COMBINATION OF SMALL MOLECULE INHIBITOR OF THE PD-1/PD-L1 INTERACTION AND ANTI-PD-1 ANTIBODY FOR TREATING CANCER

Abstract

The invention provides methods for treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a small molecule inhibitor of the PD-1/PD-L1 interaction or a pharmaceutically acceptable salt or prodrug thereof in combination with a therapeutically effective amount of an anti-PD-1 antibody, wherein the small molecule inhibitor of the PD-1/PD-L1 interaction is not a protein.

Claims

1-21. (canceled)

22. A method for treating a cancer, comprising administering to a subject in need thereof a small molecule inhibitor of the PD-1/PD-L1 interaction or a pharmaceutically acceptable salt or prodrug thereof and an anti-PD-1 antibody, wherein the small molecule inhibitor is not protein.

23. The method as described in claim 22, wherein the small molecule inhibitor is an aromatic vinyl or aromatic ethyl derivative; and/or, the small molecule inhibitor has a molecular weight (MW) of less than 1500 Daltons; and/or, the small molecule inhibitor has an IC.sub.50 of less than 100 nM in a PD-1/PD-L1 binding assay.

24. The method as described in claim 22, wherein the small molecule inhibitor binds to PD-L1.

25. The method as described in claim 22, wherein the small molecule inhibitor is a compound selected from the compounds identified in Method 1.8, 1.9, 1.10, or 1.11, supra, in free or pharmaceutically acceptable salt form.

26. The method as described in claim 22, wherein the small molecule inhibitor is ##STR00047## in free or pharmaceutically acceptable salt form.

27. The method as described in claim 22, wherein the cancer is cervical carcinomas, renal cell carcinoma, melanomas, breast cancer, colorectal cancer, or head and neck squamous cell carcinoma (HNSCC).

28. The method as described in claim 22, wherein the cancer is breast cancer, melanomas or colorectal cancer.

29. The method as described in claim 22, wherein the small molecule inhibitor is administered orally.

30. The method as described in claim 22, wherein the small molecule inhibitor is administered at a total dose of 20-300 mg/kg or 30-240 mg/kg per day.

31. The method as described in claim 22, wherein the small molecule inhibitor is simultaneously administered with the antibody.

32. The method as described in claim 22, wherein the antibody is a monoclonal antibody.

33. The method as described in claim 22, wherein the antibody is pembrolizumab, nivolumab, cemiplimab, toripalimab, camrelizumab or sintilimab.

34. The method as described in claim 22, wherein the antibody is administered intravenously or subcutaneously.

35. The method as described in claim 22, wherein the antibody is administered at a dose of 0.1-50 mg/kg, 0.2-10 mg/kg, 0.3-5 mg/kg, 0.4-5 mg/kg, 0.5-5 mg/kg, 0.6-4 mg/kg, 0.6-3 mg/kg, 0.6-2 mg/kg, 0.8-4 mg/kg, 0.8-3 mg/kg, 0.8-2 mg/kg, 1-10 mg/kg, 1-5 mg/kg, 1-4 mg/kg, 1-3 mg/kg, 1-2 mg/kg or 2-3 mg/kg twice a week (BIW), once every week, once every two weeks, once every three weeks or once every four weeks.

36. The method as described in claim 22, wherein the subject has previously received cancer treatment and wherein the subject is not responsive to the previous cancer treatment.

37. The method as described in claim 36, wherein the previous cancer treatment is chemotherapy.

38. The method as described in claim 22, which further comprising administration of an additional anti-cancer agent.

39. The method as described in claim 37, wherein the chemotherapy comprises a platinum containing chemotherapeutic agent.

40. The method as described in claim 37, wherein the chemotherapy is platinum-containing doublet chemotherapy.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0139] The disclosure is further defined in the following Examples. It should be understood that the Examples are given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain the essential characteristics of the disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosure to various uses and conditions. As a result, the disclosure is not limited by the illustrative examples set forth herein below, but rather is defined by the claims appended hereto.

Example 1: In Vitro Binding Studies of Compound 1

[0140] Biological Assay: The ability of Compound 1 to bind to PD-L1 is investigated using a PD-1/PD-L1 Homogenous Time-Resolved Fluorescence (HTRF) binding assay.

##STR00046##

[0141] All binding studies are performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors are pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cynomolgus, or mouse are used. HTRF detection is achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies are diluted in HTRF detection buffer and 5 μl is dispensed on top of binding reaction. The reaction mixture is allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) is obtained using an EnVision fluorometer. Additional binding assays are established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated polypeptide (AISGGGGSTYYADSVKD) and human PD-L1-His are performed as follows. Inhibitors are pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 .mu.Math.l of assay buffer followed by addition of biotinylated polypeptide (0.5 nM final) in 1 .mu.Math.l of assay buffer. Binding is allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction is allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) is obtained using an EnVision fluorometer. In the HTRF assay, Compound 1 potently inhibits the binding between hPD-1 and hPD-L1 with IC.sub.50 of 19 nM.

[0142] To measure the cellular activity of the compound, a protocol of Activation of T-Cell Suppressed by PD-L1 is used. In this protocol, human Hep3B cells are stably transfected with human PD-L1. The human T cells containing PD-1 are inactivated by co-culturing with these PD-L1 transfected cells. Then anti-PD-1 antibody Keytruda® (pembrolizumab) is selected as the reference to profile the compound for its activation of PD-L1 suppressed human T-cells. In a dose-dependent manner, Compound 1 effectively restores the activation of the PD-L1 suppressed human T cells, as indicated by the increase of cytokine IFN-γ. Keytruda is used as a positive control.

Example 2: In Vivo Test of Anti-Tumor Efficacy of Compound 1 in the Subcutaneous 4T1 Murine Breast Cancer Model in BALB/c Mice

[0143] The 4T1 murine mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic, invasive and able spontaneously to metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone.

[0144] Materials required for the experiment: Antibody: mouse PD-1 antibody, Product specifications: 7.09 mg/mL (50 mg/mL), Lot No.: 695318A1 purchased from BioXcell, storage at 4° C. Experiment animal: 60 BALB/C mice, female, 6-8 weeks old, 20-23 g, purchased from Shanghai Lingchang Biotechnology Co. Ltd. Formulation material: castor oil (Cremophor RH40), CAS No.: 61788-85-0, Lot No.: 29761847G0, purchased from Shanghai Xietai Chemical Co. Ltd.; β-cyclodextrin (SBE-β-CD), CAS No.: 128446-35-5, Lot No.: 20180110, purchased from Shanghai Shaoyuan Chemical Co. Ltd.; RPMI-1640 culture medium, Art. No.: 1869036, Lot No.: 11875-093, purchased from Gibco Co. Ltd.; PBS, Art. No.: SH30256.01, Lot No.: AB10141338, purchased from HyClone Co. Ltd.; Fetal bovine serum: CAS No.: 10099-141, Lot No.: 1966174C, purchased from Gibco Co. Ltd.

[0145] Cell preparation and implantation: 4T1 cells (CRL2539™) are cultured with RPMI 1640 supplemented with 10% heat inactivated FBS at 37° C. in 5% CO.sub.2 incubator. Cells are passaged 3 times a week. Cells are harvested, counted and passaged, inoculated when around 70% confluent.

[0146] Tumor cell inoculation and group administration: The 50 uL cell suspension containing 1×10.sup.5 4T1 tumor cells (cells suspended in base RPMI-1640 medium) is inoculated into the fourth fat pad of the left abdomen of mice. On the second day after inoculation, according to the order of tumor inoculation, stratified randomization is used to group and start the administration on the day of grouping.

[0147] Preparation of test substances: Preparation of formulation: 490 mL of sterile water is added into the volumetric flask with magnetic stirring to have a vortex. 100 g of castor oil (Cremophor RH40) is added with a spoon slowly into the vortex and the solution is kept stirring. 200 g of β-cyclodextrin (SBE-β-CD) is added while the solution is kept stirring until the solution is clear, and the total volume is set to 1000 mL, which contained 10% (w/v) Cremophor RH40+20% (w/v) an aqueous solution of SBE-β-CD.

[0148] Preparation of Compound Suspension: 178.88 mg compound is weighed and 14.817 mL 10% (w/v) Cremophor RH40+20% (w/v) SBE-β-CD aqueous solution are added. The suspension solution with a concentration of 12.0 mg/mL is obtained by fully mixing with magnetic stirring. 7.0 mL of the compound suspension solution with concentration of 12.0 mg/mL is pipetted and 7.0 mL aqueous formulation solution is added. The suspension solution with a concentration of 6.0 mg/mL is obtained by fully mixing with magnetic stirring. 7.0 mL of the compound suspension solution with concentration of 6.0 mg/mL is pipetted and 7.0 mL aqueous formulation solution is added. The suspension solution with a concentration of 3.0 mg/mL is obtained by fully mixing with magnetic stirring. 7.0 mL of the compound suspension solution with concentration of 3.0 mg/mL is pipetted and 7.0 mL aqueous formulation solution is added. The suspension solution with a concentration of 1.5 mg/mL is obtained by fully mixing with magnetic stirring. The compound suspension solution is prepared once a day.

[0149] Preparation of mPD-1 Antibody: 0.339 mL mPD-1 antibody (7.09 mg/mL) original solution is pipetted and 2.061 mL PBS solution is added. The solution is fully mixed and the final concentration of 1 mg/mL solution is obtained.

[0150] Procedure: The mice in the vehicle group are weighed and recorded in the electronic balance according to their numbers. The mice in the vehicle group are given prepared formulation solution twice a day by oral administration according to their body weight with a capacity of 0.1 mL/10 g.

[0151] The mice in the antibody (10 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in the antibody group are given prepared antibody solution twice a week by IP administration according to their body weight with a capacity of 0.1 ml/10 g.

[0152] The mice in the compound (15 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0153] The mice in the compound (30 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0154] The mice in the compound (60 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1m/10 g.

[0155] The mice in the compound (120 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0156] Tumors are measured with digital vernier calipers three times a week and calculating the volume of tumors. Euthanasia is imposed if the size of the tumor exceeds 2000 mm.sup.3, or when the animal has serious disease, pain, or is unable to freely eat and drink water. The body weight of the animals is measured by electronic balance every day. Euthanasia is required when the animal is obviously thin and its weight is reduced by more than 20%. The experiment ends 20 days after compound is administered.

[0157] The tumor inhibition rate is calculated as:

TGI (%)=(1−(the volume of the tumor on the day of administration−the volume of the tumor on the first day of administration(treatment group))/(the volume of the tumor on the day of administration−the volume of the tumor on the first day (vehicle group)))×100%.

[0158] With GraphPad Prism 5.0 software, the tumor volume changes in mice are analyzed by Two-way ANOVA and compared with the vehicle group according to the Bonferroni posttests method, P<0.05 is considered to be significantly different.

[0159] In the assay, Compound 1 and mPD-1 antibody demonstrated similar efficacy in tumor growth inhibition (TGI). Furthermore, the minimum effective dose of Compound 1 is 30 mpk (p<0.05). The results are summarized in Table 1.

TABLE-US-00001 TABLE 1 Results of In vivo test of anti-tumor efficacy of Compound 1 in the subcutaneous 4T1 murine breast cancer model in BALB/c mice. Groups Tumor Volume (mm.sup.3) TGI (%) p-Value Vehicle Control 860.89 ± 42.52 — — mPD-1 antibody, 700.96 ± 39.56 18.58 <0.001 10 mg/kg, IP, BIW Compound 1, 689.03 ± 43.97 19.96 <0.001 15 mg/kg, PO, BID Compound 1,  573.9 ± 43.18 33.34 <0.001 30 mg/kg, PO, BID Compound 1, 548.24 ± 31.39 36.32 <0.001 60 mg/kg, PO, BID Compound 1, 503.16 ± 32.93 41.55 <0.001 120 mg/kg, PO, BID

Example 3 In Vivo Test of Anti-Tumor Efficacy of Compound Land mPD-1 Antibody in the B16F10 Models

[0160] Materials required for the experiment: Antibody: mouse PD-1 antibody, Product specifications: 7.09 mg/mL (50 mg/mL), Lot No.: 695318A1 purchased from BioXcell, storage at 4° C. Experimental animals 60 C57BL/6 mice, female, 6-8 weeks old, 17-21 g, purchased from Shanghai Lingchang Biotechnology Co. Ltd. Formulation materials: castor oil (Cremophor RH40), CAS No.: 61788-85-0, Lot No.: 29761847G0, purchased from Shanghai Xietai Chemical Co. Ltd.; β-cyclodextrin (SBE-β-CD), CAS No.: 128446-35-5, Lot No.: 20180110, purchased from Shanghai Shaoyuan Chemical Co. Ltd.; DMEM culture medium, Art. No.: 11995-065, Lot No.: 2025378, purchased from Gibco Co. Ltd.; PBS, Art. No.: SH30256.01, Lot No.: AB10141338, purchased from HyClone Co. Ltd.; Fetal bovine serum: Art. No.: 04-002-1A, Lot No.: 1625436, purchased from Boehringer Ingelheim Co. Ltd.; Methyl cellulose (MC), Art. No.: M7027-250G, Lot No.: 079K0054V, purchased from Sigma.

[0161] Cell preparation and implantation: The B16-F10 tumor cells (ATCC CRL-6475™) are maintained in vitro as a monolayer culture in DMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO.sub.2 in air. The tumor cells are routinely subcultured three times weekly by trypsin-EDTA treatment. The cells growing to a confluency around 70%-80% are harvested and counted for tumor inoculation.

[0162] Tumor cell inoculation and group administration: The 100 uL cell suspension containing 1×10.sup.6 B16F10 tumor cells (cells suspended in base DMEM medium) is inoculated into the right subcutaneous of mice. On the second day after inoculation, according to the order of tumor inoculation, stratified randomization is used to group and start the administration on the day of grouping.

[0163] Preparation of test substances: Preparation of formulation: 700 mL of sterile water is added into the volumetric flask with magnetic stirring to have a vortex. 100 g of castor oil (Cremophor RH40) is added with a spoon slowly into the vortex and the solution is kept stirring. 200 g of β-cyclodextrin (SBE-β-CD) is added while the solution is kept stirring until the solution is clear, and the total volume is set to 1000 mL, which contained 10% (w/v) Cremophor RH40+20% (w/v) an aqueous solution of SBE-β-CD.

[0164] Preparation of compound suspension: 169.16 mg compound is weighed, 14.012 mL 10% (w/v) Cremophor RH40+20% (w/v) SBE-β-CD aqueous solution are added, and the suspension solution with a concentration of 12.0 mg/mL is obtained by fully mixing with magnetic stirring. 6.0 mL of the compound suspension solution with concentration of 12.0 mg/mL is pipetted and 6.0 mL aqueous formulation solution is added. The suspension solution with a concentration of 6.0 mg/mL is obtained by fully mixing with magnetic stirring. 6.0 mL of the compound suspension solution with concentration of 6.0 mg/mL is pipetted and 6.0 mL aqueous formulation solution is added. The suspension solution with a concentration of 3.0 mg/mL is obtained by fully mixing with magnetic stirring. The compound suspension solution is prepared once a day.

[0165] Preparation of mPD-1 antibody: 0.564 mL mPD-1 antibody (7.09 mg/mL) original solution is pipetted and 3.307 mL PBS solution is added. The solution is fully mixed and the final concentration of 1 mg/mL solution is obtained.

[0166] Procedure: The mice in the vehicle group are weighed and recorded in the electronic balance according to their numbers. The mice in the vehicle group are given prepared formulation solution twice a day by oral administration according to their body weight with a capacity of 0.1 mL/10 g.

[0167] The mice in the antibody (10 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in the antibody group are given prepared antibody solution twice a week by IP administration according to their body weight with a capacity of 0.1 ml/10 g.

[0168] The mice in the compound (30 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0169] The mice in the compound (60 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0170] The mice in the compound (120 mg/kg) group are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g.

[0171] The mice in the combo group (compound, 60 mg/kg; mPD-1, 10 mg/kg) are weighed and recorded in the electronic balance according to their numbers. The mice in this group are given prepared compound suspension twice a day by oral administration according to their body weight with a capacity of 0.1 ml/10 g and mouse antibody solution twice a week by IP administration according to their body weight with a capacity of 0.1 ml/10 g.

[0172] Tumors are measured with digital vernier calipers three times a week and calculating the volume of tumors. Euthanasia is imposed if the size of the tumor exceeds 2000 mm.sup.3, or the animal has serious disease, pain, or is unable to freely eat and drink water. The body weight of the animals is measured by electronic balance every day. Euthanasia is required when the animal is obviously thin and its weight is reduced by more than 20%. The experiment ends 20 days after compound is administered.

[0173] The tumor inhibition rate is calculated as:

TGI (%)=(1−(the volume of the tumor on the day of administration−the volume of the tumor on the first day of administration (treatment group))/(the volume of the tumor on the day of administration−the volume of the tumor on the first day (vehicle group)))×100%.

[0174] Using GraphPad Prism 5.0 software, the tumor volume changes in mice are analyzed by Two-way ANOVA and compared with the vehicle group according to the Bonferroni posttests method, P<0.05 is considered to be significantly different.

[0175] The results show that Compound 1 significantly inhibits the growth of subcutaneous transplanted melanoma cell line in mice, and it is well tolerated in C57BL/6 mice without obvious adverse reactions. Furthermore, the results show that the combination of Compound 1 and mouse PD-1 antibody is significantly more effective in inhibiting tumor growth than either drug alone. The results are summarized in Table 2.

TABLE-US-00002 TABLE 2 Results of in vivo tests of anti-tumor efficacy of the compound in the B16F10 models Groups Tumor Volume (mm.sup.3) TGI (%) p-Value Vehicle Control 1235.87 ± 220.28  — — mPD-1 antibody, 693.74 ± 272.23 43.87 0.0007 10 mg/kg, IP, BIW Compound 1, 30 mg/kg, 795.99 ± 112.92 35.59 0.0098 PO, BID Compound 1, 60 mg/kg, 755.48 ± 155.85 38.87 0.0037 PO, BID Compound 1, 120 mg/kg, 650.67 ± 157.53 47.35 0.0002 PO, BID mPD-1antibody, 10 487.05 ± 131.39 60.59 <0.0001 mg/kg, IP, BIW + Compound 1, 60 mg/kg, PO, BID

Example 4: In Vivo Test of Anti-Tumor Efficacy of Compound 1 and Human PD-1 Antibodies in the 4T1 Humanized Mouse Model

[0176] 4T1 tumor cells (ATCC, CRL-2539™) are maintained in vitro as a monolayer culture in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO.sub.2 in air. The tumor cells are routinely subcultured three times weekly by trypsin-EDTA treatment. Each mouse is inoculated subcutaneously at the fourth mammary pad with 1×10.sup.5 in 0.05 mL base medium for tumor development.

[0177] Compound 1 plus Opdivo (nivolumab): 40 BALB/c mice are inoculated subcutaneously at the fourth mammary pad with 4T1 cells for tumor development. On the six days of post inoculation, the mice are assigned into 4 groups using stratified randomization with 10 mice in each group based upon their tumor volume. The treatments are started from the day of randomization, and the groups receive the following treatments:

Group 1: Vehicle control;
Group 2: Opdivio, 10 mg/kg, i.p., BIW;
Group 3: Compound 1, 60 mg/kg, p.o., BID and
Group 4: Compound 1, 60 mg/kg, p.o., BID+Opdivio, 10 mg/kg, i.p., BIW respectively.

[0178] The tumor sizes are measured three times per week during the treatment.

[0179] The tumor growth inhibition effect of the combination of Compound 1 and Opdivo is examined. The tumor inhibition rate is calculated as:

TGI (%)=(1−(the volume of the tumor on the day of administration−the volume of the tumor on the first day of administration (treatment group))/(the volume of the tumor on the day of administration−the volume of the tumor on the first day (vehicle group)))×100%.

[0180] The results summarized in Table 3 show the tumor growth inhibition (%) of four groups at day 13. The tumor growth inhibition (%) of the combination group (Opdivo+Compound 1) is 34.65% at day 14, while the tumor growth inhibitions (%) of the Opdivo group and Compound 1 group are 21.88% and 21.70% respectively at day 13. The results show that the combination of Compound 1 and Opdivo is significantly more effective in inhibiting tumor growth than either drug alone at day 13. NB: Opdivo is a human antibody to human PD-1, and it has some toxicity in mice. After the 5th dose of Opdivo at day 14, mice are found dead within 1 hour of dosing. Thus, data are collected only up to day 13.

TABLE-US-00003 TABLE 3 Results of in vivo test of anti-tumor efficacy of the compound and PD-1 antibody Opdivo in the 4T1 humanized mice model TGI (%) Groups at day 13 p-Value Vehicle Control — — Opdivo(anti-hPD-1), 21.88 <0.0001 10 mg/kg, IP, BIW Compound 1, 60 mg/kg, 21.70 <0.0001 PO, BID Compound 1, 60 mg/kg, PO, 34.65 <0.0001 BID + Opdivo(anti-hPD-1), 10 mg/kg, IP, BIW

[0181] Compound 1 plus Keytruda (pembrolizumab): 40 BALB/c mice are inoculated subcutaneously at the fourth mammary pad with 4T1 cells for tumor development. On the six days of post inoculation, the mice were assigned into 4 groups using stratified randomization with 10 mice in each group based upon their tumor volume. The treatments were started from the day of randomization, and the groups receive the following treatments:

Group 1: Vehicle control;
Group 2: Keytruda, 10 mg/kg, i.p., BIW;
Group 3: Compound 1, 60 mg/kg, p.o., BID, and
Group 4: Compound 1, 60 mg/kg, p.o., BID+Keytruda, 10 mg/kg, i.p., BIW respectively.

[0182] The tumor sizes are measured three times per week during the treatment.

[0183] The tumor growth inhibition effect of the combination of Compound 1 and Keytruda is examined. The tumor inhibition rate is calculated as:

TGI (%)=(1−(the volume of the tumor on the day of administration−the volume of the tumor on the first day of administration (treatment group))/(the volume of the tumor on the day of administration−the volume of the tumor on the first day (vehicle group))×100%.

[0184] The results summarized in Table 4 shows the tumor growth inhibition (%) of four groups at day 10. The tumor growth inhibition (%) of the combination group (Keytruda+Compound 1) is 23.85% at day 10, while the tumor growth inhibitions (%) of the Keytruda group and Compound 1 group are 9.12% and 22.36% respectively at day 10. The results showed that the combination of Compound 1 and Keytruda is more effective in inhibiting tumor growth than either drug alone at day 10. NB: Keytruda is a humanized antibody to human PD-1, and it has some toxicity in mice. After the 4th dose of Keytruda at day 10, mice are found dead within 1 hour of dosing. Thus, data are collected before the dosing of Keytruda on day 10.

TABLE-US-00004 TABLE 4 Results of in vivo test of anti-tumor efficacy of the compound and PD-1 antibody Keytruda in the 4T1 humanized mice model TGI (%) Groups at day 10 p-Value Vehicle Control — — Keytruda (anti-hPD-1), 9.12 >0.5 10 mg/kg, IP, BIW Compound 1, 60 mg/kg, 22.36 <0.0001 PO, BID Compound 1, 60 mg/kg, PO, 23.85 <0.0001 BID + Keytruda (anti-hPD- 1), 10 mg/kg, IP, BIW

[0185] While the present invention has been described with reference to embodiments, it will be understood by those skilled in the art that various modifications and variations may be made therein without departing from the scope of the present invention as defined by the appended claims.