Combination therapy for treatment of solid tumors

Abstract

The present invention concerns the use of a COX2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, and an immune checkpoint inhibitor, such as those selected from the group of atezolizumab, avelumab, durvalumab, envafolimab, cosibelimab, socazolimab, MEDI4736, STI-3031, and LY3300054, in the treatment of solid tumors.

Description

[0001] COMBINATION THERAPY FOR TREATMENT OF SOLID TUMORS

[0002] The present invention concerns the use of a cyclooxygenase inhibitor and an immune checkpoint inhibitor in the treatment of solid tumors, including triple negative breast cancer, particularly combinations utilizing indomethacin and a PD-L1 inhibitor.

[0003] BACKGROUND OF THE INVENTION

[0004] Solid tumors represent approximately ninety percent of adult human cancers and about forty percent of childhood cancers and have been the subject of many therapeutic developments, including recent advances in immunotherapies. Several types, including adoptive cell transfer (ACT) and immune checkpoint inhibitors (ICIs) have resulted in durable clinical responses, but their efficacies vary, and only subsets of cancer patients can benefit from them (Zhang et al., Cell Mol Immunol, 2021 Aug; 17(8): 807-821).

[0005] While the recent approval of immune checkpoint inhibitors (ICIs), atezolizumab or pembrolizumab plus paclitaxel-protein bound (nab-palcitaxel), as therapy for the triple negative breast cancer (TNBC) will significantly improve “standard of care”, there is a huge scope of improving patient outcome since the median overall survival remain under two years for TNBC patients even with the approval of ICI. The tumor associated macrophages (TAMs) debilitate therapy response in breast cancer due to their CD8+T cell suppressing function whereas targeting TAMs, in combination with chemotherapy or immune checkpoint inhibitors, in preclinical tumor models result in significant tumor burden reduction in a CD8+T cell dependent manner. Thus, we hypothesize that select myelomonocytic targeting drugs that synergize with PD1 / PD-L1 inhibitors to increase CD8+T cell activity can improve ICI outcome in TNBC.

[0006] TNBC represents one of the most aggressive forms of all breast cancer subtypes with a very high rate of recurrence and dissemination to distant organs. Immune checkpoint inhibitor (ICI) targeting PD1 is recently approved for TNBC patients, which increases median overall survival by ~7 months in treated cohort. While this is a highly sought-after improvement for breast cancer patients, due to the addition of a non-chemotherapy treatment to complement chemotherapy for a clinically meaningful benefit to the patients, the median overall survival is still under two years even with the use of ICI leaving a huge scope of the improvement. Moreover, nearly 50% of TNBC patients did not show improved outcome and this phenomenon was correlated with lower percentage of PD-L1 expressing cells in the tumors. The burgeoning research towards understanding the mechanism of ICI response has led to the identification of the myeloid dependent inflammation as a significant debilitating phenomenon since it critically impacts CD8+T cell recruitment, expansion, and activation in tumors. Furthermore, preclinical studies have also assessed synergy between myeloid-targeted drugs and ICBs: in murine models of human cancer, preclinical synergy has been reported when primary tumor-bearing mice are treated with combinations of a-PD-1 or a-PD-L1 blocking monoclonal antibodies (mAb), with inhibitors of myeloid-based pathways regulated by CSF1 / CSF1 R (colony stimulating factor 1 and its receptor), phosphoinositide 3-kinase gamma (PI3KY), Bruton’s tyrosine kinase (BTK), complement receptors, etc. Based on these preclinical findings, clinical trials are now evaluating similar combinations in patients with advanced cancers with the goal of improving efficacy of I Cl s in responder patients as well as producing meaningful clinical benefits in otherwise a nonresponder patient population (ref.). However, a big fraction of ongoing trials is using targeted therapies that are not exclusively identified for their activity as a combination partner with ICI and are dependent on our current “state of knowledge” of tumor associated inflammation, which creates a significant “window of opportunity” in using unbiased phenotypic screens for discovering myeloid cell targeted therapies for the use as combination therapy partner with ICI.

[0007] There remains a need for more effective methods of treatment for many types of solid tumors, including triple negative breast cancer.

[0008] SUMMARY OF THE INVENTION

[0009] Here we report a novel drug discovery platform using macrophage and T cell co-culture assay to mimic macrophage dependent T cell suppression in cancer. Using this platform, we discovered commonly used pain-relieving C0X2 medicines, including Indomethacin, act as potent myelomonocytic reprogramming agents that also possess a significant synergy with anti PD-L1 to increase T cell expansion. Interestingly, the COX2 expression, a target molecule such as indomethacin, is highly correlated with PD-L1 expression and TH1 markers in tumor microenvironment (TME) and was strictly expressed in myelomonocytic cells. The use of these C0X2 inhibiting agents and anti PD-L1 combination in syngeneic TNBC tumor models led to significant tumor regression and improved overall survival, which was CD8+T cell dependent. Altogether, our studies describe a rationalized approach to identify combination therapy regimen to compliment ICI and report the discovery of selective C0X2 inhibitors indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib to repurpose as a combination therapy for improved outcome of ICI in treating solid tumors, including TNBC. One embodiment provides a method of enhancing the therapeutic effect of an immune checkpoint inhibitor in the treatment of triple negative breast cancer, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib or a pharmaceutically acceptable salt, cocrystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0010] Another embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: a) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and b) a pharmaceutically effective amount of an immune checkpoint inhibitor.

[0011] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, such as an anti- PD-L1 antibody.

[0012] BRIEF DESCRIPTION OF THE MANY VIEWS OF THE DRAWINGS

[0013] FIGURE 1 provides an illustration of the ex-vivo assay used to analyze macrophage mediated T-cell suppression described herein.

[0014] FIGURE 2 provides a schematic representation of ex-vivo screening of immunomodulators using an automated high-throughput method.

[0015] FIGURE 3 presents a schematic representation of the assay principle.

[0016] FIGURE 4 presents bar graphs representing results of a surrogate viability assay for detection of T-cell activity.

[0017] FIGURE 5 presents representations of activities seen in macrophage culture and coculture that lead to “hit” compounds.

[0018] FIGURE 6A presents a pie chart representing the high-throughput screening results. FIGURE 6B presents a distribution graph of 66 hits identified in the screening process.

[0019] FIGURE 7 presents a graph of proliferation (fold change) hits obtained from secondary screening.

[0020] FIGURE 8 presents a representation of the confirmation of the activity of C0X2 inhibitor indomethacin with the anti-PD-L1 antibody.

[0021] FIGURE 9 presents an overview of the 4T1 syngeneic TNBC model.

[0022] FIGURE 10 presents a representation of in vivo confirmation of indomethacin activity as combination therapy using anti-PD1 antibody.

[0023] FIGURE 11 represents in vivo confirmation of indomethacin activity as combination therapy using an anti-PD-L1 antibody.

[0024] FIGURE 12 further represents in vivo confirmation of indomethacin activity as combination therapy using an anti-PD-L1 antibody.

[0025] FIGURE 13 provides bar graphs representing immune complex analysis of 4T1 tumor samples.

[0026] FIGURE 14 provides bar graphs representing immune complex analysis of 4T1 tumor samples.

[0027] FIGURE 15A graphs data of tumor size over time from our PY8119 xenograft study

[0028] FIGURE 15B represent tumor weight data from our PY8119 xenograft study.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] Another embodiment provides a method of enhancing the activity of a PD-L1 inhibitor in the treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a COX2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof, followed by administration to the subject in need thereof a pharmaceutically effective amount of the PD-L1 inhibitor. Another embodiment provides a method of enhancing the activity of a PD-L1 inhibitor in the treatment of triple negative breast cancer in a human subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof, followed by administration to the subject in need thereof a pharmaceutically effective amount of the PD-L1 inhibitor.

[0031] Another embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: a) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and b) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab, envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), MEDI4736, STI-3031 , and LY3300054.

[0032] Another embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: c) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and d) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab, envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), MEDI4736, STI-3031 , and LY3300054. Another embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: e) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and f) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab, envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), MEDI4736, STI-3031 , and LY3300054.

[0033] Another embodiment provides a method of treatment of triple negative breast cancer in a human subject, the method comprising administering to the subject in need thereof: g) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and h) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab, envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), MEDI4736, STI-3031 , and LY3300054.

[0034] In some embodiments herein, the PD-L1 inhibitor in the method in question is an anti- PD-L1 antibody selected from the group of atezolizumab, avelumab, durvalumab.

[0035] Another embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: a) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and b) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab (MEDI4736), envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), STI-3031 , and LY3300054; and c) a pharmaceutically effective amount of one or more additional chemotherapeutic agents selected from the group of nab-paclitaxel, paclitaxel, docetaxel, cisplatin, and carboplatin, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0036] Another embodiment provides a method of treatment of triple negative breast cancer in a human subject, the method comprising administering to the subject in need thereof: d) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and e) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab (MEDI4736), envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), STI-3031 , and LY3300054; and f) a pharmaceutically effective amount of one or more additional chemotherapeutic agents selected from the group of nab-paclitaxel, paclitaxel, docetaxel, cisplatin, and carboplatin, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0037] A further embodiment provides a method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof; a) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and b) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab, avelumab, durvalumab; and c) a pharmaceutically effective amount of one or more additional chemotherapeutic agents selected from the group of nab-paclitaxel, paclitaxel, docetaxel, cisplatin, and carboplatin, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0038] A further embodiment provides a method of treatment of triple negative breast cancer in a human subject, the method comprising administering to the subject in need thereof; d) a pharmaceutically effective amount of a C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and e) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab, avelumab, durvalumab; and f) a pharmaceutically effective amount of one or more additional chemotherapeutic agents selected from the group of nab-paclitaxel, paclitaxel, docetaxel, cisplatin, and carboplatin, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0039] In some regimens, administration of the C0X2 inhibitor may begin shortly before administration of each dose of a PD-L1 inhibiting agent, such as 30 minutes or one hour prior to administration of the PD-L1 inhibiting agent. In other embodiments, the continual administration of the COX2 inhibitor may be initiated days before the first PD-L1 inhibitor is administered, such as one week or two weeks prior. In some embodiments, administration of the COX2 inhibitor may be continual throughout the administration cycle of the PD-L1 inhibitor (alone or in combination with other agents, such as nab-paclitaxel, paclitaxel, cisplatin, docetaxel, and carboplatin). In other regimens where the PD-L1 inhibitor is administered in a cycled regimen, such as on days 1 and 15 (± 3 days) of every 28-30 cycle, administration of the COX2 inhibitor may be equally staggered in conjunction to PD-L1 inhibitor administration. For example, the COX2 inhibitor may be administered beginning one week prior to the PD-L1 inhibitor and maintained for one week following each PD-L1 inhibitor administration.

[0040] In some embodiments in which the COX2 inhibitor is indomethacin, indomethacin may be administered to the subject in need thereof at an effective dose of from about 25 mg per day to about 250 mg per day in a single dose or in divided doses, such as in 2, 3, or 4 divided doses per day. In separate embodiments, the indomethacin may be administered to the subject in need thereof, respectively, at an effective dose of a) from about 50 mg to about 200 mg; b) from about 75 mg to about 150 mg; c) from about 80 mg to 120 mg; d) from about 50mg to about 100 mg; e) from about 100 mg to about 150 mg; f) from about 150 mg to about 200 mg; from about 200 mg to about 250 mg; each administered as a single daily dose or in divided doses, such as in 2, 3, or 4 divided doses per day.

[0041] In some embodiments in which the COX2 inhibitor is diclofenac, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 1 mg to about 400 mg, in a single or divided doses. In other embodiments, diclofenac is administered to the subject in need thereof at a of from about 5 mg / day to about 300 mg / day in one dose or divided doses. In still other embodiments, the daily dose of diclofenac is from about 10 mg to about 250 mg. In further embodiments, the daily dose of diclofenac is from about 20 mg to about 250 mg. In still further embodiments, the daily dose of diclofenac is from about 40 mg to about 250 mg. In some embodiments, diclofenac is administered for a period of from about 3 days to about 7 days at the beginning of each three-week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of diclofenac, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 100 mg, 120 mg, 150 mg, 175 mg, 200 mg, 220 mg, 240 mg, and 250 mg.

[0042] In some embodiments in which the COX2 inhibitor is lornoxicam, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 1 mg to about 40 mg, in a single or divided doses. In other embodiments, lornoxicam is administered to the subject in need thereof at a of from about 5 mg / day to about 30 mg / day in one dose or divided doses. In still other embodiments, the daily dose of lornoxicam is from about 10 mg to about 30 mg. In further embodiments, the daily dose of lornoxicam is from about 10 mg to about 25 mg. In still further embodiments, the daily dose of lornoxicam is from about 15 mg to about 25 mg. In some embodiments, lornoxicam is administered for a period of from about 3 days to about 7 days at the beginning of each three week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of lornoxicam, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, and 40 mg, administered as a single dose or divided doses, such as twice daily (b.i.d.), three times daily (t.i.d.), or four times daily (q.i.d. or QID).

[0043] In some embodiments in which the COX2 inhibitor is tenoxicam, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 1 mg to about 50 mg, in a single or divided doses. In other embodiments, tenoxicam is administered to the subject in need thereof at a of from about 5 mg / day to about 40 mg / day in one dose or divided doses. In still other embodiments, the daily dose of tenoxicam is from about 10 mg to about 35 mg. In further embodiments, the daily dose of tenoxicam is from about 10 mg to about 30 mg. In still further embodiments, the daily dose of tenoxicam is from about 10 mg to about 25 mg. In some embodiments, tenoxicam is administered for a period of from about 3 days to about 7 days at the beginning of each three week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of tenoxicam, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, and 50 mg.

[0044] In some embodiments in which the COX2 inhibitor is meloxicam, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 1 mg to about 40 mg, in a single or divided doses. In other embodiments, meloxicam is administered to the subject in need thereof at a of from about 1 mg / day to about 30 mg / day in one dose or divided doses. In still other embodiments, the daily dose of meloxicam is from about 1 mg to about 25 mg. In further embodiments, the daily dose of meloxicam is from about 5 mg to about 25 mg. In still further embodiments, the daily dose of meloxicam is from about 10 mg to about 20 mg. In some embodiments, meloxicam is administered for a period of from about 3 days to about 7 days at the beginning of each three week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of meloxicam, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, and 40 mg.

[0045] In some embodiments in which the COX2 inhibitor is tolfenamic acid, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 20 mg to about 2500 mg, in a single or divided doses. In other embodiments, tolfenamic acid is administered to the subject in need thereof at a of from about 50 mg / day to about 2500 mg / day in one dose or divided doses. In still other embodiments, the daily dose of tolfenamic acid is from about 100 mg to about 2500 mg. In further embodiments, the daily dose of tolfenamic acid is from about 200 mg to about 2500 mg. In still further embodiments, the daily dose of tolfenamic acid is from about 400 mg to about 2500 mg. In some embodiments, tolfenamic acid is administered for a period of from about 3 days to about 7 days at the beginning of each three week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of tolfenamic acid, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 20 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 1000 mg, 1200 mg, 1500 mg, 1750 mg, 2000 mg, 2200 mg, and 2400 mg.

[0046] In some embodiments in which the COX2 inhibitor is etoricoxib, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 10 mg to about 200 mg, in a single or divided doses. In other embodiments, etoricoxib is administered to the subject in need thereof at a daily dose of from about 25 mg / day to about 120 mg / day in one dose or divided doses. In still other embodiments, the daily dose of etoricoxib is from about 50 mg to about 120 mg. In further embodiments, the daily dose of etoricoxib is from about 50 mg to about 120 mg. In still further embodiments, the daily dose of etoricoxib is from about 75 mg to about 110 mg. In some embodiments, etoricoxib is administered for a period of from about 3 days to about 7 days at the beginning of each three week or 28-30-day regimen of administration in combination with the immune checkpoint inhibitor. Individual daily doses of etoricoxib, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, and 150 mg.

[0047] In some embodiments in which the COX2 inhibitor is parecoxib, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 10 mg to about 150 mg, in a single or divided doses. In some embodiments, it is administered to the subject in need thereof at a daily dose of from about 20 mg to about 140 mg, in a single or divided doses. In additional embodiments, it is administered to the subject in need thereof at a daily dose of from about 30 mg to about 120 mg, in a single or divided doses. In further embodiments, it is administered to the subject in need thereof at a daily dose of from about 40 mg to about 120 mg, in a single or divided doses. Individual daily doses of parecoxib, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, and 150 mg. In some embodiments, parecoxib is administered in two divided doses per day (every twelve hours).

[0048] In some embodiments in which the COX2 inhibitor is rofecoxib, or a pharmaceutically acceptable salt thereof, it is administered to the subject in need thereof at a daily dose of from about 1 mg to about 50 mg, in a single or divided doses. In some embodiments, it is administered to the subject in need thereof at a daily dose of from about 5 mg to about 40 mg, in a single or divided doses. In additional embodiments, it is administered to the subject in need thereof at a daily dose of from about 5 mg to about 30 mg, in a single or divided doses. In further embodiments, it is administered to the subject in need thereof at a daily dose of from about 10 mg to about 30 mg, in a single or divided doses. Individual daily doses of rofecoxib, or a pharmaceutically acceptable salt thereof, in different embodiments may comprise, respectively, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, and 50 mg. In some embodiments, rofecoxib is administered in two divided doses per day (every twelve hours).

[0049] Atezolizumab may be administered to the subject in need thereof at an effective dose of from about 500 mg to about 1 ,400 mg via infusion on days 1 and 15 days (± 3 days) of each 28- 30-day cycle. In some embodiments, Atezolizumab may be administered to the subject in need thereof by intravenous infusion at an effective dose of from about 600 mg to about 1 ,400 mg once every three weeks. In other embodiments, Atezolizumab may be administered to the subject in need thereof by intravenous infusion at an effective dose of about 1 ,200 mg once every three weeks.

[0050] Avelumab may be administered to the subject in need thereof at an effective dose of from about 5 mg / kg to about 20 mg / kg via intravenous infusion on days 1 and 15 days (± 3 days) of each 28-30-day cycle. In some embodiments, this administration of avelumab does not exceed 1 ,000 mg / day. In other embodiments, avelumab may be administered to the subject in need thereof at a dose of 500 mg to about 1 ,000 mg once every two weeks in combination with other agents described herein. In some embodiments, avelumab may be administered to the subject in need thereof at a dose of about 800 mg once every two weeks in combination with other agents described herein.

[0051] Durvalumab may be administered to the subject in need thereof at an effective dose of from about 500 mg to about 2,000 mg via intravenous infusion on day 1 of each 28-30-day cycle. In some embodiments, Durvalumab may be administered at an effective dose of from about 1 ,000 mg to about 1 ,600 mg via intravenous infusion on day 1 of each 28-30-day cycle. In other embodiments, Durvalumab may be administered at an effective dose of from about 1 ,500 mg via intravenous infusion on day 1 of each 28-30-day cycle (once every four weeks). In still other embodiments, durvalumab is administered to the subject in need thereof at an intravenous dose of from about 10 mg / kg up to a maximum daily dose of about 1 ,500 mg on day one of each 28- 30-day cycle. In still other embodiments, durvalumab is administered at an intravenous dose of from about 10 mg / kg to about 25 mg / kg up to a maximum daily dose of about 1 ,500 mg once every three weeks. In some embodiments, durvalumab is administered at an intravenous dose of from about 20 mg / kg up to a maximum daily dose of about 1 ,500 mg once every three weeks.

[0052] In some embodiments, envafolimab may be administered to the subject in need thereof at an effective dose of from about 100 mg to about 500 mg via intravenous infusion once every three weeks. In other embodiments envafolimab may be administered to the subject in need thereof at an effective dose of from about 100 mg to about 500 mg via intravenous infusion on the first day of each 28-30 administration cycle. In still other embodiments, envafolimab may be administered to the subject in need thereof at an effective dose of from about 100 mg to about 500 mg via intravenous infusion on the first and fifteenth days of the first 28-30-day cycle of a treatment regimen, following by administration of from about 100 mg to about 500 mg via intravenous infusion on the first day of each subsequent 28-30 administration cycle. For additional embodiments following each of the dosing cycles / regimens just described for envafolimab administration, a fixed dose of 300 mg is utilized for each administration.

[0053] In some embodiments, cosibelimab is administered to the subject in need thereof via intravenous infusion at a dose of from about 300 mg to about 1 ,500 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen. In some embodiments, cosibelimab is administered to the subject in need thereof at a dose of from about 500 mg to about 1 ,000 mg once every two weeks. In some embodiments, cosibelimab is administered to the subject in need thereof at a dose of about 800 mg once every two weeks. In still other embodiments, cosibelimab is administered to the subject in need thereof via intravenous infusion at a dose of from about 1 ,000 mg to about 1 ,500 mg once every three weeks. In additional embodiments, cosibelimab is administered to the subject in need thereof via intravenous infusion at a dose of about 1 ,200 mg once every three weeks.

[0054] In some embodiments, socazolimab is administered to the subject in need thereof via intravenous infusion at a dose of from about 1 mg to about 25 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen. In separate embodiments, the socazolimab is administered to the subject in need thereof, respectively, at a daily dose of 5 mg, 10 mg, 15 mg, 20 mg, and 25 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen.

[0055] In some embodiments, STI-3031 is administered to the subject in need thereof via intravenous infusion at a dose of from about 1 mg to about 40 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen (every two weeks). In other embodiments, STI-3031 is administered to the subject in need thereof via intravenous infusion at a dose of from about 10 mg to about 30 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen (every two weeks). In other embodiments, STI-3031 is administered to the subject in need thereof via intravenous infusion at a dose of about 20 mg on the first and fifteenth days of a 28-30-day cycle treatment regimen (every two weeks).

[0056] In some embodiments, LY3300054 is administered by intravenous infusion or intraperitoneal injection at a daily dose of from about 50 mg to about 1 ,000 mg. In other embodiments, LY3300054 is administered by intravenous infusion or intraperitoneal injection at a daily dose of from about 500 mg to about 1 ,000 mg once every two weeks.

[0057] In some embodiments, paclitaxel may be administered to the subject in need thereof in the methods herein at an effective dose of from about 50 mg / m2to about 250 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle. In other separate embodiments, the paclitaxel may be administered to the subject in need thereof at doses, respectively, of a) from about 50 mg / m2to about 200 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle; b) from about 50 mg / m2to about 150 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle; c) from about 50 mg / m2to about 100 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle; d) from about 100 mg / m2to about 200 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle; and e) from about 150 mg / m2to about 200 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle. In still other embodiments, the paclitaxel may be administered at a dose of from about 100 mg / m2to about 200 mg / m2once every three weeks. In some embodiments, the paclitaxel may be administered at a dose of from about 100 mg / m2to about 150 mg / m2once every three weeks. In still other embodiments, the paclitaxel may be administered at a dose of from about 150 mg / m2to about 200 mg / m2once every three weeks.

[0058] Nab-paclitaxel may be administered to the subject in need thereof in the methods herein at an effective dose of from about 50 mg / m2to about 300 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle. Nab-paclitaxel may be administered to the subject in need thereof in the methods herein at an effective dose of from about 50 mg / m2to about 150 mg / m2on days 1 , 8, and 15 of every 28-30-day cycle. In other embodiments, the nab-paclitaxel is administered at a dose of from about 150 mg / m2to about 300 mg / m2once every two weeks. In further embodiments, the nab-paclitaxel is administered at a dose of from about 200 mg / m2to about 300 mg / m2once every two weeks. In other embodiments, the nab-paclitaxel is administered at a dose of from about 150 mg / m2to about 300 mg / m2once every two weeks. In even further embodiments, the nab-paclitaxel is administered at a dose of from about 250 mg / m2to about 275 mg / m2once every two weeks. In other embodiments, the nab-paclitaxel is administered at a dose of about 135 mg / m2once every three weeks. In additional embodiments, the nab- paclitaxel is administered at a dose of about 175 mg / m2once every three weeks.

[0059] Carboplatin may be administered to the subject in need thereof in the methods herein at an effective dose of from about AUC 1 to about AUC 6. In additional embodiments, the carboplatin is administered at an effective dose, respectively, of a) from about AUC 2 to about AUC 5; b) from about AUC 3 to about AUC 5; c) from about AUC 4 to about AUC 5; d) from about AUC 5 to about AUC 6; e) from about AUC 1 to about AUC 2; and f) from about AUC 2 to about AUC 3. In some embodiments, the cited AUC doses are administered on day one of each three-week (21-day) dosing cycle. In other embodiments, the cited AUC doses are administered on day one and on day eight of each three-week (21-day) dosing cycle. In still additional embodiments, the cited AUC doses are administered respectively, a) on day one of each three- week (21 day) dosing cycle; and b) on day one of each four week (one month, or 28-30-days) dosing cycle.

[0060] In other embodiments, carboplatin may be administered via intravenous infusion at a dose of from about 100 mg / m2to about 400 mg / m2on the first day of each 28-to-30-day administration cycle. In some embodiments, the carboplatin is administered at a rate to achieve an area under the free carboplatin plasma concentration versus time curve (AUC) of from about 3 to about 6 mg / ml / min. In some embodiments, the carboplatin is administered at an effective dose of from about 50 mg / m2to about 300 mg / m2. In other embodiments, the carboplatin is administered at doses of, respectively, a) from about 50 mg / m2to about 250 mg / m2; b) from about 50 mg / m2to about 200 mg / m2; c) from about 50 mg / m2to about 150 mg / m2; d) from about 50 mg / m2to about 100 mg / m2; e) from about 100 mg / m2to about 150 mg / m2; f) from about 150 mg / m2to about 200 mg / m2; and g) from about 200 mg / m2to about 250 mg / m2.

[0061] Docetaxel may be administered to the subject in need thereof in the methods herein at an effective dose of from about 25 mg / m2to about 200 mg / m2administered intravenously once every three weeks. In some embodiments, the docetaxel may be administered intravenously once every three weeks at a dose of from about 50 mg / m2to about 100 mg / m2. In other embodiments docetaxel may be administered intravenously once every three weeks at a dose of about 75 mg / m2.

[0062] Cisplatin may be administered to the subject in need thereof in the methods herein at an effective dose of from about 25 mg / m2to about 200 mg / m2administered intravenously once every three weeks. In some embodiments, the cisplatin may be administered intravenously once every three weeks at a dose of from about 50 mg / m2to about 100 mg / m2. In other embodiments cisplatin may be administered intravenously once every three weeks at a dose of about 75 mg / m2.

[0063] Definitions

[0064] The term “solid tumor” as used herein refers to abnormal masses of tissue, particularly malignant masses and includes advanced solid tumors, metastatic solid tumors, refractory solid tumors of any cellular, tissue, or organ origin. Solid tumors referred to in the methods and other descriptions herein include, but are not limited to: a) breast cancers, including triple negative breast cancer, inflammatory breast cancer, ductal or lobular carcinoma, ductal carcinoma in situ, invasive breast cancer, Paget disease of the breast, angiosarcomas of the breast, and Phyllodes tumors; b) thyroid cancers, including papillary, follicular, medullary, anaplastic thyroid cancers, and Hurthle cell cancer and parathyroid cancers (including parathyroid adenomas); c) mesotheliomas, including pleural mesothelioma, peritoneal mesothelioma, pericardial mesotheliomas, epitheliod mesotheliomas, sarcomatoid mesotheliomas, biphasic mesotheliomas, and dermoplastic mesotheliomas; d) head & neck cancers, including salivary gland cancer, glioblastoma multiforme, paragangliomas, retinoblastomas, laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancers, nasopharyngeal cancer, oral and oropharyngeal cancer; e) ovarian cancers, including germ cell tumors, stromal cell tumors, epithelial ovarian carcinomas, and borderline ovarian tumors; f) cervical cancers, including squamous cell carcinoma, cervical adenocarcinomas, small cell cervical cancer (SCCC), and large cell cervical cancer (LCCC); g) vaginal cancers, including vaginal adenocarcinomas, verrucous carcinoma, clear cell adenocarcinoma, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, vaginal sarcomas, vaginal embryonal rhabdomyosarcoma, vaginal melanomas, small cell cancer of the vagina, and vaginal lymphoma; h) skin cancers, including squamous cell cancers of the skin, basal cell carcinoma (BCC) large cell carcinomas, Merkel Cell carcinoma, and cutaneous T-cell lymphoma; i) neuroendocrine malignancies, including carcinoid tumors of the lung, Gl tract, or thymus; pancreatic neuroendocrine tumors and islet cell cancer; medullary thyroid carcinoma; Merkel cell carcinoma; pheochromocytoma of the adrenal gland; adrenal cancer; small cell carcinomas, and large cell carcinoid tumors; j) melanomas, including superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma; k) multiple myelomas, including light chain myeloma, non-secretory melanoma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma; l) lung cancers, including non-small cell lung cancer, small cell lung cancer, and carcinoid lung cancer; m) renal cancers, including renal cell carcinoma and Wilms tumors; n) prostate cancers, including androgen-independent prostate cancer, hormone-refractory prostate cancer (Al PC)), sarcoma tumors of the prostate, prostate small cell carcinoma, transitional cell carcinoma, prostate squamous cell carcinoma, and adenocarcinomas of the prostate, such as acinar adenocarcinoma and ductal adenocarcinoma of the prostate; o) pancreatic cancers, including exocrine / nonendocrine pancreatic cancer, pancreatic adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, colloid carcinoma, pancreatic neuroendocrine tumors, and benign precancerous pancreatic lesions; 1 p) bladder cancers, including urothelial or transitional cell bladder cancer, squamous cell bladder cancer, adenocarcinomas of the bladder, muscle invasive bladder cancer, non-muscle invasive bladder cancer, papillary bladder cancer, carcinoma in situ of the bladder, high grade T 1 bladder tumors, and secondary cancers of the bladder originating in the prostate, rectum, ovary, cervix, womb, or other organs or tissues; q) gastrointestinal cancers, including gastric cancer, gastrointestinal stromal tumors, colorectal cancer, esophageal cancers, neuroblastoma, anal cancer, small intestine cancer, stomach cancer, gallbladder cancer r) hepatocellular cancers, including liver cancer and bile duct cancer; s) adenocystic cancers, including cribiform, tubular, and solid adenoid cystic carcinomas; t) adrenal cancers, including adrenocortical carcinoma, adenomas, neuroblastomas, and pheocromocytomas; u) sarcomas, including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, alveolar soft- part sarcoma, clear cell sarcoma, chondrosarcoma, dermatofibrosarcoma protuberans, epitheliod sarcoma, fibrosarcoma, gastrointestinal stromal tumors (GIS), Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, synovial cell sarcoma, undifferentiated pleomorphic sarcoma (UPS) / malignant fibrous histiocytoma (MFH), uterine sarcoma, vascular sarcomas, and desmoplastic small round cell tumors (DSRCT); v) solid lymphomas, including Hodgkin lymphoma, non-Hodgkin lymphoma; w) testicular cancers, including classic seminoma, spermatocytic seminoma, testicular stromal tumors (Leydig cell tumors and Sertoli cell tumors), and nonseminomatous germ cell tumors; x) central nervous system cancers, including astrocytomas, medulloblastoma, neuroectodermal tumors, gliomas, oligodendrogliomas, meningioma, germ cell tumors, diffuse intrinsic pontine glioma / brain stem glioma, ependymomas, pineal gland and pituitary gland tumors, primary CNS lymphoma, craniopharyngioma, and Schwannoma.

[0065] It is understood that for each method described herein for the treatment of a solid tumor, there is a counterpart method for the treatment of a solid tumor of each type of cancer listed above using the methods and pharmaceutical agents referenced for the particular method of treatment in question. For instance, there are individual methods for treating each of triple negative breast cancer, inflammatory breast cancer, and ductal or lobular carcinoma of the breast, wherein each method comprises administering to a subject in need thereof a combination of pharmaceutically effective amounts of the C0X2 inhibitor indomethacin and the PD-L1 inhibitor atezolizumab. Similarly, there are methods of treatment of each of triple negative breast cancer, inflammatory breast cancer, and ductal or lobular carcinoma of the breast, wherein each method comprises administering to a subject in need thereof a combination of pharmaceutically effective amounts of a) the C0X2 inhibitor indomethacin, b) the PD-L1 inhibitor atezolizumab, and c) the additional chemotherapeutic agent, nab-paclitaxel.

[0066] The term “refractory” used herein in regard to a cancer refers to a cancer that does not respond to one or more treatments. In some embodiments, the cancer does not respond to one or more chemotherapeutic agents. In other embodiments, the cancer does not respond to radiation therapy. In other embodiments, the refractory cancer does not respond to one or more chemotherapeutic agents and radiation therapy. A refractory cancer may be resistant at the beginning of such treatments or may become resistant over the course of one or more treatments.

[0067] “Androgen-independent prostate cancer” or “hormone-refractory prostate cancer (Al PC)” is prostate cancer that progresses after primary androgen-ablation therapy, either from orchiectomy or a gonadotropin-releasing hormone (LHRH) agonist, followed by addition and subsequent withdrawal of an antiandrogen. In some embodiments hormone-refractory prostate cancer is defined as 2-3 consecutive rises in prostate-specific antigen (PSA) levels obtained at intervals of greater than 2 weeks and / or documented disease progression based on findings from CT scan and / or bone scan, bone pain, or obstructive voiding symptoms. In some embodiments, the PSA level does not rise at diagnosis or throughout the entire course of the disease. In some embodiments, the prostate cancer is an advanced prostate cancer. In some embodiments, the prostate cancer or metastatic prostate cancer is resistant to treatments with hormone-blocking therapies, such as abiraterone (ZYTIGA®), enzalutamide (XTANDI®), bicalutamide (CASODEX®), flutamide (DROGENIL®), or cyproterone acetate (CYPROSTAT®).

[0068] The term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount that is sufficient to effect treatment, as defined below, when administered to a subject (e.g., a mammal, such as a human) in need of such treatment. The therapeutically or pharmaceutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For example, a "therapeutically effective amount" or a "pharmaceutically effective amount" of a compound as described herein, or a pharmaceutically acceptable salt or co-crystal thereof, is an amount sufficient to modulate target expression or activity, and thereby treat a subject (e.g., a human) suffering an indication, or to ameliorate or alleviate the existing symptoms of the indication. For example, a therapeutically or pharmaceutically effective amount may be an amount sufficient to decrease a symptom of a disease or condition responsive to inhibition of COX-2 or PD-L1 activity.

[0069] The term “pharmaceutically acceptable salt” herein refers to salts prepared by conventional methods. These include basic salts of inorganic and organic acids, such as, without limitation, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, and mandelic acid. "Pharmaceutically acceptable salts" of the presently disclosed isotopic compounds also include those formed from cations such as, without limitation, sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reaction of the free acid with a suitable organic or inorganic base. Any chemical isotopic compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts are also inclusive of the free acid, base, and zwitterionic forms of the disclosed isotopic compounds. Descriptions of exemplary pharmaceutically acceptable salts can be found in Stahl and Wermuth, Eds., Handbook of Pharmaceutical Salts; Properties, Selection and Use, Wiley VCH (2008). When the isotopic compounds disclosed herein include an acidic group such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include, without limitation, alkaline, alkaline earth, ammonium, and quaternary ammonium cations. Such salts are known to those of skill in the art. Similarly, when the isotopic compounds disclosed herein include a basic group such as an amino group, then suitable pharmaceutically acceptable anion pairs for the basic group are similarly well known and include halide, hydroxide, perhalate, halite, hypohalite, sulfate, sulfite, phosphate, phosphite, nitrate, nitrite, and others known to those of skill in the art. For additional examples of pharmacologically acceptable salts, see Berge et al, J. Pharm. Sc / . 66, 1 (1977). As example, in embodiments herein, indomethacin and diclofenac are utilized are used in their sodium salt form.

[0070] In instances herein, reference to a particular agent may be limited for brevities sake to the generic name of the compound or the generic name and pharmaceutically acceptable salts thereof. It is understood that each reference, use, method, formation, etc. includes any pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug of the referenced agent.

[0071] Each of the C0X2 inhibiting agents and anti-PD-L1 agents described herein may be administered in pharmaceutical compositions or formulations comprising them known in the art, including those for oral, intravenous, and intramuscular routes of administration.

[0072] Antibody agents described herein may be administered in injectable and infusible compositions known in the art, including, but not limited to, sterile aqueous solutions and sterile sodium chloride solutions optionally containing ingredients such as glacial acetic acid, polysorbate 20, polysorbate 80, sucrose, dextrose, buffers (such as a sodium-acetate buffer, citrate-phosphate buffer, histidine-arginine buffer, etc.), and the like.

[0073] The terms "treatment" or "treating" herein refer to an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of the following: (i) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition, and / or diminishing the extent of the disease or condition); (ii) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition, and / or preventing or delaying the spread (e.g., metastasis) of the disease or condition); and / or (iii) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival).

[0074] The phrases “enhancing the effect of” and “enhancing the activity of’ refer to a first drug or agent’s ability to increase the pharmaceutical effect obtained by a second drug, agent, or combined drug therapy than is seen with the administration of the second drug, agent, or combined drug therapy, by itself. A “C0X2 inhibitor” herein refers to a pharmaceutical agent capable of inhibiting the activity of the cyclooxygenase-2 enzyme, whether or not it also inhibits the activity of one or more other enzymes, such as cyclooxygenase- 1. Examples of C0X2 inhibitors include indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib; or a or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0075] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with measurement of the particular quantity). In some embodiments the term “about” refers to the amount indicated, plus or minus 10%. In some embodiments the term “about” refers to the amount indicated, plus or minus 5%.

[0076] All ranges disclosed and / or claimed herein are inclusive of the recited endpoint and independently combinable. For example, the ranges of "from 2 to 10”, “2 through 10”, and “2- 10” are inclusive of the endpoints, 2 and 10, and all the intermediate values between in context of the units considered. For instance, reference to “Claims 2-10” or “C2-C10 alkyl” includes units 2, 3, 4, 5, 6, 7, 8, 9, and 10, as claims and atoms are numbered in sequential numbers without fractions or decimal points, unless described in the context of an average number. The context of “pH of from 5-9” or “a temperature of from 5°C to 9°C”, on the other hand, includes whole numbers 5, 6, 7, 8, and 9, as well as all fractional or decimal units in between, such as 6.5 and 8.24.

[0077] Material and Methods

[0078] Ex vivo macrophage T cell co-culture assay

[0079] Bone marrow derived macrophages were prepared 5 days prior to setting up a macrophage T cell co-culture assay. Briefly, as illustrated in Figure 1 , bone marrow cells were isolated tibia and femur of hind leg bones of C57BL6 mice using syringe. The cells were then cultured in DMEM (high glucose) with 10% FBS and 10ng / ml CSF1 for 5 days. Adherent cell in bone marrow cell culture after 5 days of CSF1 incubation were used as bone marrow derived macrophages (BMDM). Splenic CD4+or CD8+T cells were isolated by negative selection using antibody cocktails provided in a CD8+T cell or CD4+T cell selection kit (Stem cell inc.). Either a 96 microwell plate or a 384 microwell plate was used for the co-culture assay, which was precoated with CD3 or a combination of CD3 and CD28 antibodies at 5pg / ml concentration at 4 degrees for at least 16 hrs. The CD4+T cells, CD8+T cells, and BMDMs were co-cultured in a 1 :1 :1 ratio in pre-coated microwell plates. These co-cultures were incubated for another 5 days followed by a flow cytometry-based analysis. The iNOS inhibitor, 1400W, N-(3- (aminomethyl)benzyl)acetamide, was used as a positive control for relieving macrophage mediated T cell suppression in the high throughput screening (HTS) assay at 50pM. The indomethacin was used at 1nM. The Prostaglandin E2 (PGE2) (Tocris / R&D) was used at 40 M to analyze its impact on T cell proliferation in the absence of BMDMs.

[0080] HTS assay for screening small molecule library

[0081] A surrogate T cell proliferation assay was derived from the macrophage T cell co-culture assay using luminescence signal generated by ATP release from the cellular content of each well in a microwell plate to perform a high throughput screening (HTS) using a 3D CELLTITER-GLO® Cell Viability Assay (Promega). This luminescence signal was then analyzed to calculate relative change from untreated co-culture wells. 3 replicates were used in the HTS screen to identify “hits”, which increased T cell proliferation in similar manner as the positive control. A macrophage-only plate was utilized to rule out any macrophage dependent spike in the luminescence signal. Cell viability data was collected and % viability was calculated compared to control. Hits were identified using following selection strategy: a. compounds producing no activity in coculture and only activity in macrophage culture were rejected; b. compounds producing tested activity in both the coculture and macrophage-only culture were also rejected; and c. compounds producing the tested activity in coculture but producing no activity in only macrophage culture were accepted as a ‘Hit’ compound.

[0082] Animal tumor models for in vivo studies

[0083] A select group of compounds / drugs were tested in the in vivo mouse tumor models for their anti-tumor activity as well as synergistic action when combined with a PD-L1 inhibiting antibody, RecombiMAb andti-mouse PD-L1 (B7-H1) Clone 10F.9G2-CP168 (BioXCell Catalog No. #CP168, seen online at https: / / bioxcell.com / catalogsearch / result / ?q=10F.9G2-CP168%20). Three different syngeneic orthotopic tumor models were used in these studies; 1) EMT6, 2) 4T1 , and 3) PyMT. The female BalBc mice were used for EMT6 and 4T 1 tumor models whereas female C57BL6 mice were used for the PyMT tumor model. 1 x 106tumor cells were implanted in mammary fat pad and drug treatment began 7 days post tumor implantation. Indomethacin was formulated in 20% captisol and administered by oral gavage 5 days a week at a dose of 3mg / kg of animal body weight. For anti PD-L1 experiments, mice were injected i.p. with aPD-L1 (10F.9G2, Bioxcell) or isotype lgG2b (LTF-2, BioXcell) control antibody at a dose of 250 pg every 4 days. For CD8+T cell depletion experiments mice were injected i.p. with aCD8 antibody (YTS-169.4, Bioxcell), at a dose of 250 pg twice in three days prior to chemotherapy treatment and followed by 250 pg every four days. T umor burden was analyzed using a caliper every 3rdday by measuring longest and shortest length. Tumor sizes were measured using caliper and tumor volume was calculated using formula tumor volume= 0.5 x L * W2. Tumor burden was also measured as weight after euthanasia and tissue harvest. For survival experiments, mice were weighed at the time of randomization into experimental group. Mice were weighed daily while on therapy. Survival endpoints were defined in accordance with OHSU IACUC requirements. A survival endpoint was considered reached when any of the following conditions were met: 1) weight gain or weight loss of > 15% or baseline body weight; 2) development of abdominal swelling; 3) respiratory distress defined as respiratory rate > 50% of baseline; 4) or lack of movement within the cage. Upon reaching a survival endpoint, mice were sacrificed as described above.

[0084] Tissue harvest and multiplexed spectral flow cytometry

[0085] To prepare single cell suspensions of mouse orthotopic mammary tumors, tissues were minced manually and then digested at 37°C for 45 minutes in a solution of 2.0 mg / mL Collagenase A (Roche) and 50 units / mL DNase I (Roche). Cells were filtered through a 70 pm nylon filter and pelleted by centrifugation at 1,200 rpm x 5 minutes, followed by washing 1 x with PBS. Nearly 106cells were then incubated on ice for 30 min in a solution consisting of a 1 : 10 dilution of Fc Receptor Binding Inhibitor (eBiosciences) and 1 :500 Live / Dead Aqua stain (Invitrogen) diluted in PBS. Cells were then incubated with fluorescently-labeled monoclonal antibodies as previously described in a solution of PBS + 5% FCS + 1.0 mM EDTA (FACS buffer). After 30 min incubation on ice, cells were washed 1 x with FACS buffer followed by intracellular staining. The stained cells were treated with permeabilization / fixation buffer (eBioscience) on ice for 10 minutes followed by 1X wash using permeabilization buffer (eBioscience). Cells were then incubated with fluorescently- labeled monoclonal antibodies for intracellular staining in a solution of PBS + 5% FCS + 1.0 mM EDTA (FACS buffer) for 30 minutes on ice. The flow cytometry data was acquired on Cytek Aurora (Cytek biosciences) spectral flow cytometer and data was analyzed using FlowJo software v9.5. Statistical methods

[0086] Statistical analyses were performed using Prism 9 for iMac. Specific tests included Mann- Whitney (unpaired, nonparametric two-tailed), unpaired t test, one-way ANOVA, and Wilcoxon rank-sum.

[0087] Results

[0088] A high throughput screening assay of macrophage dependent T cell suppression ex vivo.

[0089] The TAMs are frequently shown to cause T cell suppression in multiple tumor models where genetic ablation of myeloid genes or inhibition of these gene products led to reversal of T cell suppression. These tumor phenotypes have also been analyzed in highly specific ex vivo assays where adding bone marrow derived macrophages (BMDM) to splenic CD8+T cells cause T cell suppression. These assays have helped in identifying activating or inhibitory cytokines to T cell function in the presence of myelomonocytic cells. We have exploited such an assay to screen drugs, which can relieve myelomonocytic T cell suppression and help expand CD8+T cells. We utilized BMDMs differentiated under CSF1 supplemented culture conditions for 5 days prior to coculturing with spleen derived CD4+and CD8+T cells. After 5 days of co-culturing, CD8+T cells are several thousand-fold less in the presence of BMDMs when compared with CD4+and CD8+T cells co-culture. This suppression was highly reversible as the use of 1400, an iNOS inhibitor, reverted this suppression and augmented it even further. This indicates that if a rate-limiting T cell suppression pathway in BMDM is inhibited, BMDM can accelerate T cell proliferation reminiscent of how dendritic cell activating or myeloid reprogramming therapies enhance T cell response in vivo. These cell number differences in different culture conditions were sufficient to produce significant change in luminescence signal, which we used to convert our flow cytometry-based co-culture assay analysis into a semi-automated high throughput screening assay to analyze -1400 different drugs at 7 different concentrations and in 3 biological replicates. We used BMDM only culture also to screen this library to rule out any signal derived from BMDM expansion. This helped us define a cherry-picking strategy prior to the screening where increased luminescence intensity in BMDM T cell co-cultures and either no change or decrease in signal in BMDM was considered as the “hit”. Using this strategy, we identified 66 compounds out of 1400 drugs, which increased signal in the BMDM-T cell co-cultures and it was either unchanged or decreased in the BMDM only cultures. This library of 1400 different drug is diverse in terms of different pathways these drugs are known to modulate in order to generate therapeutic activity; only -11 % of all drugs in the library are known to be inflammation modulators (Fig. 1). However, - 50% the 66 compounds that were identified using this strategy turned out to be inflammation modulators. This significant enrichment of the activity in inflammation modulators is indicative of the specificity of the assay in displaying myelomonocytic T cell suppression.

[0090] COX2 inhibitors constitute the major class of drugs that increase T cell expansion when combined with anti PD-L1

[0091] We next improved our screen to a) confirm the activity of 66 drugs identified in our primary HTS screen and b) to further identify the drugs, which amplifies this signal further. Out of these 66 drugs, 20 drugs showed an increase of 2-fold or higher. The enrichment of inflammation modulator was further increased in this secondary screen in combination with anti PD-L1 (14 drugs, 70%). Of these 20 drugs, 9 were COX2 inhibitors commonly used for pain relief or to manage hypertension. This indicates that our assay has a greater activity in identifying COX2 dependent changes as well as may yield high efficiency in identifying COX2 modulators. Of all these 20 drugs, Indomethacin was found to be the strongest signal inducer with ~3.5-fold increase in signal. The other major classes of drugs that showed synergy with anti PD-L1 were nucleic acid metabolizing enzymes, JAK inhibitors, oncogenic signaling inhibitors, HDAC inhibitor, or steroids. Indomethacin, among all COX2 inhibitors and other drugs, is the strongest inducer of the signal in the HTS assay in combination with anti PD-L1. We next confirmed that increase signal is also reflective of increased CD8+T cells in the co-culture by flow cytometry. To test this, we used Indomethacin at an HTS assay determined concentration, which generated maximal activity in the HTS assay, to treat BMDM-T cell co-culture and analyzed by flow cytometry. Indomethacin could increase the CD8+T cells in the co-culture in the presence of BMDM as a single agent treatment as well as enhanced it further in combination with anti PD-L1.

[0092] Indomethacin and anti PD-L1 combination therapy regress tumors in TNBC models.

[0093] After ascertaining synergy of Indomethacin and anti PD-L1 in the HTS assay, we decided to test the therapy combination as a cancer treatment in vivo. To do this we used two different syngeneic and orthotopic triple negative breast cancer models, namely 4T 1 and EMT6, widely used for analyzing anti-cancer therapies response. Both indomethacin and anti PD-L1 therapy treatment resulted no significant change as a single agent treatment in 4T1 tumors whereas indomethacin showed significant single agent efficacy in EMT6 tumors. However, the combination of Indomethacin and anti PD-L1 cause significant tumor regression in both tumor models when compared to untreated as well as single agent treatment cohorts. In addition, there was a significant increase in overall survival by combination therapy in both tumor models whereas the longest increase in survival was observed in EMT6.

[0094] Indomethacin and anti PD-L1 combination therapy increases CD8+T cell function in tumors to cause tumor shrinkage

[0095] To analyze whether the mechanism of Indomethacin and anti PD-L1 combination therapy response in vivo is aligned with our drug screening strategy, we assessed two key properties highlighted from our screen results 1) whether myelomonocytic cells express the COX2 and 2) the blockade of C0X2 and PD-L1 should increase CD8+T cell function in tumors. We used a highly multiplexed spectral flow cytometry using 27 different immune cell markers to analyze immune complexity of the TME of 4T1 and EMT6 tumors. To address which cell type express C0X2 the most, we first defined CD45' and CD45+leukocytes and subsequently identified all immune subpopulations by canonical gating (Fig.). The 4T1 tumors show a very high frequency (~ 50% of all CD45+leukocytes) of immature myelomonocytic cells expressing Ly6C and Ly6G and F4 / 80+TAMs being the second most frequent subpopulation of CD45+leukocytes. Interestingly, COX2 expression was found to be the highest in F4 / 80+TAMs among all different cell types indicating that COX2 dependent tissue change in tumor can indeed be primarily emanating from TAMs (Fig.). Next, we analyzed the change in immune complexity of 4T1 and EMT tumors by Indomethacin and anti PD-L1 treatment as single agent or the combination. Indomethacin increased total CD8+T cells in 4T1 tumors as a single agent but surprisingly not by the combination therapy. However, both the GzmB expressing and TCF1 expressing CD8+T cells were significantly increased by the combination therapy. We also found significant changes in the frequency of key innate immune cells; an increase in Ly6+ monocytes by combination therapy, a decrease in Ly6G+ granulocytes by indomethacin, a significant increase in monocytic dendritic cells by the combination therapy. Interestingly, anti PD-L1 significantly increased the frequency of F4 / 80+TAMs that was subverted by the indomethacin treatment. Indomethacin also changes TAM phenotype to align with TH1 characteristics of immune activation where we observed an increase in the expression of PD-L1 , MHCII, and COX2. These changes led us to hypothesize that therapeutic benefits of combination blockade of COX2 and PD-L1 in vivo are dependent on CD8+T cells. To test this hypothesis, we critically ablated CD8+T cells in EMT6 tumor bearing animals using anti CD8 antibody. The CD8+T cells depletion did not affect tumor growth by itself but completely reverted benefits of the combination therapy and EMT6 tumor showed no regression whatsoever in CD8+T cell deficient animals. Myelomonocytic cells regulate T cell function by COX2-PGE2 pathway

[0096] Since we identified indomethacin in our HTS assay due to increase in T cell activity in the presence of the BMDMs, we hypothesized that indomethacin is inhibiting C0X2 activity in macrophages and thereby decreasing the PGE2 production and its release in co-cultures. If this hypothesis is true, it would mean that PGE2 would directly inhibit CD8+T cell function in the similar manner as BMDMs inhibit CD8+T cells in the HTS assay. Therefore, instead of adding BMDMs to the CD4+ and CD8+ T cell co-cultures we added purified prostaglandin to the co-culture and analyzed T cell expansion over the period of 5 days. Prostaglandin treatment was equally powerful in reducing any T cell expansion in these co-cultures in response to CD3 stimulation or even a stronger and more robust stimulation by CD28 and IL2 supplementation. When we analyzed C0X2 expression in BMDMs, it was only detectable in the classically activated macrophages stimulated either by a TLR4 ligand or interferon gamma (IFNg). This indicates that BMDMs elevates C0X2 expression in co-culture and regulate T cells expansion in a feed-back manner. Indeed, BMDM increases TH1 dependent gene expression in T cell co-cultures when compared to BMDM grown by themselves. Interestingly, TAMs in both human and mouse tumor models show high degree of C0X2 expression that is also correlated with increased MHCII expression and PD-L1 expression suggestive of that indeed C0X2 dependent T cell regulation could be induced in TME in a feed-back manner in response an intertumoral T cell infiltration and activation. In human TNBC patient cohort C0X2 was also strongly correlated with PD-L1 expression and further showed positive association with improved OS. This is similar to the findings of recent trial that led to the ICI approval that the PD-L1 expression is the highest in the patient, which responded better with the use of ICI.

[0097] Discussion

[0098] A slow but steady development and approval of targeted therapies for TNBC have curated a combination therapy regimen, which is increasing life expectancy of TNBC patients and the use of ICI will help in extending it further. However, these developments remain well below the expectations of modern cancer medicine to cure cancer. We hope to improve these outcomes by identifying combination therapy partners for the new “standard of care” of TNBC consist of an ICI. Our simplified yet targeted HTS assay, which mimic macrophage dependent T cell suppression in cancer is important in identifying such combination with ICI due to 1) the direct approach of the HTS assay to screen drugs in combination setting with anti PD-L1 to discover synergistic agents for relieving macrophage dependent T cell suppression, 2) the scope of identifying the novel regulatory mechanisms in macrophages that orchestrate T cell function in tumors, and 3) the high fidelity of the signal to noise ratio to be associated with increased T cell activity rather than due to myelomonocytic cell expansion. The high fidelity of the screen to identify synergistic myeloid targeting agents to combine with anti PD-L1 accelerate the discovery process while also increase the probability of success in in vivo testing of these agents as combination therapy with PD1 / PD- L1 inhibitors. When applied to a diverse set of drugs or small molecules modulating known molecular mechanisms and signaling pathways, such HTS assay can also help discover a novel molecular biology by tracking the knowledge associated with each drug in the library that may produce signal. These 3 attributes of our HTS assay helped us in narrowing upon the candidate for the combination therapy with anti PD-L1 ; since Indomethacin, our top hit identified from the combination therapy screen, indeed show a deep and durable response in reducing tumor burden in TNBC models when combined with anti PD-L1.

[0099] Indomethacin is a non-steroidal anti-inflammatory drug (NSAID), which inhibits C0X1 and C0X2 enzymes involved in lipid metabolism of the cell and thereby inhibit the release of paracrine mediators known as prostaglandins in extracellular tissue microenvironment. Similar to TAMs, C0X1 / 2 are shown to be involved in tumorigenesis and cancer progression. Several studies predicted the use of NSAIDS as preventive medicine however with variable outcomes of the controlled prospective clinical testing of the aspirin and Ibuprofen (ref.). More recently, prostaglandin receptors were shown to be expressed on PD1 expressing dysfunctional CD8+T cells in an LCMV model and shown to be rate-limiting in causing T cell dysfunction by PD1. These changes however are not seen in the context of the immuno-oncology and the source of prostaglandin remain unidentified in both viral and tumor immunology context. Interestingly, when tested as ad-hoc analysis of ICI treated cancer patients, patients who used NSAIDS for different reasons e.g., pain show significantly improved ICI response. Our study not only identify the COX2 biology as important regulators of anti PD-L1 therapy response but also identify key C0X2 targeting drugs that can be used for this purpose. Moreover, we show that a TH1 oriented tumor microenvironment harbors COX2 expressing myelomonocytic cells indicative of the feedback regulation of the T cell response. Indeed, C0X2 expression is tightly regulated by the activation of interferon and toll like receptor (TLR) signaling in macrophages and its activity is further dependent upon nitric oxide production: nitric oxide synthase is another key enzyme strongly associated with classical activation of the macrophages (ref.). Interestingly, C0X2 expression in cancer in general and TNBC is associated with TH1 signature and PD-L1 expression with strong correlation with improved OS. Interestingly, in the checkmate trial...the best response was seen in the patient with high percent of PD-L1 expression in the TME. Therefore, in the wake of our results from present study and previously published analyses, we predict that TNBC patients who show high PD-L1 and CCOX2 expression will be benefited with the use of combination targeting of COX2 using Indomethacin and PD1 / PD-L1 inhibiting ICI. This study shows how a rationalized screening of drugs targeting ICI resistance pathways can unearth new combination therapy partners where identifying rate-limiting cell interaction that regulate cytotoxic T cell response may be the key to success in developing a high yield HTS assay. Moreover, our study opens new frontier in explicitly showing enzymes expressed in TH1 conditions, and not necessarily immunosuppressive T 2 conditions, as targets for combination therapy with ICI where the enzymes such as COX2 or the upstream regulators e.g., NOS2 can provide critical therapy alternatives to harness the maximal immune activity for anti-cancer therapy.

[0100] Detailed Description of the Figures

[0101] Figure 1 presents a schematic representation of the collection of cells and creation of the macrophage and macrophage / CD4 / CD8 cultures as described above in detail.

[0102] Figure 2 presents a schematic representation of the assay principle of ex vivo screening of immunomodulator agents using an automated high-throughput method. In the co-culture of bone-marrow-derived macrophages with spleen-derived CD4 and CD8 cells, bone marrow derived macrophages (BMDMs) suppress CD8 cell proliferation. Treatment with small molecules, such as 1400W, may reverse the suppression.

[0103] Figure 3 presents a further schematic representation of the assay principle of an ex vivo assay to analyze macrophage-mediated T-Cell suppression. The co-culture of bone-marrow derived macrophages with spleen-derived CD4 and CD8 cells, BMDMs suppress CD8 cell proliferation, which, as just described, may be reversed with small molecule suppression, such as that of 1400W. This provides a platform to launch a chemical biology approach for identifying molecular targets and novel therapeutics.

[0104] Figures 4A and 4B concern the replacement of a flow cytometry analysis with a cell viability assay to convert the co-culture assay from low throughput to high throughput. The basic principle we adopted is that, in drug treatment, the macrophage-induced suppression is removed, then T cells will start proliferating. This proliferation can be captured using ATP-based cell viability assay (Cell-titer gio assay). The graph in Figure 4A represent the results of cell viability assay. Co-culture of CD4 and CD8 cells lead to the expansion of T-cell cells and the addition of BMDMs suppresses the T-cell proliferation. The graph in Figure 4B demonstrates that 1400W treatment reverses the BMDMs induced suppression and uses cell viability-based readout using Cell-titer gio to depict the outcome closer to flow cytometry-based analysis. Figure 5 presents a cherry-picking strategy using T-cell co-cultures with macrophages and collections of macrophages, alone. Agents presenting no activity in the co-culture, with the only activity seen in the macrophage culture are rejected. Also rejected are agents presenting activity in both the co-culture and macrophage culture. Agents were accepted as hits when they presented activity in the co-culture, but none in the macrophage culture.

[0105] Figures 6A and 6B represent the high-throughput screening results. Figure 6A presents a pie chart classifying 1430 FDA-approved drugs in a library which is divided into the nine broader drug classes indicated in the table below. 66 drugs were identified from primary screening, with the distribution graph of 66 hits representing the enrichment of each class in output results indicated in Figure 6B.

[0106] Figure 7 represents a secondary screen used to confirm the “hits” and to identify combination therapy partner agents (screened 66 “hits”). In this secondary co-culture assay, 66 hits were identified in the primary screening with 1430 FDA-approved drugs on BMDMs+CD4+CD8 coculture. In secondary screening, coculture was treated with anti-PDL1 antibodies along with 66 FDA-approved drugs. Figure 7 presents a graph of proliferation (fold change) hits obtained from secondary screening. The graph represents FDA-approved drugs that sensitize the coculture for anti-PDL1 treatment. Bars are in ascending order of sensitizing effect.

[0107] Figure 8 presents a brief representation of the confirmation of the activity of C0X2 inhibitor indomethacin with the anti-PD-L1 antibody.

[0108] Figure 9 presents an overview of the 4T1 syngeneic TNBC model. In total, 1 x 1054T1 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1 . The line graph presents tumor growth kinetics: tumor volumes for each group’s tumors. Data are shown as the mean ± SD; *P< 0.05, **P< 0.01 and ***p< 0.001 significantly different compared with the corresponding control.

[0109] Figure 10 presents a representation of in vivo confirmation of indomethacin activity as combination therapy using anti-PD1 antibody. In total, 1 x 1054T1 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGa antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PD1 250 ug / mouse or COXi + Anti-PD1. The upper panel provides a schematic representation of treatment plan and the line graph represents tumor growth kinetics using tumor volumes for each group’s tumors.

[0110] Figure 11 represents in vivo confirmation of indomethacin activity as combination therapy using an anti-PD-L1 antibody. For the survival study, in total, 1 x 1054T1 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1. At the end of treatment, each mouse was followed until they rechies to euthanasia point. Overall survival curves in mice treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1. Survival expressed in percentage and time in days.

[0111] Figure 12 further represents in vivo confirmation of indomethacin activity as combination therapy using an anti-PD-L1 antibody. In total, 1 x5EMT6 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti- PDL1 . The upper panel provides a schematic representation of treatment plan and the line graph represents tumor growth kinetics through tumor volumes for each group’s tumors.

[0112] Figure 13 provides bar graphs representing immune complex analysis of 4T1 tumor samples: 1 * 1054T1 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1 . At the end of treatment tumors were collected and single cells were isolated. Single cell suspension was stained with 27 antibodies panel and analyzed using flow cytometer. Graph represent the % population of each immune cell type present in each treatment group.

[0113] Figure 14 provides bar graphs representing immune complex analysis of 4T 1 tumor samples: 1 x 1054T1 cells were implanted subcutaneously in 4thmammary fat pad of female Balb / c mice. Mice were randomized and divided into four groups. Each group was treated with Anti-IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1 . At the end of treatment tumors were collected and single cells were isolated. Single cell suspension was stained with 27 antibodies panel and analyzed using flow cytometer. Graph represent the % population of each immune cell type present in each treatment group.

[0114] Figures 15A and 15B represent data from our PY8119 xenograft study. In total, 1 x 105PY8119 cells were implanted subcutaneously in 4thmammary fat pad of female C57BL / 6 mice. Mice were randomized and divided into four groups. Each group was treated with Anti- IgGb antibody 250 ug / mouse or COXi (indomethacin) 3 mg / kg & anti-IgGa or Anti-PDL1 250 ug / mouse or COXi + Anti-PDL1. The line graph plots tumor growth kinetics: tumor volumes for each group’s tumors. Data are shown as the mean ± SD; *P< 0.05, **P< 0.01 and ***p< 0.001 significantly different compared with the corresponding control.

[0115] Additional Material and Methods

[0116] Ex vivo macrophage T cell co-culture assay

[0117] Bone marrow-derived macrophages (BMDM) were prepared 5 days prior to establishing a macrophage-T cell co-culture assay, as previously described (Weischenfeldt and Porse, 2008). Briefly, bone marrow cells were isolated from the tibia and femur of hind legs of C57BL6 mice using a syringe. Cells were then cultured in DMEM (high glucose) with 10% FBS and 10 ng / ml CSF-1 for 5 days. Adherent cells were then used as BMDMs. Splenic CD4+or CD8+T cells were isolated from C57BL6 mice by negative selection using antibody cocktails provided in a CD8+T cell or CD4+T cell selection kit. Either a 96 microwell plate or a 384 microwell plate were used for co-culture assays, where plates were pre-coated with aCD3 or a combination of aCD3 and aCD28 antibodies at 5.0 pg / ml concentration at 4°C for at least 16 hrs prior to cell addition. CD4+T cells, CD8+T cells, and BMDMs were then co-cultured in a 1 :1 :1 ratio in these pre-coated microwell plates at 37°C for another 5 days followed by flow cytometry-based analyses. The iNOS inhibitor, 1400W, was used as a positive control for relieving macrophage-mediated T cell suppression in the HTS assay at 50 pM, as previously reported(Doedens et al., 2010). Indomethacin was used at 1.0 nM. PGE2 (Tocris) was used at 40 pM to analyze its impact on T cell proliferation in the absence of BMDMs.

[0118] HTS assay for screening small molecule library

[0119] A surrogate T cell proliferation assay was developed utilizing the macrophage-T cell co-culture assay. Luminescence signal generated by ATP release from cellular contents of each well in a microwell plate for high-throughput screening (HTS) using 3D cell titre gio (Promega); luminescence signal was then analyzed to calculate relative change from untreated co-culture wells. Three replicates were used in the HTS screen to identify “hits” defined as increased T cell proliferation, in a similar manner as the 1400w-treated conditions. A BMDM only plate was utilized to rule out any BMDM-dependent spike in luminescence signal. Data for cell viability was collected and % viability calculated compared to control. ‘Hits’ were identified using the following selection strategy: 1) no activity in coculture and only BMDM culture (rejected); 2) activity in both coculture and only BMDM culture (rejected); 3) Activity in coculture with no activity in BMDM only culture (accepted as ‘Hit’). Cells were plated on day 1 on CD3-precoated white 384 well plates using a BioTek Multiflo FX fluid dispenser and incubated overnight at 37°C. On day 2, cells were drugged with SCREEN-WELL FDA-approved drug library V2 (Enzo Life Sciences Inc. # BML-2843) using Sciclone ALH3000 liquid handler with 384-well mandrel and Z-8. The library was in quantitative high-throughput screening (qHTS) format with 8 half-log dilutions from 10 pM to 3.0 nM. During the secondary screening, cells were drugged using HP D300e digital dispenser using T8+ dispense cassettes. After five days of incubation, 10 pL Cell titer Gio was added to each well using a BioTek Multiflo FX fluid dispenser and incubated for 10 minutes at 37°C. Luminescence signals were measured using Twister II microplate handlers with an integrated barcode reader, and BioTek Synergy 4 microplate reader using Gen5 software.

[0120] Animal tumor models for in vivo studies

[0121] 4T1 (Cat. No. # CRL-2539) and EMT-6 (Cat. No. # CRL-2755) were purchased from ATCC, USA. 4T1 cells were cultured in RPMI-1640 growth medium supplemented with 10% FBS and 1 % PSA at 5% CO2 and 37°C. EMT-6 cells were cultured in Waymouth's MB 752 / 1 medium supplemented with 2.0 mM L-glutamine, 15% FBS and 1% PSA at 5% CO2 and 37°C. Animal studies were approved by OHSU’s Institutional Animal Care and Use Committee (elACUC number: IP00003247). To generate all in vivo data, wild-type female Balb / cJ mice were purchased from the Jackson Laboratory and maintained within the OHSU animal care barrier facility according to IACUC procedures. Two different syngeneic orthotopic tumor models were used in these studies, e.g., EMT6 and 4T1 , where 1.0 x 105tumor cells were implanted in mammary fat pads and drug treatment began 7 days post tumor implantation. Indomethacin was formulated in 20% captisol and administered by oral gavage 5 days a week at a dose of 3.0 mg / kg of animal body weight. For O.PD-L1 mAb studies, mice were injected i.p. with aPD-L1 (10F.9G2, BioXcell) or isotype lgG2b (LTF-2, BioXcell) control antibody at a dose of 250 pg every 4 days until endpoint. For CD8+T cell depletion, mice were injected i.p. with aCD8 mAb (YTS-169.4, BioXcell), at a dose of 250 pg twice in three days prior to treatment. Tumor burden was analyzed using electronic calipers every 3rdday post implantation by measuring longest and shortest tumor lengths. Tumor volumes were calculated using the formula tumor volume= 0.5 x L x w2. Terminal tumor burden was measured as weight after euthanasia and tissue harvest. For survival studies, mice were weighed at the time of randomization into experimental groups and weighed daily while on therapy. Survival endpoints were defined in accordance with OHSU IACUC requirements. A survival endpoint was considered reached when any of the following conditions were met: 1) weight gain or weight loss of > 15% of baseline body weight; 2) development of abdominal swelling; 3) respiratory distress defined as respiratory rate > 50% of baseline; 4) diminished cage mobility. Upon reaching a surrogate survival endpoint (per IACUC guidelines), mice were sacrificed per OHSU IACUC protocols.

[0122] Tumor harvest and multiplexed spectral flow cytometry

[0123] T 0 prepare single cell suspensions from murine orthotopic mammary tumors, tissues were minced manually and then digested at 37°C for 45 minutes in a solution of 2.0 mg / mL Collagenase A (Roche) and 50 units / mL DNase I (Roche). Cells were filtered through a 70 p.m nylon filter and pelleted by centrifugation at 1 ,200 rpm x 5 minutes, followed by washing 1x with PBS. Nearly 106cells were then incubated on ice for 30 min in a solution consisting of a 1 : 10 dilution of Fc Receptor Binding Inhibitor (eBiosciences) and 1 :500 Live / Dead Aqua stain (Invitrogen) diluted in PBS. Cells were then incubated with fluorescently-labeled monoclonal antibodies as previously described(Ruffell et al., 2012) in a solution of PBS, 5% FCS, 1.0 mM EDTA (FACS buffer). After 30 min incubation on ice, cells were washed 1 x with FACS buffer followed by intracellular staining. The stained cells were treated with permeabilization / fixation buffer (eBioscience) on ice for 10 minutes followed by 1x wash using permeabilization buffer (eBioscience). Cells were then incubated with fluorescently labeled monoclonal antibodies for intracellular staining in a solution of PBS, 5% FCS, 1 .0 mM EDTA (FACS buffer) for 30 minutes on ice. Flow cytometry data was acquired on a Cytek Aurora (Cytek biosciences) spectral flow cytometer and data analyzed using FlowJo software v9.5.

[0124] Statistical Analysis

[0125] Statistical analyses were performed using Prism 9 for Mac. Specific tests included Mann-Whitney (unpaired, nonparametric two-tailed), unpaired t test, one-way ANOVA, and Wilcoxon rank-sum as indicated in Figure Legends.

Claims

What is claimed:

1. A method of treatment of a solid tumor in a human subject, the method comprising administering to the subject in need thereof: i) a pharmaceutically effective amount of at least one C0X2 inhibitor selected from the group of indomethacin, meloxicam, tenoxicam, lornoxicam, diclofenac, tolfenamic acid, etoricoxib, parecoxib, and rofecoxib, or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof; and j) a pharmaceutically effective amount of a PD-L1 inhibitor selected from the group of atezolizumab (MPDL3280A), avelumab, durvalumab, envafolimab (KN035 or ASC22), cosibelimab (CK-301), socazolimab (ZKAB001), MEDI4736, STI-3031 , and LY3300054.

2. The method of Claim 1 , wherein the at least one C0X2 inhibitor is indomethacin.

3. The method of Claim 2, wherein the indomethacin is administered to the subject in need thereof at an effective daily dose of from about 25 mg per day to about 250 mg per day in a single dose or in divided doses.

4. The method of Claim 3, wherein the PD-L1 inhibitor is selected from the group of atezolizumab, avelumab, durvalumab.

5. The method of Claim 4, wherein the PD-L1 inhibitor is atezolizumab administered to the subject in need thereof at an effective daily dose of from about 500 mg to about 1 ,400 mg.

6. The method of Claim 3, wherein the PD-L1 inhibitor is avelumab administered to the subject in need thereof at an effective daily dose of from about 5 mg / kg to about 20 mg / kg.

7. The method of Claim 6, wherein avelumab is administered to the subject in need thereof at an effective daily dose of 500 mg to about 1 ,000 mg.

8. The method of Claim 3, wherein the PD-L1 inhibitor is durvalumab administered to the subject in need thereof at an effective daily dose of from about 500 mg to about 2,000 mg.

9. The method of Claim 1 , wherein the solid tumor is a cancerous solid tumor selected from the group of cancers including:a) breast cancers, including triple negative breast cancer, inflammatory breast cancer, ductal or lobular carcinoma, ductal carcinoma in situ, invasive breast cancer, Paget disease of the breast, angiosarcomas of the breast, and Phyllodes tumors; b) thyroid cancers, including papillary, follicular, medullary, anaplastic thyroid cancers, and Hurthle cell cancer and parathyroid cancers (including parathyroid adenomas); c) mesotheliomas, including pleural mesothelioma, peritoneal mesothelioma, pericardial mesotheliomas, epitheliod mesotheliomas, sarcomatoid mesotheliomas, biphasic mesotheliomas, and dermoplastic mesotheliomas; d) head & neck cancers, including salivary gland cancer, glioblastoma multiforme, paragangliomas, retinoblastomas, laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancers, nasopharyngeal cancer, oral and oropharyngeal cancer; e) ovarian cancers, including germ cell tumors, stromal cell tumors, epithelial ovarian carcinomas, and borderline ovarian tumors; f) cervical cancers, including squamous cell carcinoma, cervical adenocarcinomas, small cell cervical cancer (SCCC), and large cell cervical cancer (LCCC); g) vaginal cancers, including vaginal adenocarcinomas, verrucous carcinoma, clear cell adenocarcinoma, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, vaginal sarcomas, vaginal embryonal rhabdomyosarcoma, vaginal melanomas, small cell cancer of the vagina, and vaginal lymphoma; h) skin cancers, including squamous cell cancer of the skins, basal cell carcinoma (BCC) large cell carcinomas, Merkel Cell carcinoma, and cutaneous T-cell lymphoma; i) neuroendocrine malignancies, including carcinoid tumors of the lung, Gl tract, or thymus; pancreatic neuroendocrine tumors and islet cell cancer; medullary thyroid carcinoma; Merkel cell carcinoma; pheochromocytoma of the adrenal gland; adrenal cancer; small cell carcinomas, and large cell carcinoid tumors; j) melanomas, including superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma; k) multiple myelomas, including light chain myeloma, non-secretory melanoma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undeterminedsignificance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma, and immunoglobulin E (IgE) myeloma; l) lung cancers, including non-small cell lung cancer, small cell lung cancer, and carcinoid lung cancer; m) renal cancers, including renal cell carcinoma and Wilms tumors; n) prostate cancers, including androgen-independent prostate cancer, hormone-refractory prostate cancer (Al PC)), sarcoma tumors of the prostate, prostate small cell carcinoma, transitional cell carcinoma, prostate squamous cell carcinoma, and adenocarcinomas of the prostate, such as acinar adenocarcinoma and ductal adenocarcinoma of the prostate; o) pancreatic cancers, including exocrine / nonendocrine pancreatic cancer, pancreatic adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, colloid carcinoma, pancreatic neuroendocrine tumors, and benign precancerous pancreatic lesions; p) bladder cancers, including urothelial or transitional cell bladder cancer, squamous cell bladder cancer, adenocarcinomas of the bladder, muscle invasive bladder cancer, non-muscle invasive bladder cancer, papillary bladder cancer, carcinoma in situ of the bladder, high grade T 1 bladder tumors, and secondary cancers of the bladder originating in the prostate, rectum, ovary, cervix, womb, or other organs or tissues; q) gastrointestinal cancers, including gastric cancer, gastrointestinal stromal tumors, colorectal cancer, esophageal cancers, neuroblastoma, anal cancer, small intestine cancer, stomach cancer, gallbladder cancer r) hepatocellular cancers, including liver cancer and bile duct cancer; s) adenocystic cancers, including cribiform, tubular, and solid adenoid cystic carcinomas; t) adrenal cancers, including adrenocortical carcinoma, adenomas, neuroblastomas, and pheocromocytomas; u) sarcomas, including Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, alveolar soft- part sarcoma, clear cell sarcoma, chondrosarcoma, dermatofibrosarcoma protuberans, epitheliod sarcoma, fibrosarcoma, gastrointestinal stromal tumors (GIS), Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, synovial cell sarcoma, undifferentiated pleomorphic sarcoma (UPS) / malignant fibroushistiocytoma (MFH), uterine sarcoma, vascular sarcomas, and desmoplastic small round cell tumors (DSRCT); v) solid lymphomas, including Hodgkin lymphoma, non-Hodgkin lymphoma; w) testicular cancers, including classic seminoma, spermatocytic seminoma, testicular stromal tumors (Leydig cell tumors and Sertoli cell tumors), and nonseminomatous germ cell tumors; x) central nervous system cancers, including astrocytomas, medulloblastoma, neuroectodermal tumors, gliomas, oligodendrogliomas, meningioma, germ cell tumors, diffuse intrinsic pontine glioma / brain stem glioma, ependymomas, pineal gland and pituitary gland tumors, primary CNS lymphoma, craniopharyngioma, and Schwannoma.

10. The method of Claim 9, wherein the solid tumor is a breast cancer solid tumor.

11. The method of Claim 10, wherein the solid tumor is a triple negative breast cancer solid tumor.

12. The method of Claim 9, wherein the solid tumor is a head and neck cancer solid tumor.

13. The method of Claim 9, wherein the solid tumor is an ovarian cancer solid tumor.

14. The method of Claim 9, wherein the solid tumor is a melanoma solid tumor.

15. The method of Claim 9, wherein the solid tumor is a non-small cell lung cancer solid tumor.

16. The method of Claim 9, wherein the solid tumor is a prostate cancer solid tumor.

17. The method of Claim 9, wherein the solid tumor is a gastrointestinal cancer solid tumor.

18. The method of Claim 9, wherein the solid tumor is a colorectal cancer solid tumor.

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