) Hereinafter, the contents of the present invention will be described in more detail by examples. However, the scope of the present invention is not limited by the following examples.

Example 1. Cell Culture and Preparation of Allograft Mouse Model

(10) 1-1. Culture of Breast Cancer Cell Line

(11) A triple negative breast cancer cell line (4T1-luc) was cultured in DMEM (Dulbecco's Modified Eagle's Medium, CORNING™) medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) Penicillin-Streptomycin, using a cell culture flask, in a thermostat cell culture device under the condition of 37° C., 5% CO.sub.2.

(12) 1-2. Preparation of Allograft Mouse Model

(13) 6×10.sup.5 4T1-luc triple negative breast cancer cells prepared in Example 1-1 were injected to the right hindlimb of a BALB/c female age standard (8 weeks) mice. At the 10th day after injecting each of the cancer cells, the mice with the tumor volume up to 40 mm.sup.3 were selected, and were used for later experiments.

Example 2. Measurement of Size Change of Tumor and Survival Rate According to Treatment Methods

(14) 2-1. Measurement of Size Change of Tumor

(15) The triple negative breast cancer (TNBC) mouse model prepared by the method of Example 1-2 was assigned to 7 groups in total from the 10th day after cancer cell transplantation, and each treatment method was performed, and it was grouped into 7 groups so that the tumor mean volume of 7 mice was 40 mm.sup.3 per group, and were used for later experiments.

(16) The 7 groups were composed of control group (no-treatment group), radiotherapy group, PI3Kγ δ inhibitor (duvelisib) treatment group, combined treatment group of PI3Kγ δ inhibitor and radiotherapy, PD-1 blockade treatment group, combined treatment group of PD-1 blockade and radiotherapy, and triple combined treatment group of PI3Kγ δ inhibitor, PD-1 blockade and radiotherapy, respectively. Each treatment was progressed for 31 days after tumor transplantation.

(17) The radiotherapy used electron beam, and 24 Gy in total was irradiated in 3 fractions, once per 2 days or 3 days (Monday, Wednesday and Friday), for 1 week (8 Gy×3). The PD-1 blockade and the PI3Kγ δ inhibitor (duvelisib) were administered by injection at doses of 10 mg/kg and 4 mg/kg, respectively, 6 times in total, once per 2 days or 3 days (Monday, Wednesday and Friday), over 2 weeks in total. On the day where the radiotherapy and drug administration overlapped, the drug was administered after confirming that the mice recovered from anesthesia in 4 hours after radiotherapy.

(18) On the day of each treatment, the size of tumor, the length and width were measured using Vernier caliper, and then the volume (mm.sup.3) of tumor was calculated by the method of the following equation 1.
Volume (mm.sup.3)=length (mm)×width (mm).sup.2×0.5  [Equation 1]

(19) FIG. 1a showed the graph of the tumor volume change over time according to each treatment method, and the following Table 1 showed the mean value of the tumor volume of each treatment group at the 31.sup.st day of the experiment.

(20) TABLE-US-00001 TABLE 1 Mean tumor volume Classification (mm.sup.3) Control group (no-treatment) 1383.08 RT 949.64 PI3K γ δ inhibitor 786.59 PI3K γ δ inhibitor + RT 311.05 PD-1 blockade 1107.81 PD-1 blockade + RT 483.15 PI3K γ δ inhibitor + 143.30 PD-1 blockade + RT

(21) As could be confirmed in the Table 1 and FIG. 1, the excellent inhibitory effect of tumor growth was confirmed compared to dual combined therapy, as the tumor volume was about 143.30 mm.sup.3 in case of the triple combined therapy of the PI3Kγ δ inhibitor, PD-1 blockade and RT inhibitor. More specifically, this final tumor volume was just a size of 0.10 times compared to the control group, 0.15 times compared to the single treatment of radiotherapy, 0.18 times compared to the single treatment of the PI3Kγ δ inhibitor, 0.13 times compared to the PD-1 single treatment group, 0.46 times compared to the combination of the PI3Kγ δ inhibitor and radiotherapy, and 0.30 times compared to the combination of the PD-1 blockade and radiotherapy, and thus it was confirmed that the inhibitory effect of the pharmaceutical composition comprising the PD-1 blockade and PI3Kγ δ inhibitor combined with radiotherapy, provided by the present invention, was very excellent.

(22) 2-2. Survival Rate According to Treatment Methods

(23) While performing the Example 2-1, the survival rate of each group was measured. For survival studies, mice were culled when tumors reached an average volume of 1000 mm.sup.3 or they died.

(24) FIG. 1B showed the graph of the survival rate over time according to each treatment method.

(25) Over a period of more than 42 days, the survival rate was not decreased in case of the triple combined therapy of the PI3Kγ δ inhibitor, PD-1 blockade and RT inhibitor, and increase of the survival rate was statistically significant.

Example 3. Confirmation of Immunoregulatory Effect Using FACS

(26) After completing the experiment of Example 2, tumor and spleen, and tumor draining lymph nodes were extracted from each mouse. For the tumor draining lymph nodes, inguinal lymph nodes which were the closest to the hindlimb calf site in which tumor was seeded were extracted. Then, each tissue was subject to single cell isolation, and thereafter, the immunoregulatory effect was confirmed using flow cytometric analysis (FACS).

(27) More specifically, on the 31th day after tumor cell injection, each mouse was administered euthanasia, and then the spleen and tumor were extracted, respectively.

(28) The tumor tissue was excised and subjected to fine chopping mechanical processing using a sterilized razor blade, and was treated with DNase 0.1 mg/ml and collagenase 1 mg/ml, respectively, and reacted at 36° C. for 30 minutes to obtain a single cell suspension.

(29) The spleen tissue was excised and subjected to the mechanical processing by the same method as the tumor tissue, and then red blood cells were lysed by single cell suspension method (See Standard Recommended Sample Preparation Procedures of Thermofisher company) and the single cell isolation was completed.

(30) After completing the single cell isolation from the tumor tissue and spleen tissue, to analyze infiltrated leukocytes, 1×10.sup.6 cells per FACS tube were stained by FITC, PE, PerCP/Cy5.5, APC fluorescence, using CD3 antibody, CD8b antibody, Ly6G antibody, CD11b antibody, CD25 antibody, CD4 antibody, Ly6G antibody, CD25 antibody, Ly6C antibody, CD8a antibody, CD25 antibody, CD127 antibody and CD206 antibody, respectively.

(31) Staining of each cell surface marker was conducted by reacting on ice for 30 minutes, and intracellular FOXP3 staining was progressed by the manual of eBioscience company.

(32) For samples that finished each staining, using BD Bioscience FACSCALIBUR machines, fractions of regulatory T cells (T.sub.regs or T.sub.reg), myeloid-derived suppressor cells (MDSCs), M1 or M2 tumor-associated macrophages (TAMs: M1 TAM or M2 TAM), and CD8.sup.+ cytotoxic T cells, from the tumor microenvironment and spleen were calculated respectively. The analysis of FACS results used FLOWJO software, version 10, and the results are shown in FIG. 2a to FIG. 4c. Specifically, FIG. 2a to FIG. 2c represent the FACS analysis results in the spleen tissue, and FIG. 3a to FIG. 3c represent the FACS analysis results in the tumor draining lymph nodes, and FIG. 4a to FIG. 5 represent the FACS analysis results in the tumor tissue (tumor microenvironment).

(33) The composition change of immunocytes in the draining lymph nodes and the spleen is a prerequisite for treatment to lead to adaptive resistance to inhibit tumor elsewhere. The change of immunocytes in the tumor draining lymph nodes (FIG. 3a to FIG. 3b) and spleen (FIG. 2a to FIG. 2b) confirmed in FIG. 2a to FIG. 4c, demonstrated that the combination of the pharmaceutical composition and radiotherapy provided by the present invention could go beyond the role of enhancing immunity in the tumor microenvironment of the simple primary anatomical location or anti-tumor inhibition of immunocytes, and generate adaptive resistance in order to have a function of suppressing tumor formation even for metastasis of other anatomical location other than the primary site or recurrent tumor.

(34) The ratios of T.sub.reg, MDSC(PMN_MDSC) and CD8+ T ells in the spleen and tumor draining lymph nodes and tumor tissue are shown in the following Tables 2 to 4.

(35) TABLE-US-00002 TABLE 2 T.sub.reg ratio (%) Tumor draining Classification Spleen lymph nodes Tumor Control group 6.46 5.43 12.62 RT 16.77 7.36 16.23 PI3K γ δ inhibitor 2.93 2.33 3.63 PI3K γ δ inhibitor + RT 3.64 2.61 7.15 PD-1 blockade 5.85 5.69 8.27 PD-1 blockade + RT 15.95 6.46 11.51 PI3K γ δ inhibitor + PD-1 2.69 1.89 4.25 blockade + RT

(36) TABLE-US-00003 TABLE 3 PMN-MDSC ratio (%) Tumor draining Classification Spleen lymph nodes Tumor Control group 58.70 3.63 26.55 RT 63.37 3.30 30.67 PI3K γ δ inhibitor 37.33 2.01 19.20 PI3K γ δ inhibitor + RT 36.75 2.77 21.98 PD-1 blockade 62.82 3.86 25.88 PD-1 blockade + RT 65.70 3.32 27.75 PI3K γ δ inhibitor + PD-1 37.63 2.00 19.48 blockade + RT

(37) TABLE-US-00004 TABLE 4 CD8.sup.+ T cell ratio (%) Tumor draining Classification Spleen lymph nodes Tumor Control group 13.87 15.93 1.64 RT 25.37 22.87 10.10 PI3K γ δ inhibitor 14.00 19.20 4.85 PI3K γ δ inhibitor + RT 22.02 22.33 11.23 PD-1 blockade 20.62 16.15 3.33 PD-1 blockade + RT 23.40 25.90 12.13 PI3K γ δ inhibitor + PD-1 25.75 28.60 27.17 blockade + RT

(38) As could be confirmed in FIG. 5, when combining radiotherapy and the PD-1 blockade and PI3K inhibitor, the ratio of M1 TAM was the highest compared to radiotherapy or single or dual combination of each inhibitor, and the M1 TAM ratio compared to the control group (no-treatment group) increased about 1.8 times. M2 TAM showed about ⅓ of M2 TAM ratio in case of the triple combination compared to the control group, and showed the significantly low M2 TAM ratio compared to radiotherapy or single or dual combination of each inhibitor (Table 5).

(39) TABLE-US-00005 TABLE 5 M1 TAM ratio M2 TAM ratio Classification (%) (%) Control group 30.27 4.68 RT 35.72 5.22 PI3K γ δ inhibitor 40.98 2.43 PI3K γ δ inhibitor + RT 27.18 3.20 PD-1 blockade 26.13 4.49 PD-1 blockade + RT 31.27 6.75 PI3K γ δ inhibitor + PD-1 53.92 1.50 blockade + RT

(40) M1 TAM inhibits tumor in the tumor microenvironment by secreting proinflammatory cytokines on the contrary to M2 TAM. Accordingly, as could be confirmed in the Table 5 and FIG. 5, it was confirmed that the immunotherapy efficiency of the pharmaceutical composition with the combined treatment of radiotherapy, comprising the PI3K inhibitor and PD-1 blockade of the present invention, in which M1 TAM widely increased and M2 TAM significantly decreased, was excellent.

Example 4. Confirmation of Increase in Abscopal Effect

(41) 4-1. Bioluminescence Imaging

(42) An animal model was prepared by injecting tumor cells to the right hindlimb in the same manner as in Example 1-2, and transplanting secondary tumor to the left flank. The animal model was grouped into 7 groups and treated in the same manner as Example 2-1. However, the radiotherapy was performed only for the right hindlimb.

(43) Bioluminescence images were obtained using the IVIS Imaging System 100 series (Xenogen Corporation) according to the manufacturer's protocol. Mice were injected with luciferin (Promega, 2.5 mg/mouse) 10 min before imaging under anaesthesia (1-2% isoflurane). The acquired images included peak luminescence signals and were recorded for 10 min. To normalize initial minor differences in tumor burden, relative tumor burden was defined as (signal value at the time of last measurement−signal value at baseline)/(signal value at starting point). The calculated values for relative tumor burden were used in the statistical analysis.

(44) The imaging results are shown in FIG. 6a to FIG. 6c.

(45) The relative tumor burden in the secondary tumor confirmed in FIG. 6c demonstrated that the combination of the pharmaceutical composition and radiotherapy provided by the present invention could prevent or treat metastatic tumor or recurrent tumor by enhancing abscopal effect.

(46) 4-2. Size of Tumor after Treatment

(47) After performing Example 4-1, tumor tissues were extracted from each group. The macroscopic images are shown in FIG. 7.

(48) The group treated by triple combination of the PI3K inhibitor, PD-1 blockade and radiotherapy showed the smallest tumor size (less than 10 mm diameter). In particular, the size of secondary tumor was significantly decreased even though the secondary tumor was not treated with radiation in case of triple combined therapy of the PI3Kγ δ inhibitor, PD-1 blockade and RT inhibitor.

(49) The macroscopic observation result demonstrates the triple combination treatment of the present invention could significantly inhibit growth of secondary tumor as well as primary tumor.

Comparative Example 1. Size of Tumor in Immunodeficient Mouse

(50) An immunodeficient animal model was prepared in substantially the same manner as in Example 4-1 by using nude mouse (BALB/c-nude female mice, CAnN.Cg-Foxn1nu/Cr1jOri; Orient Bio, Inc.). The animal model was grouped into 7 groups and treated in the same manner as Example 4-1.

(51) The macroscopic images of primary and secondary tumors in immunodeficient mouse model are shown in FIG. 7b. The secondary tumor size was not changed according to treatment methods in immunodeficient mouse model.

(52) The result demonstrates that the triple combined therapy of the PI3K-γ or PI3K-δ inhibitor, PD-1 blockade and RT inhibitor have systemic effect by regulating immune cells in tumor microenvironment.