WO2022125829A1 - Treatments for advanced and/or metastatic triple negative breast cancer - Google Patents

Treatments for advanced and/or metastatic triple negative breast cancer Download PDF

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WO2022125829A1
WO2022125829A1 PCT/US2021/062696 US2021062696W WO2022125829A1 WO 2022125829 A1 WO2022125829 A1 WO 2022125829A1 US 2021062696 W US2021062696 W US 2021062696W WO 2022125829 A1 WO2022125829 A1 WO 2022125829A1
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administered
patient
gemcitabine
carboplatin
trilaciclib
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French (fr)
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Jessica A. SORRENTINO
Andrew BEELEN
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G1 Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention is in the area of improved treatments for a select group of difficult to treat patients with advanced/metastatic triple negative breast cancer (TNBC), including patients whose TNBC has advanced while receiving an immune checkpoint inhibitor or whose TNBC is PD-L1 negative.
  • TNBC advanced/metastatic triple negative breast cancer
  • TNBC Triple negative breast cancer
  • onset at a younger age has been characterized by several aggressive clinicopathologic features, including onset at a younger age; large, high-grade tumors; and a propensity for visceral metastasis (Cheang et al., Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 2008; 14(5): 1368-76; Foulkes et al., Triple-negative breast cancer. N Engl J Med. 2010 Nov 11;363(20): 1938-48).
  • Treatments which are effective for hormone receptor-positive breast cancer and human epidermal growth factor receptor 2 (HER2)-positive breast cancer, such as endocrine therapy or HER2-targeted therapies are not effective in TNBC, which lacks these markers; as such, chemotherapy remains the mainstay of treatment for TNBC.
  • chemotherapies that target deoxyribonucleic acid (DNA) repair e.g., platinum compounds
  • cell proliferation e.g., taxanes and anthracyclines, like doxorubicin
  • Toxicity issues include chemotherapy-induced immunosuppression and myelosuppression (CIM), which may also affect anti-tumor efficacy due to an inability of the host immune system to effectively mount a response against the cancer.
  • CIM myelosuppression
  • Targeted therapies have greatly improved outcomes for hormone receptor-positive and HER2 positive breast cancer, however, there is a paucity of effective targeted therapies in TNBC.
  • pharmacological blockade of programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor has been at the forefront of immunotherapy for various cancers, as it facilitates efficacious anti-tumor immune responses by reinvigorating previously exhausted T cells.
  • the PD-L1 positive subset lived for an average of 25 months when treated with atezolizumab plus nab-paclitaxel, compared with 18 months when given placebo plus nab- paclitaxel.
  • the efficacy improvement observed with the addition of atezolizumab to nab-paclitaxel was associated with immune related adverse events that occurred at relatively low frequencies, which could cause significant morbidity and mortality.
  • Pembrolizumab (KEYTRUDA®) monotherapy has also demonstrated improved objective response rates (ORR) in patients with PD-L1 positive TNBC tumors ranging from 18.5% to 21.4%, compared with response rates ranging from 5.3% to 9.6% in patients with PD-L1 negative tumors.
  • ORR objective response rates
  • Major factors contributing to PD-1/PD-L1 blockade resistance include constitutive PD-L1 expression in cancer cells, lack of tumor antigens, ineffective antigen presentation, activation of oncogenic pathways, mutations in IFN-y signaling, and factors within the tumor microenvironment including exhausted T cells, Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) (Bai et al., Regulation of PD-1/PD-L1 pathway and resistance to PD-1/PD-L1 blockade. Oncotarget. 2017;8(66): 110693-110707).
  • ICI resistance Additional mechanisms include genetic, epigenetic, and cellular signaling alterations that dysregulate neoantigen presentation/processing and disrupt cytotoxic T cells activity as well as mechanisms in which non-cancerous stromal or immune cells promote growth and resistance to ICIs (Liu et al., Mechanisms of resistance to immune checkpoint blockade. Am J Clin Dermatol. 2019;20(l):41-54; Barrueto et al., Resistance to checkpoint inhibition in cancer immunotherapy. Transl. One. 2020;13 : 100738; Jenkins et al., Mechanisms of resistance to immune checkpoint inhibitors. Brit J Cancer.
  • the present invention provides improved methods for treating advanced/metastatic triple negative breast cancer (TNBC) in specific, select patient subgroups by administering the short acting, selective, and reversible cyclin dependent kinase (CDK) 4/6 inhibitor trilaciclib, or a pharmaceutically acceptable salt thereof, in a specifically timed therapeutic protocol with select chemotherapeutic agents.
  • TNBC triple negative breast cancer
  • CDK cyclin dependent kinase
  • the administration of the selective CDK4/6 inhibitor trilaciclib provides for improved survival outcomes, including overall survival (OS) and/or progression free survival (PFS) for these difficult to treat patients.
  • OS overall survival
  • PFS progression free survival
  • the improved treatments are for a select group of difficult to treat patients with advanced/metastatic TNBC who have prior exposure to an immune checkpoint inhibitor (ICI), for example, a programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who have developed therapeutic resistance to the immune checkpoint inhibitor leading to disease progression after an initial response.
  • an immune checkpoint inhibitor for example, a programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor
  • ICI immune checkpoint inhibitor
  • PD-1 programmed cell death protein-1
  • PD-L1 programmed death-ligand- 1
  • the patient has a TNBC tumor that is PD-L1 positive.
  • ICI administration to patients with PD-L1 -status positive advanced/metastatic TNBC represents a recent evolution in TNBC treatment options.
  • Mechanisms of ICI resistance include genetic, epigenetic, and cellular signaling alterations that dysregulate neoantigen presentation/processing and disrupt cytotoxic T cell activity as well as mechanisms in which non- cancerous stromal or immune cells promote growth and resistance to ICIs.
  • the development of ICI resistance with resultant TNBC progression renders such patients particularly difficult to treat, with limited additional targeted therapies available.
  • ICI resistance and disease progression can be overcome, resulting in increased antigen presentation (major histocompatibility complex (MHC) class I), enhanced T cell clonality and tumor infiltration, inhibition of regulatory T cell proliferation, decreased expression of T cell exhaustion markers (programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain 3 (TIM3)), stabilized expression of PD-L1 on tumor cells, promotion of dendritic cell migration, or increased T-effector cell function through high interferon -gamma (IFN-y) production.
  • MHC major histocompatibility complex
  • the immunosuppressive tumor microenvironment in the patient’s tumor which renders the previously administered ICI ineffective or less effective and allows the tumor to progress — can be significantly overcome, improving the ability of the patient’s immune system to reduce or control tumor burden, improving quality of life, and improving overall survival in these difficult to treat subsets of patients.
  • the improved methods of treatment in a patient in need thereof include the administration of an effective amount of trilaciclib in combination with an effective amount of gemcitabine and carboplatin (or alternatively, an alternative platinum chemotherapeutic agent, including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), which is administered to a select patient with advanced/metastatic TNBC who has prior exposure to an immune checkpoint inhibitor (ICI), for example, a programmed cell death protein-1 (PD-1) inhibitor and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who has developed therapeutic resistance to the ICI leading to disease progression.
  • ICI immune checkpoint inhibitor
  • PD-1 inhibitor programmed cell death protein-1
  • PD-L1 inhibitor programmed death-ligand- 1
  • TNBC patients undergoing second-line therapy have had a very poor prognosis (PFS duration of 2 to 3 months and OS duration of 9 to 12 months). It has been discovered that trilaciclib in combination with gemcitabine and carboplatin provides a novel therapeutic option for these patients who have minimal options available, including the recently emergent subgroup of patients having PD-Ll-status positive TNBC who have progressed following first-line treatment with an ICI, for example nivolumab, atezolizumab, avelumab, durvalumab, or pembrolizumab, or other ICI. In prior studies evaluating the use of trilaciclib in TNBC chemotherapeutic protocols (e.g., Clinical Trial NCT02978716), this newly emergent subset of patients did not form part of the study analysis.
  • an ICI for example nivolumab, atezolizumab, avelumab, durvalumab, or pembrolizumab, or other ICI.
  • a method of treating a patient in a second-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC comprising administering to the patient an effective amount of trilaciclib in a chemotherapeutic protocol, wherein the chemotherapeutic protocol further comprises the administration of gemcitabine and carboplatin (GC), and wherein the patient’s TNBC is PD-L1 status positive and the patient’s tumor has advanced or progressed following administration of an immune checkpoint inhibitor in a first- line setting.
  • GC gemcitabine and carboplatin
  • the method comprises administering to the patient in a 21- day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine.
  • the 21 -day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.
  • the TNBC protocol incorporates a platinum-containing chemotherapeutic agent other than carboplatin, for example, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.
  • the platinum containing chemotherapeutic used is cisplatin.
  • the platinum containing chemotherapeutic used is oxaliplatin.
  • the patient being treated in the second-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC.
  • the patient being treated in the second-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC, defined as >1% tumor-infiltrating immune cells as confirmed by an in vitro diagnostic (IVD) assay, for example the Ventana SP-142 in vitro diagnostic (IVD) assay or use I IHC 22C3 pharmDx PDL1 assay.
  • IVD in vitro diagnostic
  • the patient being treated in the second-line chemotherapeutic protocol has previously been treated with an ICI for a minimum duration of 8 weeks in the advanced/metastatic TNBC treatment setting.
  • the patient being treated in the second-line chemotherapeutic protocol has received the ICI as the most recent TNBC therapy.
  • the ICI is a PD-1 inhibitor.
  • the PD- 1 inhibitor is pembrolizumab.
  • the PD-1 inhibitor is nivolumab.
  • the PD-1 inhibitor is cemiplimab.
  • the PD-1 inhibitor is CS1003.
  • the PD-1 inhibitor is tislelizumab.
  • the PD-1 inhibitor is toripalimab.
  • the PD-1 inhibitor is sintilimab.
  • the PD-1 inhibitor is camrelizumab. In some embodiments, the PD-1 inhibitor is pidilizumab. In some embodiments, the PD-1 inhibitor is retifanlimab. In some embodiments, the ICI is a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-L1 inhibitor is avelumab. In some embodiments, the PD-L1 inhibitor is durvalumab. In some embodiments, the PD-L1 inhibitor is sugemalimab. In some embodiments, the PD-L1 inhibitor is utomilumab. In some embodiments, the ICI is a CTLA-4 inhibitor.
  • the CTLA-4 inhibitor is ipilimumab. In some embodiments, the CTLA-4 inhibitor is tremelimumab. In some embodiments, the ICI is a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is relatlimab. In some embodiments, the LAG-3 inhibitor is eftilagimod alpha. . In some embodiments, the ICI is a B7-H3 inhibitor. In some embodiments, the B7-H3 inhibitor is enoblituzumab. In some embodiments, the B7-H3 inhibitor is MGD009. In some embodiments, the ICI is a PD-l/Lag-3 specific DART molecule, for example, tebotelimab.
  • the ICI is a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is sabatolimab. In some embodiments, the ICI is a CD137 inhibitor. In some embodiments, the CD 137 inhibitor is urelumab. In some embodiments, the previous first-line chemotherapeutic protocol received by the patient for the treatment of TNBC did not include the administration of gemcitabine and carboplatin. In some embodiments, the patient has not received an ICI, for example a PD-1 inhibitor or PD-L1 inhibitor, within at least 14 days from the administration of trilaciclib. In some embodiments, the patient has a TNBC that is CDK4/6-positive. In some embodiments, the TNBC to be treated is CDK4/6-negative.
  • the TNBC to be treated has at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as i) homozygous deletion, ii) a frameshift mutation, or iii) a stop-gained mutation (i.e., a mutation that results in a premature termination codon (a stop codon was gained)).
  • the TNBC to be treated is CDK4/6-positive.
  • the TNBC is CDK4/6 indeterminate.
  • a method of treating a patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC comprising administering to the patient an effective amount of trilaciclib in a chemotherapeutic protocol, wherein the chemotherapeutic protocol further comprises the administration of gemcitabine and the platinum containing chemotherapeutic carboplatin (GC) (or alternatively, an alternative platinum containing chemotherapeutic agent including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), and wherein the patient is ICI treatment naive.
  • GC platinum containing chemotherapeutic carboplatin
  • Trilaciclib enhances immune activation and promotes anti-tumor immunity by differentially arresting cytotoxic and regulatory T-cell subsets followed by a faster recovery of cytotoxic T lymphocytes than regulatory T cells in tumors.
  • This differential alteration of cell cycle kinetics between cytotoxic T lymphocytes and regulatory T cells results in a higher proportion of cytotoxic T lymphocytes than regulatory T cells, enhancement of T cell activation, and a decrease in regulatory T cell-mediated immunosuppressive functions. Together, these events promote the cytotoxic T lymphocyte-mediated clearance of tumor cells, which can be achieved without the administration of an ICI and the associated side-effects which accompany such ICI use.
  • the patient has a PD-L1 negative tumor.
  • the patient has a PD-L1 positive tumor.
  • the method comprises administering to the patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21 -day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting.
  • the 21-day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.
  • the platinum containing chemotherapeutic used is selected from cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • the platinum containing chemotherapeutic used is cisplatin.
  • the platinum containing chemotherapeutic used is oxaliplatin.
  • the patient has a PD-L1 negative tumor. In some embodiments, the patient has a PD-L1 positive tumor.
  • the method comprises administering to the patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21 -day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting, and wherein the patient’s TNBC is classified as PD-L1 negative.
  • the 21-day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.
  • the platinum containing chemotherapeutic used is selected from cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • the platinum containing chemotherapeutic used is cisplatin.
  • the platinum containing chemotherapeutic used is oxaliplatin.
  • the method comprises administering to the patient in a first- line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21-day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting, and wherein the patient’s TNBC is classified as PD-L1 positive.
  • the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC.
  • the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC, defined as >1% tumor-infiltrating immune cells as confirmed by the Ventana SP-142 in vitro diagnostic (IVD) assay or I H4C 22C3 pharmDx PDL1 assay.
  • the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status negative TNBC.
  • the select patient subgroup with advanced/metastatic TNBC to be treated according to the methods described herein has a T-cell clonal peripheral diversity score at or above the median for the TNBC patient population.
  • the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC with a threshold Simpson clonality score. In one aspect, the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC with a threshold Simpson clonality score of less than about 0.08.
  • the administration of a treatment regimen described herein to the patient subgroups described herein provides enhanced anti-tumor efficacy in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin or oxaliplatin, or nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin) without trilaciclib.
  • the administration of a treatment regimen described herein in the particular patient subgroups described above provides improved progression free survival (PFS) and/or overall survival (OS) in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative) without trilaciclib.
  • PFS progression free survival
  • OS overall survival
  • an improvement in PFS is based on per Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1).
  • the administration of a treatment regimen described herein to a TNBC patient subgroups described above provides improved my el opreservation of hematopoietic stem and progenitor cells (HSPCs) and immune effector cells such as lymphocytes including T- lymphocytes.
  • the administration of a treatment regimen described herein to the TNBC patient subgroups described above provides reduced chemotherapy-induced myelosuppression (CIM).
  • CIM chemotherapy-induced myelosuppression
  • the administration of a treatment regimen described herein provides myelopreservation of the neutrophil lineage in patients compared to those receiving the chemotherapy without trilaciclib.
  • the administration of a treatment regimen described herein provides a reduction in the duration of severe (Grade 4) neutropenia in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative) without trilaciclib.
  • the administration of a treatment regimen described herein provides a reduction in severe neutropenia events, a reduction in granulocyte-colony stimulating factor (G- CSF) treatment, or a reduction in febrile neutropenia (FN) adverse events (AEs).
  • the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 decreased hemoglobin laboratory values, red blood cell (RBC) transfusions, or erythropoiesis-stimulating agent (ESA) administration.
  • the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 decreased platelet count laboratory values and/or the number of platelet transfusions.
  • the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 hematologic laboratory values.
  • the administration of a treatment regimen to the TNBC patient subgroup described herein provides a reduction in all-cause dose reductions or cycle delays and relative dose intensity of gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin or oxaliplatin).
  • the administration of a treatment regimen described herein provides a reduction in i) hospitalizations, including but not limited to those due to all causes, febrile neutropenia/neutropenia, anemia/RBC transfusion, thrombocytopenia/bleeding and infections) or ii) antibiotic use, including but not limited to intravenous (IV), oral, and oral and IV administered antibiotics.
  • the administration of a treatment regimen described herein provides a reduction of chemotherapy-induced fatigue (CIF) in patients compared to those receiving chemotherapy without trilaciclib.
  • the reduction of CIF is a reduction in the time to first confirmed deterioration of fatigue (TTCD-fatigue), as measured by the Functional Assessment of Cancer Therapy -Fatigue (FACIT-F).
  • the administration of a treatment regimen described herein provides an improvement to one or more of: Functional Assessment of Cancer Therapy-General (FACT- G) domain scores (physical, social/family, emotional, and functional well-being); Functional Assessment of Cancer Therapy-Anemia (FACT-An); 5-level EQ-5D (EQ-5D-5L); Patient Global Impression of Change (PGIC) fatigue item; or Patient Global Impression of Severity (PGIS) fatigue item.
  • FACT- G Functional Assessment of Cancer Therapy-General
  • FACT-An Functional Assessment of Cancer Therapy-Anemia
  • EQ-5D-5L 5-level EQ-5D
  • PGIC Patient Global Impression of Change
  • PGIS Patient Global Impression of Severity
  • Fig. 1 is a schematic of a human clinical trial assessing the effect of trilaciclib on overall survival (OS) compared with placebo in patients receiving first-line gemcitabine and carboplatin (GC) for advanced/metastatic triple negative breast cancer (Cohort 1) and patients receiving GC as second-line therapy after treatment with a PD-1/PD-L1 inhibitor in advanced/metastatic triple negative breast cancer (Cohort 2).
  • the study will include 3 study phases: Screening Phase, Treatment Phase, and Survival Follow up Phase.
  • the Treatment Phase begins on the day of the first dose with study treatment and completes at the Safety Follow-up Visit.
  • the first Survival Follow-up assessment should occur 3 months after the Safety Follow-up Visit.
  • GC Gemcitabine and Carboplatin
  • DC discontinuation
  • PD Progressive disease
  • PI Principal Investigator
  • WD W ithdrawal .
  • Fig. 2 is a bar graph showing the median treatment effect (25% and 75% quartiles) of Trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on Simpson clonality score.
  • the x-axis represents the timing of the measurement.
  • C1D1 cycle 1, day 1;
  • C3D1 cycle 3, day 1;
  • C5D1 cycle 5, day 1.
  • the y-axis represents the Simpson clonality score ranging from 0-0.25. There was a significant decrease in Simpson clonality among patients who received Trilaciclib prior to GCb compared with GCb alone (P interactional.012).
  • Fig. 3 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median Simpson clonality score.
  • the x-axis depicts months from randomization and number of patients at risk.
  • the y-axis depicts the probability of survival.
  • Fig. 4 is a bar graph showing the median treatment effect (25% and 75% quartiles) of Trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on Fraction of newly detected expanded clones.
  • the x-axis represents response to treatment (Yes or No) and the y-axis represents the median value (25% and 75% quartiles) of the fraction of newly detected expanded clones.
  • Fig. 5 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of newly detected expanded clones.
  • the x-axis depicts months from randomization and number of patients at risk.
  • the y-axis depicts the probability of survival.
  • For Kaplan-Meier estimates of probability of survival patients were stratified by high (equal or above median, solid lines) and low (below median, dashed lines). Although not statistically significant, when patients were stratified above or below median fraction of newly detected expanded clones, OS was improved among patients with a higher fraction of newly detected expanded clones who received trilaciclib.
  • Fig. 6 is a bar graph showing the median treatment effect (25% and 75% quartiles) of trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on T-cell diversity.
  • the x-axis represents the timing of the measurement.
  • C1D1 cycle 1, day 1;
  • C3D1 cycle 3, day 1;
  • C5D1 cycle 5, day 1.
  • the y-axis represents the number of T-cell clones (T-cell clonality).
  • Fig. 7 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of peripheral diversity as measured by the number of T-cell clones (T-cell clonality).
  • the x-axis depicts months from randomization and number of patients at risk.
  • the y-axis depicts the probability of survival. For Kaplan-Meier estimates of probability of survival, patients were stratified by high (equal or above median) and low (below median).
  • Fig. 8 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of peripheral diversity as measured by the number of T cell clones (T-cell clonality) across the different treatment group.
  • the x-axis depicts months from randomization and number of patients at risk.
  • For Kaplan-Meier estimates of probability of survival patients were stratified by high (equal or above median, solid lines) and low (below median, dashed lines).
  • Fig. 9 is a table of ORR, PFS and OS by Prior Line of Therapy for Recurrent/Metastatic TNBC as assessed in a Phase 2 clinical trial, a.
  • PFS and ORR based on data with cutoff date of 17 May2019; OS results and duration of survival follow-up are updated based on final database lock on 17Jul2020.
  • Fig. 10 is table of ORR, PFS and OS by Prior Line of Therapy (pooled trilaciclib + G/C + SOC).
  • PFS and ORR based on data with cutoff date of 17 May2019, OS updated with final database lock on 17Jul2020.
  • the two-sided p-value is calculated using stratified exact CMH method to account for liver involvement [Yes or No] as the stratification factor,
  • the two-sided p-value was calculated using stratified log-rank test to account for liver involvement [Yes or No] as the stratification factor, d.
  • the compound may be in the form of a racemate, enantiomer, mixture of enantiomers, diastereomer, mixture of diastereomers, tautomer, N-oxide, or isomer, such as a rotamer, as if each is specifically described unless specifically excluded by context.
  • the terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.
  • the term “or” means “and/or”.
  • the compound may be in the form of a tautomer, N-oxide, or isomer, such as a rotamer, as if each is specifically described unless specifically excluded by context.
  • an “effective amount” as used herein means an amount which provides a therapeutic or prophylactic benefit.
  • a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease, disorder, or side-effect experienced by a patient (i.e. palliative treatment) or to decrease a cause or effect of the disease, disorder (i.e. disease-modifying treatment), or side effect experienced by a patient as a result of the administration of a therapeutic agent.
  • salts of the present compounds further include solvates of the compounds and of the compound salts.
  • Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n- COOH where n is 0-4, and the like, or using a different acid that produces the same counterion.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric
  • Prodrugs can be used to achieve any desired effect, including to enhance properties of the parent drug or to improve the pharmaceutic or pharmacokinetic properties of the parent.
  • Prodrug strategies exist which provide choices in modulating the conditions for in vivo generation of the parent drug, all of which are deemed included herein.
  • Nonlimiting examples of prodrug strategies include covalent attachment of removable groups, or removable portions of groups, for example, but not limited to acylation, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation or anhydride, among others.
  • a “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition/combination that is generally safe, and neither biologically nor otherwise inappropriate for administration to a host, typically a human.
  • trilaciclib can be used in a form that has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched.
  • Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons.
  • isotopes that can be incorporated into trilaciclib for use in the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine and iodine such as 2H, 3H, 11C, 13C, 14C, 15N, 18F 3 IP, 32P, 35S, 36CI, and 1251 respectively.
  • isotopically labelled compounds can be used in metabolic studies (with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18F labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
  • isotopes of hydrogen for example, deuterium ( 2 H) and tritium ( 3 H) may be used anywhere in described structures that achieves the desired result.
  • isotopes of carbon e.g., 13 C and 14 C, may be used.
  • Isotopic substitutions for example deuterium substitutions, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with deuterium.
  • the isotope is 90, 95, or 99% or more enriched in an isotope at any location of interest. In one non-limiting embodiment, deuterium is 90, 95, or 99% enriched at a desired location.
  • Trilaciclib for use in the present invention may form a solvate with solvents (including water). Therefore, in one non-limiting embodiment, the invention includes a solvated form of trilaciclib.
  • solvate refers to a molecular complex of trilaciclib (including a salt thereof) with one or more solvent molecules.
  • solvents are water, ethanol, dimethyl sulfoxide, acetone and other common organic solvents.
  • hydrate refers to a molecular complex comprising a compound of the invention and water.
  • Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent may be isotopically substituted, e.g., D2O, d6-acetone, d6-DMSO.
  • a solvate can be in a liquid or solid form.
  • patient or “subject” treated is typically a human patient, although it is to be understood the methods described herein are effective with respect to other animals, such as mammals. More particularly, the term patient can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.
  • animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.
  • hematopoietic stem and progenitor cells includes, but are not limited to, long term hematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs), hematopoietic progenitor cells (HPCs), multipotent progenitors (MPPs), oligodendrocyte pre-progenitors (OPPs), monocyte progenitors, granulocyte progenitors, common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), granulocyte progenitors, monocyte progenitors, and megakaryocyte-erythroid progenitors (MEPs), megakaryocyte progenitors, erythroid progenitors, HSC/MPPs (CD45dim/CD34+/CD38-), OPPs
  • LT-HSCs long term hematopo
  • Immune effector cell generally refers to an immune cell that performs one or more specific functions.
  • Immune effector cells are known in the art and include for example, but are not limited to, T-cells, including Naive T-cells, Memory T-cells, Activated T-cells (Thelper (CD4+) and Cytotoxic T cells (CD8+)), TH1 activated T-cells, TH2 activated T-cells, TH17 activated T-cells, Naive B cells, Memory B cells, plasmablasts, dendritic cells, monocytes, and natural killer (NK) cells.
  • T-cells including Naive T-cells, Memory T-cells, Activated T-cells (Thelper (CD4+) and Cytotoxic T cells (CD8+)
  • TH1 activated T-cells TH2 activated T-cells
  • TH17 activated T-cells
  • Naive B cells Memory B cells
  • Memory B cells plasmablasts
  • dendritic cells
  • ICI immune checkpoint inhibitor
  • ICIs include those targeting immune checkpoint proteins such as programmed cell death- 1 protein (PD-1), PD-1 Ligand- 1 (PD-L1), PD-1 Ligand- 2 (PD-L2), CTLA-4, LAG-3, TIM-3, and V-domain Ig suppressor of T-cell activation (VISTA), B7-H3/CD276, indoleamine 2, 3 -dioxygenase (IDO), killer immunoglobulin-like receptors (KIRs), carcinoembryonic antigen cell adhesion molecules (CEACAM) such as CEACAM-1, CEACAM- 3, and CEACAM-5, sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and B and T lymphocyte attenuator (BTLA) protein, and the like.
  • Immune checkpoint inhibitors are known in the art.
  • TNBC Triple-negative Breast Cancer
  • TNBC is a highly aggressive breast cancer subtype that accounts for 15-20% of breast cancer cases annually and 25% of all breast cancer deaths.
  • TNBC has been characterized by several aggressive clinicopathologic features, including onset at a younger age; large, high-grade tumors; and a propensity for visceral metastasis (Cheang et al., Basal-like breast cancer defined by five biomarkers has superior prognostic value than triplenegative phenotype.
  • Clin Cancer Res. 2008;14(5): 1368-76. Foulkes et al., Triple-negative breast cancer. N Engl J Med. 2010 Nov 11;363(20): 1938-48).
  • a breast cancer is generally classified as TNBC based on local ER-negative, progesterone receptor-negative, HER2-negative status, which can be determined through a histological or cytological hormone receptor immunohistochemistry (IHC) assessment for estrogen and progesterone (defined as ⁇ 1% nuclei staining), and by IHC [0 or 1+] OR in situ hybridization [ratio ⁇ 2.0] OR average gene copy number of ⁇ 4 signals/nucleus) for HER2-negative, non- overexpression (per 2018 American Society of Clinical Oncology and the College of American Pathologists (ASCO CAP) criteria).
  • IHC histological or cytological hormone receptor immunohistochemistry
  • the patient is receiving a second-line therapy in the advanced/metastatic TNBC setting following failure of or advancement on an ICI inhibitor and the TNBC to be treated is PD-L1 positive.
  • the patient is receiving a first- line therapy in the advanced/metastatic TNBC setting and the TNBC to be treated is PD-L1 positive.
  • the patient is receiving a first-line therapy in the advanced/metastatic TNBC setting and the TNBC to be treated is PD-L1 negative.
  • PD-L1 is a transmembrane protein that down-regulates immune responses through binding to its two inhibitory receptors, programmed death-1 (PD-1) and B7.1.
  • PD-1 is an inhibitory receptor expressed on T cells following T-cell activation, which is sustained in states of chronic stimulation such as in chronic infection or cancer (Blank, C and Mackensen, A, Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol. Immunother., 2007. 56(5): p. 739-745). Binding of PD-L1 with PD-1 inhibits T cell proliferation, cytokine production and cytolytic activity, leading to the functional inactivation or exhaustion of T cells.
  • B7.1 is a molecule expressed on antigen presenting cells and activated T cells.
  • PD-L1 binding to B7.1 on T cells and antigen presenting cells can mediate down-regulation of immune responses, including inhibition of T-cell activation and cytokine production (see Butte MJ, Keir ME, Phamduy TB, et al. Programmed death- 1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity. 2007;27(l): 111-122).
  • PD-L1 expression has been observed in immune cells and tumor cells. See Dong H, Zhu G, Tamada K, Chen L.
  • B7-H1 a third member of the B7 family, co-stimulates T- cell proliferation and interleukin- 10 secretion.
  • Nature. 2014;515(7528):563-567 Aberrant expression ofPD-Ll on tumor cells has been reported to impede anti-tumor immunity, resulting in immune evasion.
  • PD-L1 expression can be determined by methods known in the art.
  • PD-L1 expression can be detected using PD-L1 IHC 22C3 pharmDx, the FDA-approved in vitro diagnostic immunohistochemistry (IHC) test developed by Dako and Bristol -Meyers Squibb as a companion test for treatment with pembrolizumab.
  • IHC in vitro diagnostic immunohistochemistry
  • This is qualitative assay using Monoclonal Mouse Anti-PD-Ll, Clone 22C3 PD-L1 and EnVision FLEX visualization system on Autostainer Lin 48 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human cancer tissue.
  • Expression levels can be measured using the tumor proportion score (TPS), which measures the percentage of viable tumor cells showing partial or complete membrane staining. Staining can show PD-Ll expression from l% to 100%.
  • TPS tumor proportion score
  • PD-L1 expression can also be detected using PD-L1 IHC 28-8 pharmDx, the FDA- approved in vitro diagnostic immunohistochemistry (IHC) test developed by Dako and Merck as a companion test for treatment with nivolumab.
  • IHC in vitro diagnostic immunohistochemistry
  • This qualitative assay uses the Monoclonal rabbit anti-PD-Ll, Clone 28-8 and EnVision FLEX visualization system on Autostainer Lin 48 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human cancer tissue.
  • FFPE paraffin-embedded
  • PD-L1 detection include the Ventana SP263 assay (developed by Ventana in collaboration with AstraZeneca) that utilizes monoclonal rabbit anti- PD-Ll, Clone SP263 and the Ventana SP142 Assay (developed by Ventana in collaboration with Genentech/Roche) that uses rabbit monoclonal anti-PD-Ll clone SP142. Determination of PD- L1 status is indication-specific, and evaluation is based on either the proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells (% IC) of any intensity or the percentage of PD-L1 expressing tumor cells (% TC) of any intensity.
  • % IC proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells
  • % TC percentage of PD-L1 expressing tumor cells
  • PD-L1 positive status in TNBC is considered > 1% IC.
  • the TNBC has a PD-L1 positive status > 1% IC.
  • the TNBC has a PD-L1 positive status > 1% IC as determined by the Ventana SP142 Assay.
  • the TNBC has a PD-L1 positive status ⁇ 1% IC. In some embodiments, the TNBC has a PD-L1 positive status ⁇ 1% IC as determined by the Ventana SP142 Assay.
  • CDK4/6 replication independent cancers generally have a retinoblastoma gene (Rbl) aberration.
  • the gene product of Rbl — Rb-protein — is a downstream target of CDK4/6.
  • RBI is commonly dysregulated in cancer cells through deletion, mutation or epigenetic modification resulting in loss of RB expression, as well as by aberrant CDK kinase activity leading to excessive phosphorylation and inactivation of RB function (Chen et al. Novel RBI -Loss Transcriptomic Signature Is Associated with Poor Clinical Outcomes across Cancer Types. Clin Cancer Res. 2019;25(14); Sherr, C.J., and McCormick, F. The RB and p53 pathways in cancer.
  • CDK4/6 genetic signature analysis involves the utilization of tumor tissue collected from a patients’ biopsy (TNBC primary or metastatic site) and are described in Shapiro GI. Genomic biomarkers predicting response to selective CDK4/6 inhibition: Progress in an elusive search. Cancer Cell. 2017;32(6):721-3 and Gong et al. Genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4 and CDK5 inhibitor abemaciclib. Cancer Cell. 2017;32(6):761-76.
  • the patient has previously been administered a dual PD-L1/PD-1 inhibitor.
  • LAG-3 immune checkpoint inhibitors include, but are not limited to, relatlimab (BMS-986016; Bristol-Myers Squibb), GSK2831781 (GlaxoSmithKline), eftilagimod alpha (IMP321; Prima BioMed), leramilimab (LAG525; Novartis), MK-4280 (Merck), REGN3767 (Regeneron), TSR-033 (Tesaro), BI754111 (Bohringer Ingelheim), Sym022 (Symphogen).
  • relatlimab BMS-986016; Bristol-Myers Squibb
  • GSK2831781 GaxoSmithKline
  • eftilagimod alpha IMP321; Prima BioMed
  • leramilimab LAG525; Novartis
  • MK-4280 Merck
  • REGN3767 Registered Node
  • TSR-033 Tesaro
  • BI754111 Bohringer Ingel
  • the patient has previously been administered a TIGIT (T cell immunoreceptor with Ig and ITIM domains) immune checkpoint inhibitor.
  • TIGIT immune checkpoint inhibitors include, but are not limited to, MK-7684 (Merck), Etigilimab /OMP- 313 M32 (OncoMed), Tiragolumab/MTIG7192A/RG-6058 (Genentech), BMS-986207 (BMS), AB-154 (Arcus Biosciences), and ASP-8374 (Potenza).
  • the patient has previously been administered an immune checkpoint inhibitor including, for example, but not limited to, a B7-H3/CD276 immune checkpoint inhibitor such as enoblituzumab (MGA217, Macrogenics) MGD009 (Macrogenics), 13 H-8H9/omburtamab (Y-mabs), and I-8H9/omburtamab (Y-mabs), an indoleamine 2,3-dioxygenase (IDO) immune checkpoint inhibitor such as Indoximod and INCB024360, a killer immunoglobulin-like receptors (KIRs) immune checkpoint inhibitor such as Lirilumab (BMS-986015), a carcinoembryonic antigen cell adhesion molecule (CEACAM) inhibitor (e.g., CEACAM-1, -3 and/or -5).
  • a B7-H3/CD276 immune checkpoint inhibitor such as enoblituzumab (MGA217, Macrogenics) MGD00
  • the select patient subgroup with advanced/metastatic TNBC, to be treated according to the methods described herein has a T-cell clonal peripheral diversity score at or above the median for the TNBC patient population.
  • the patient to be treated has a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones.
  • Trilaciclib (2'-((5-(4-methylpiperazin-l -yljpyri din-2 -yl)amino)-7',8'-dihydro-6'H- spiro(cyclohexane-l,9'-pyrazino(r,2':l,5)pyrrolo(2,3-d)pyrimidin)-6'-one) is a highly selective CDK4/6 inhibitor having the structure:
  • trilaciclib or its pharmaceutically acceptable salt, composition, isotopic analog, or prodrug thereof is administered in a suitable carrier.
  • Trilaciclib is described in US 2013-0237544, incorporated herein by reference in its entirety.
  • Trilaciclib can be synthesized as described in US 2019-0135820, incorporated herein by reference in its entirety.
  • Trilaciclib can be administered in any manner that achieves the desired outcome, including systemically, parenterally, intravenously, intramuscularly, subcutaneously, or intradermally.
  • Trilaciclib can be administered intravenously as described herein.
  • trilaciclib is in the form of a dihydrochloride optionally as a hydrate.
  • trilaciclib can be used in the present invention as a dihydrochloride, dihydrate or as a pharmaceutical composition formed from trilaciclib dihydrochloride, dihydrate.
  • lerociclib is administered as a pharmaceutically acceptable salt, for example, the dihydrocloride salt.
  • the CDK4/6 inhibitor has the structure: or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, which is described in US 2013-0237544, incorporated herein by reference in its entirety, and can be synthesized as described in US 2019-0135820, incorporated herein by reference in its entirety.
  • Gemcitabine is a nucleoside metabolic inhibitor. Descriptions of formulations of commercially available gemcitabine can be found in current available prescribing information.
  • Gemcitabine is generally administered by intravenous injection on day 1 and day 8 of each 21-day cycle after administration of trilaciclib. As provided herein, administration of gemcitabine should not be longer than 4 hours. In the methods provided herein, gemcitabine can be administered according to institutional guidelines. In some embodiments, gemcitabine can be administered at its standard of care dose. In some embodiments, gemcitabine is administered at a dose of between about 600 mg/m 2 and 1250 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 600 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 800 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 1000 mg/m 2 .
  • gemcitabine is administered at a dose of about 1200 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 1250 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 600 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 800 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 1000 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 1200 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 1250 mg/m 2 .
  • gemcitabine is associated with bone marrow suppression: neutropenia, thrombocytopenia, and anemia, including Grade 3 or 4 hematologic toxicity. Additional risks associated with the use of gemcitabine include: capillary leak syndrome; hemolytic uremic syndrome: may lead to renal failure and dialysis (including fatalities); hepatotoxicity; hypersensitivity: anaphylaxis and allergic reactions (including bronchospasm and anaphylactoid reactions) have been observed; posterior reversible encephalopathy syndrome (PRES): may manifest with blindness, confusion, headache, hypertension, lethargy, seizure, and other visual and neurologic disturbances; and, pulmonary toxicity: including adult respiratory distress syndrome, interstitial pneumonitis, pulmonary edema, and pulmonary fibrosis.
  • PRES posterior reversible encephalopathy syndrome
  • Carboplatin is a platinum coordination compound. Descriptions of formulations of commercially available carboplatin can be found in current available prescribing information.
  • the chemical name for carboplatin is platinum, diamine [l,l-cyclobutane-dicarboxylato(2-)-0,0']-,(SP- 4-2), and carboplatin has the following structural formula:
  • Carboplatin like cisplatin, produces predominantly inter-strand DNA cross-links rather than DNA-protein cross-links. This effect is apparently cell-cycle nonspecific.
  • the aquation of carboplatin which is thought to produce the active species, occurs at a slower rate than in the case of cisplatin.
  • carboplatin and cisplatin induce equal numbers of drug-DNA cross-links, causing equivalent lesions and biological effects.
  • the differences in potencies for carboplatin and cisplatin appear to be directly related to the difference in aquation rates.
  • AUC target area under the curve
  • carboplatin is associated with: bone marrow suppression: leukopenia, neutropenia, and thrombocytopenia; nephrotoxic potential: concomitant treatment with aminoglycosides has resulted in increased renal and/or audiologic toxicity; emesis; peripheral neurotoxicity: observed infrequently, but its incidence is increased in patients older than 65 years and in patients previously treated with cisplatin; Loss of vision; allergic reactions, including anaphylaxis; abnormal liver function tests; and, fetal harm.
  • cisplatin has been linked to its ability to crosslink with the purine bases on the DNA; interfering with DNA repair mechanisms, causing DNA damage, and subsequently inducing apoptosis in cancer cells.
  • the use of cisplatin is associated with: nausea, vomiting, nephrotoxicity, low blood counts, blood test abnormalities, thrombocytopenia, neutropenia, ototoxicity (especially in children), myelosuppression, anaphylaxis and alopecia.
  • cisplatin is used in combination with gemcitabine.
  • Oxaliplatin is a platinum-based chemotherapy drug in the same family as cisplatin and carboplatin. Compared to cisplatin the two amine groups are replaced by cyclohexyldiamine for improved antitumour activity. The chlorine ligands are replaced by the oxalato bidentate derived from oxalic acid in order to improve water solubility. It is also used off-label for indications including TNBC. Descriptions of formulations of commercially available oxaliplatin can be found in current available prescribing information.
  • Oxaliplatin is an antineoplastic agent with the molecular formula CsHi4N2O4Pt and the chemical name of cis-[(l R,2 R)-l,2- cyclohexanediamine-N,N'] [oxalato(2)-O,O'] platinum.
  • Oxaliplatin is an organoplatinum complex in which the platinum atom is complexed with 1,2-diaminocyclohexane (DACH) and with an oxalate ligand as a leaving group with a structural formula of:
  • oxaliplatin is associated with peripheral neuropathy, nausea and vomiting, diarrhea, mouth sores, low blood counts, fatigue, loss of appetite, constipation, fever, generalized pain, headache, cough, temporary increases in blood tests measuring liver function, and anaphylaxis.
  • oxaliplatin is used in combination with gemcitabine.
  • an alternative platin is used in combination with gemcitabine, for example, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.
  • TNBC as described herein are administered trilaciclib, or a pharmaceutically acceptable salt thereof, in combination with the chemotherapeutic agents gemcitabine and carboplatin in a specifically timed administrative protocol.
  • a method of treating a human patient with advanced/metastatic TNBC comprising: i) administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure: or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii) administering to the patient an effective amount of gemcitabine; and, iii) administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered prior to the initiation of administration of the gemcitabine and carboplatin, and wherein the patient is receiving either 1) first-line treatment for advanced/metastatic TNBC and is immune-checkpoint inhibitor treatment-naive or, alternatively ⁇ ) second-line treatment for advanced/metastatic TNBC and has had prior exposure to an immune checkpoint inhibitor, for example, a programmed cell
  • trilaciclib is administered less than 4 hours or prior to the administration of gemcitabine and carboplatin. In some embodiments, trilaciclib is administered about one hour or less, for example, about 45 minutes, about 40 minutes, about 35 minutes, or about 30 minutes, prior to administration of gemcitabine and carboplatin.
  • trilaciclib is administered to the patient intravenously between about 190 and 280 mg/m 2 . In some embodiments, the trilaciclib is administered at about 240 mg/m 2 .
  • gemcitabine is administered at a dose of between about 600 mg/m 2 and 1200 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 600 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 800 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 1000 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of about 1200 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 600 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 800 mg/m 2 .
  • gemcitabine is administered at a dose of at least about 1000 mg/m 2 . In some embodiments, gemcitabine is administered at a dose of at least about 1200 mg/m 2 . In some embodiments, the gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours. In some embodiments, gemcitabine is administered at a starting dose of about 1000 mg/m 2 . In some embodiments, gemcitabine is administered at about 1000 mg/m 2 for one or more doses, and at about 800 mg/m 2 for at least one dose. In some embodiments, gemcitabine is administered at 1000 mg/m 2 in one or more doses, at 800 mg/m 2 for one or more doses, and about 600 mg/m 2 for at least one dose.
  • CI continuous infusion
  • the trilaciclib/GC chemotherapy regimen is administered in 1 or more cycles, 2 or more cycles, 3 or more cycles, 4 or more cycles, 5 or more cycles, 6 or more cycles, 7 or more cycles, 8 or more cycles, 9 or more cycles, 10 or more cycles, or 11 or more cycles. In some embodiments, the trilaciclib/GC chemotherapy is administered up to 12 times.
  • the protocol comprises one or more 21 -day therapeutic cycles, wherein trilaciclib, carboplatin, and gemcitabine are administered on days 1 and 8 of each 21 -day cycle, wherein trilaciclib is administered no more than 4 hours prior to the administration of the gemcitabine and carboplatin, and wherein the trilaciclib is completely administered before the start of the administration of gemcitabine and/or carboplatin.
  • cisplatin is used instead of carboplatin.
  • oxaliplatin is used instead of carboplatin.
  • the inclusion of trilaciclib in a gemcitabine/carboplatin (GC) chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased anti-tumor efficacy compared to those patients receiving a GC chemotherapy protocol without trilaciclib.
  • Methods of accessing tumor response are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased or extended progression free survival (PFS) compared to those patients not receiving trilaciclib.
  • PFS is generally defined as the time (number of months) from date of protocol administration until the date of documented radiologic disease progression or death from any cause.
  • Methods of accessing increased PFS are well known in the art and include, for example RECIST vl. l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased or extended overall survival (OS) compared to those patients not receiving trilaciclib.
  • OS is generally calculated as the time (months) from the date of the onset of protocol administration to the date of death due to any cause.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an improved objective response rate (ORR) compared to those patients not receiving trilaciclib.
  • ORR is generally defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period.
  • Examples of an objective response (OR) includes a complete response (CR), which is the disappearance of all signs of the tumor in response to treatment and a partial response (PR), which is a decrease in the size of a tumor in response to treatment.
  • the objective response (OR) is a complete response (CR).
  • the objective response (OR) is a partial response (PR).
  • ORR is an important parameter to demonstrate the efficacy of a treatment and it serves as a primary or secondary end-point in clinical trials.
  • Methods of assessing ORR are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247) and World Health Organization (WHO) (World Health Organization. WHO Handbook for Reporting Results of Cancer Treatment. World Health Organization Offset Publication No. 48; Geneva (Switzerland), 1979).
  • Statistical methods of measuring objective response rate are well known in the art and include, for example, the Clopper-Pearson Method (Clopper, C.; Pearson, E. S. (1934). "The use of confidence or fiducial limits illustrated in the case of the binomial". Biometrika. 26 (4): 404-413. doi: 10.1093/biomet/26.4.404).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an improved Duration of Objective Response (DOR) compared to those patients not receiving trilaciclib.
  • DOR is generally defined as the time between first objective response of CR or PR and the first date that progressive disease is objectively documented or death, whichever comes first.
  • Methods of assessing improved DOR are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for T-cell immune activation against the tumor.
  • the T-cell immune activation results in T-cell receptor (TCR) modulation.
  • the T-cell activation results in increased interferon gamma (IFNy) expression.
  • the T-cell activation results in increased activation-induced expression of CD137.
  • the T-cell activation results in increased TCR diversity.
  • the T-cell activation results in a decrease in the baseline Simpson clonality score.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in improved myelopreservation of hematopoietic stem and progenitor cells (HSPCs) and immune effector cells such as lymphocytes including T-lymphocytes, as well as enhanced anti -turn or efficacy in patients compared to those receiving a chemotherapy protocol without trilaciclib.
  • HSPCs hematopoietic stem and progenitor cells
  • immune effector cells such as lymphocytes including T-lymphocytes
  • HSPCs hematopoietic stem and progenitor cells
  • immune effector cells such as lymphocytes including T-lymphocytes
  • CBC complete blood count
  • RBC red blood cell count
  • WBC platelet count
  • WBC white blood cell count
  • ANC absolute neutrophil count
  • AEs severe adverse events
  • PROs patient recorded outcomes
  • CIM commonly leads to dose reductions and delays, which limit therapeutic dose intensity and can compromise the anti-tumor efficacy benefits of chemotherapy.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in myelopreservation of the neutrophil lineage in patients compared to those receiving the chemotherapy protocol without trilaciclib.
  • Endpoints to measure myelopreservation of the neutrophil lineage include a reduction in duration of severe neutropenia, for example after cycle 1, and the reduction in the occurrence of severe neutropenia.
  • Neutropenia is generally defined as a condition that results when the body does not have enough neutrophils, an important white blood cell that fights infections. The lower the neutrophil count, the more vulnerable one is to infectious diseases. Neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 1500 per microliter (1500/pL).
  • Severe neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 500 per microliter (500/ pL).
  • ANC absolute neutrophil count
  • Methods of calculating absolute neutrophil count (ANC) are well known in the art and include multiplying the WBC count times the percent of neutrophils in the differential WBC count. The percent of neutrophils consists of the segmented (fully mature) neutrophils + the bands (almost mature neutrophils).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the duration of severe (Grade 4) neutropenia (DSN) in patients compared to those receiving the chemotherapy protocol without trilaciclib.
  • the duration of SN (DSN) is generally defined as the number of days from the date of the first ANC value of ⁇ 0.5 x 10 9 /L to the date of the first ANC value >0.5 x 10 9 /L where no additional ANC values ⁇ 0.5 x 10 9 /L are observed for the remainder of that cycle.
  • Severe neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 500 per microliter (500/ pL).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction of chemotherapy- induced fatigue (CIF) in patients compared to those receiving the chemotherapy protocol without trilaciclib.
  • the reduction of CIF is a reduction in the time to first confirmed deterioration of fatigue (TTCD-fatigue), as measured by the Functional Assessment of Cancer Therapy -Fatigue (FACIT-F).
  • FACIT-F is a 13-item subscale that measures fatigue severity and the impact of fatigue on functioning and is described in Yellen et al., Measuring fatigue and other anemia-related symptoms with the Functional Assessment of Cancer Therapy (FACT) measurement system. J Pain Symptom Manage.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in severe neutropenia events, a reduction in granulocyte-colony stimulating factor (G-CSF) treatment, or a reduction in febrile neutropenia (FN) adverse events (AEs) in patients compared to those receiving the chemotherapy protocol without trilaciclib.
  • G-CSF treatment will be utilized according to the treatment guidelines outlined in Aapro et al. 2010 update of EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours. Eur J Cancer. 2011; 47:8-32.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 decreased hemoglobin laboratory values, red blood cell (RBC) transfusions, or erythropoiesisstimulating agent (ESA) administration.
  • RBC red blood cell
  • ESA erythropoiesisstimulating agent
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 decreased platelet count laboratory values and/or the number of platelet transfusions.
  • platelets are generally transfused at a threshold of ⁇ 10,000/pL. Platelets are also generally transfused in any patient who is bleeding with a platelet count ⁇ 50,000/pL (100,000/pL for central nervous system or ocular bleeding).
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 hematologic laboratory values.
  • the use of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in all-cause dose reductions or cycle delays and relative dose intensity of a chemotherapy protocol described herein.
  • the inclusion of trilaciclib a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in i) hospitalizations, including but not limited to those due to all causes, febrile neutropenia/neutropenia, anemia/RBC transfusion, thrombocytopenia/bleeding and infections) or ii) antibiotic use, including but not limited to intravenous (IV), oral, and oral and IV administered antibiotics.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in an improvement to one or more of: Functional Assessment of Cancer Therapy -General (FACT-G) domain scores (physical, social/family, emotional, and functional well-being); Functional Assessment of Cancer Therapy- Anemia (FACT-An); 5-level EQ-5D (EQ-5D-5L); Patient Global Impression of Change (PGIC) fatigue item; or Patient Global Impression of Severity (PGIS) fatigue item.
  • FACT-G Functional Assessment of Cancer Therapy -General
  • FACT-An Functional Assessment of Cancer Therapy- Anemia
  • EQ-5D-5L 5-level EQ-5D
  • PGIC Patient Global Impression of Change
  • PGIS Patient Global Impression of Severity
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the number of severe diarrhea episodes (Grade 3 or greater) experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the development of, severity of, or episodes of mucositis experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
  • the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the development of, severity of, or episodes of stomatitis experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
  • the selected compounds of the protocols described herein or their pharmaceutically acceptable salts can be administered as the neat chemical, but is more typically administered as a pharmaceutical composition, that includes an effective amount for a patient, typically a human, in need of such treatment in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may contain a compound or salt thereof as the only active agent, or, in an alternative embodiment, the compound or its salt and at least one additional active agent for the disease to be treated.
  • the pharmaceutical composition is in a dosage form that contains from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 750 mg, from about 1 mg to about 500 mg, or from about 5, 10, 15, or 20 mg to about 250 mg of the active compound or its pharmaceutically acceptable salt.
  • dosage forms are those delivering at least 0.01, 0.05, 0.1, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 500, 600, 700, or 750 mg of active compound, or its salt.
  • the weight can refer to either the compound alone or the compound in combination with its pharmaceutically acceptable salt.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packed tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, syrup, suspensions, creams, ointments, lotions, paste, gel, spray, aerosol, foam, or oil, injection or infusion solution, a transdermal patch, a subcutaneous patch, an inhalation formulation, in a medical device, suppository, buccal, or sublingual formulation, parenteral formulation, or an ophthalmic solution, or the like, preferably in unit dosage form suitable for single administration of a precise dosage.
  • solid, semi-solid or liquid dosage forms such as, for example, tablets, suppositories, pills, capsules, powders, liquids, syrup, suspensions, creams, ointments, lotions, paste, gel, spray, aerosol, foam, or oil, injection or infusion solution, a transdermal patch, a subcutaneous patch, an inhalation formulation, in a medical device,
  • compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other pharmaceutical agents, adjuvants, diluents, buffers, and the like.
  • excipients include, but are not limited, to liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like.
  • the compound can be provided, for example, in the form of a solid, a liquid, spray dried material, a microparticle, nanoparticle, controlled release system, etc., as desired according to the goal of the therapy.
  • Suitable excipients for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).
  • a biological buffer can be any solution which is pharmacologically acceptable, and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range.
  • buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’ s buffered saline, and the like.
  • conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like.
  • compositions containing the active agents can be formulated for oral administration.
  • the composition may take the form of a tablet, capsule, a softgel capsule or can be an aqueous or nonaqueous solution, suspension or syrup.
  • Tablets and capsules are typical oral administration forms.
  • Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and com starch.
  • Lubricating agents, such as magnesium stearate, are also typically added.
  • prior systemic therapy in the advanced/metastatic setting, and patients with rapid progression within 6 months from the end of last treatment with curative intent are excluded.
  • prior PD-1/PD-L1 inhibitor treatment is not permitted in any setting, including in the neoadjuvant setting.
  • Cohort 2 comprises patients with a documented PD-L1 positive TNBC tumor and treatment with a PD 1/PD-L1 inhibitor for a minimum duration of 8 weeks as the most recent therapy in the advanced/metastatic setting.
  • Trilaciclib is supplied as a sterile, preservative-free, yellow, lyophilized cake in a singledose vial (300 mg/20 mL). Trilaciclib must be reconstituted and further diluted prior to IV infusion. Upon reconstitution, the solution must then be diluted to the calculated dose (240 mg/m 2 ) based on the body surface area (BSA) of the patient. Actual body weight (not ideal body weight) should be utilized for dose calculations.
  • Trilaciclib solution is administered as a 30-minute IV infusion no more than 4 hours prior to chemotherapy and is always administered first.
  • Gemcitabine/carboplatin GC
  • the order of administration of gemcitabine and carboplatin is per institutional standards.
  • the interval between trilaciclib administration and the first dose of chemotherapy (gemcitabine or carboplatin) administration should not be greater than 4 hours. If administration of trilaciclib is discontinued, GC therapy will also be discontinued.
  • AUC target area under the curve
  • a minimum of 0.7 mg/dL should be used.
  • “Adjusted” rather than actual body weight should be used for patients who are overweight (those with body mass index [BMI] > 25 kg/m2). Actual weight should be used for patients with BMI ⁇ 25 kg/m2.
  • Patients who have > 10% weight change from baseline or who experience CTCAE > Grade 2 renal toxicity (serum creatinine > 1.5 ULN) will require recalculation of the carboplatin dose for subsequent cycles (this would not be considered a dose reduction).
  • the creatinine at the time of the dose modification is lower than the baseline creatinine that was used, the prior (higher) creatinine value should be used. If the creatinine at the time of dose modification is higher than the baseline creatinine value, the current (higher) value should be used. This is to ensure that patients receive the intended dose reduction.
  • the maximum carboplatin dose based on target area under the curve (AUC) will be capped at 300 mg.
  • Gemcitabine is administered at 1000 mg/m 2 according to institutional standards.
  • the dose reductions for gemcitabine following a hematologic or non-hematologic toxicity are as follows: starting dose: 1000 mg/m 2 ; first dose reduction: 800 mg/m 2 ; and second dose reduction: 600 mg/m 2 . Criteria for Starting Cycle 2 and Each Subsequent Cycle
  • ANC > 1 x 10 9 /L
  • Platelet count > 100 x 10 9 /L
  • Nonhematologic toxi cities must be ⁇ Grade 2 or have returned to baseline.
  • ANC 1 x 10 9 /L
  • platelet count 100 x 10 9 /L.
  • Trilaciclib/placebo should be administered only on days that chemotherapy is administered.
  • ANC is ⁇ 0.75 x 10 9 /L OR platelets count ⁇ 75 x 10 9 /L, then all study drugs (GC and trilaciclib/placebo) will be skipped and doses of gemcitabine and carboplatin should be adjusted per Table XX.
  • the patient will return at the next planned visit (approximately 7 days later) which will be Day 1 of the next cycle to resume dosing. Note that the criteria for starting Day 1 outlined above will now apply to resumption of dosing. Dose reductions of trilaciclib are not permitted. Trilaciclib/placebo should be administered only on days that chemotherapy is administered.

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Abstract

This invention is in the area of improved treatments for a select group of patients with advanced/metastatic triple negative breast cancer (TNBC) which provide increased overall survival. In particular, the improved treatments are for a targeted group of patients receiving first- line treatment for advanced/metastatic TNBC who are checkpoint inhibitor treatment-naïve or, alternatively, in patients with advanced/metastatic TNBC who have prior exposure to an immune checkpoint inhibitor, for example, a programmed cell death protein- 1 (PD-1) and/or programmed death-ligand-1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who have developed therapeutic resistance to the immune checkpoint inhibitor leading to disease progression after an initial response.

Description

TREATMENTS FOR ADVANCED AND/OR METASTATIC TRIPLE NEGATIVE BREAST CANCER
Cross-Reference to Related Applications
This application claims the benefit of U.S. Provisional Patent Application No. 63/123,429, filed on December 9, 2020, U.S. Provisional Patent Application No. 63/127,851, filed on December 18, 2020; and U.S. Provisional Patent No. 63/136,181, filed on January 11, 2021. The entirety of each of these applications is hereby incorporated by reference for all purposes.
Field of the Invention
This invention is in the area of improved treatments for a select group of difficult to treat patients with advanced/metastatic triple negative breast cancer (TNBC), including patients whose TNBC has advanced while receiving an immune checkpoint inhibitor or whose TNBC is PD-L1 negative.
Background of the Invention
Triple negative breast cancer (TNBC) has been characterized by several aggressive clinicopathologic features, including onset at a younger age; large, high-grade tumors; and a propensity for visceral metastasis (Cheang et al., Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 2008; 14(5): 1368-76; Foulkes et al., Triple-negative breast cancer. N Engl J Med. 2010 Nov 11;363(20): 1938-48). Treatments which are effective for hormone receptor-positive breast cancer and human epidermal growth factor receptor 2 (HER2)-positive breast cancer, such as endocrine therapy or HER2-targeted therapies (e.g., trastuzumab) are not effective in TNBC, which lacks these markers; as such, chemotherapy remains the mainstay of treatment for TNBC. In particular, chemotherapies that target deoxyribonucleic acid (DNA) repair (e.g., platinum compounds) and cell proliferation (e.g., taxanes and anthracyclines, like doxorubicin) have been found to be particularly effective in TNBC; however, these treatments are limited by toxicity and eventually all patients develop resistance. Toxicity issues include chemotherapy-induced immunosuppression and myelosuppression (CIM), which may also affect anti-tumor efficacy due to an inability of the host immune system to effectively mount a response against the cancer. Targeted therapies have greatly improved outcomes for hormone receptor-positive and HER2 positive breast cancer, however, there is a paucity of effective targeted therapies in TNBC. Recently, pharmacological blockade of programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor has been at the forefront of immunotherapy for various cancers, as it facilitates efficacious anti-tumor immune responses by reinvigorating previously exhausted T cells.
In 2019, accelerated approval was granted by the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) for atezolizumab (TECENTRIQ®), a PD-L1 blocking antibody (immune checkpoint inhibitor [ICI]), in combination with nab-paclitaxel for patients with PD-L1 positive locally advanced/metastatic TNBC (see TECENTRIQ® Package Insert). The US FDA accelerated approval was based on the clinical trial results indicating improved progression-free survival (PFS) for patients with PD-L1 positive TNBC (Hazard ratio [HR]: 0.60 [0.48, 0.77]; p<0.0001; median 7.4 months vs. 4.8 months). In addition, the PD-L1 positive subset lived for an average of 25 months when treated with atezolizumab plus nab-paclitaxel, compared with 18 months when given placebo plus nab- paclitaxel. The efficacy improvement observed with the addition of atezolizumab to nab-paclitaxel was associated with immune related adverse events that occurred at relatively low frequencies, which could cause significant morbidity and mortality.
Pembrolizumab (KEYTRUDA®) monotherapy has also demonstrated improved objective response rates (ORR) in patients with PD-L1 positive TNBC tumors ranging from 18.5% to 21.4%, compared with response rates ranging from 5.3% to 9.6% in patients with PD-L1 negative tumors. Similarly, when pembrolizumab was used in combination with chemotherapy to treat TNBC, statistically significant and clinically meaningful improvements in PFS were observed for patients with PD-L1 positive tumors (overall PFS: 7.5 vs 5.6 months; combined positive score [CPS] >1 PFS: 7.6 vs 5.6; CPS>10 PFS: 9.7 vs 5.6) (Cortes J et al., KEYNOTE-355: Randomized, doubleblind, phase III study of pembrolizumab + chemotherapy versus placebo + chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer. J Clin Oncol. 2020;38(15)). These data led to a recent accelerated approval in the US of pembrolizumab in combination with chemotherapy for the treatment of patients with locally recurrent unresectable or metastatic TNBC whose tumors express PD-L1 (CPS >10) as determined by an FDA approved test (KEYTRUDA® Package Insert).
Despite the compelling clinical efficacy of ICIs, up to 50% of patients with PD-L1 positive tumors show resistance or relapse after PD-1/PD-L1 blockade (Herbst et al., Predictive correlates of response to the anti-PD-Ll antibody MPDL3280A in cancer patients. Nature. 2014; 515(7528):563-567). After an initial response to PD-1/PD-L1 blockade, acquired resistance occurs in most patients. Major factors contributing to PD-1/PD-L1 blockade resistance include constitutive PD-L1 expression in cancer cells, lack of tumor antigens, ineffective antigen presentation, activation of oncogenic pathways, mutations in IFN-y signaling, and factors within the tumor microenvironment including exhausted T cells, Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) (Bai et al., Regulation of PD-1/PD-L1 pathway and resistance to PD-1/PD-L1 blockade. Oncotarget. 2017;8(66): 110693-110707). Additional mechanisms of ICI resistance include genetic, epigenetic, and cellular signaling alterations that dysregulate neoantigen presentation/processing and disrupt cytotoxic T cells activity as well as mechanisms in which non-cancerous stromal or immune cells promote growth and resistance to ICIs (Liu et al., Mechanisms of resistance to immune checkpoint blockade. Am J Clin Dermatol. 2019;20(l):41-54; Barrueto et al., Resistance to checkpoint inhibition in cancer immunotherapy. Transl. One. 2020;13 : 100738; Jenkins et al., Mechanisms of resistance to immune checkpoint inhibitors. Brit J Cancer. 2018;118:9-16; Borcherding et al., Keeping tumors in check: A mechanistic review of clinical response and resistance to immune checkpoint blockade in cancer. J Mol Biol. 2018;430:2014-29; Gide et al., Primary and acquired resistance to immune checkpoint inhibitors in metastatic melanoma. Clin Cancer Res. 2018;24(6)). Additionally, PD- 1/PD-L1 blockade has generally not been effective in treating tumors that do not express PD-L1 (PD-L1 -negative cancers).
While the combination of ICIs with chemotherapy have provided a meaningful step forward for the treatment of patients with PD-L1 positive advanced/metastatic TNBC, it should be noted that due to the potential treatment toxicities associated with ICIs as well as the potential for resistance, not all patients with PD-L1 positive TNBC are appropriate candidates for ICI treatment and the patient population with PD-L1 negative TNBC generally does not derive benefit. As a result, overall, the TNBC patient population continues to represent an area of high unmet medical need, and novel therapeutic treatment regimens are required for TNBC patients that develop resistance to ICIs as well as treatment-naive patients regardless of PD-L1 tumor expression status, including those that are PD-L1 negative.
Because of the limited treatment available to TNBC patients who have advanced on immune-checkpoint inhibitors, novel therapies whose mechanisms broadly target these ICI mechanisms of resistance are needed following disease progression on currently clinically available immunotherapies.
Summary of the Invention
The present invention provides improved methods for treating advanced/metastatic triple negative breast cancer (TNBC) in specific, select patient subgroups by administering the short acting, selective, and reversible cyclin dependent kinase (CDK) 4/6 inhibitor trilaciclib, or a pharmaceutically acceptable salt thereof, in a specifically timed therapeutic protocol with select chemotherapeutic agents. The administration of the selective CDK4/6 inhibitor trilaciclib provides for improved survival outcomes, including overall survival (OS) and/or progression free survival (PFS) for these difficult to treat patients.
In one aspect, the improved treatments are for a select group of difficult to treat patients with advanced/metastatic TNBC who have prior exposure to an immune checkpoint inhibitor (ICI), for example, a programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who have developed therapeutic resistance to the immune checkpoint inhibitor leading to disease progression after an initial response. In particular embodiments, the patient has a TNBC tumor that is PD-L1 positive.
ICI administration to patients with PD-L1 -status positive advanced/metastatic TNBC represents a recent evolution in TNBC treatment options. Despite the compelling clinical efficacy of ICIs, it has been found that the majority of patients eventually develop therapeutic resistance leading to disease progression after an initial response. Mechanisms of ICI resistance include genetic, epigenetic, and cellular signaling alterations that dysregulate neoantigen presentation/processing and disrupt cytotoxic T cell activity as well as mechanisms in which non- cancerous stromal or immune cells promote growth and resistance to ICIs. The development of ICI resistance with resultant TNBC progression renders such patients particularly difficult to treat, with limited additional targeted therapies available.
By administering trilaciclib in combination with specific chemotherapeutic agents as described herein, one or more mechanisms leading to ICI resistance and disease progression can be overcome, resulting in increased antigen presentation (major histocompatibility complex (MHC) class I), enhanced T cell clonality and tumor infiltration, inhibition of regulatory T cell proliferation, decreased expression of T cell exhaustion markers (programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain 3 (TIM3)), stabilized expression of PD-L1 on tumor cells, promotion of dendritic cell migration, or increased T-effector cell function through high interferon -gamma (IFN-y) production. By administering trilaciclib to these difficult to treat patient subgroups, the immunosuppressive tumor microenvironment in the patient’s tumor — which renders the previously administered ICI ineffective or less effective and allows the tumor to progress — can be significantly overcome, improving the ability of the patient’s immune system to reduce or control tumor burden, improving quality of life, and improving overall survival in these difficult to treat subsets of patients.
In one aspect, the improved methods of treatment in a patient in need thereof include the administration of an effective amount of trilaciclib in combination with an effective amount of gemcitabine and carboplatin (or alternatively, an alternative platinum chemotherapeutic agent, including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), which is administered to a select patient with advanced/metastatic TNBC who has prior exposure to an immune checkpoint inhibitor (ICI), for example, a programmed cell death protein-1 (PD-1) inhibitor and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who has developed therapeutic resistance to the ICI leading to disease progression. ICI administration to patients with PD-L1- status positive advanced/metastatic TNBC represents a recent evolution in TNBC treatment options.
Historically, TNBC patients undergoing second-line therapy have had a very poor prognosis (PFS duration of 2 to 3 months and OS duration of 9 to 12 months). It has been discovered that trilaciclib in combination with gemcitabine and carboplatin provides a novel therapeutic option for these patients who have minimal options available, including the recently emergent subgroup of patients having PD-Ll-status positive TNBC who have progressed following first-line treatment with an ICI, for example nivolumab, atezolizumab, avelumab, durvalumab, or pembrolizumab, or other ICI. In prior studies evaluating the use of trilaciclib in TNBC chemotherapeutic protocols (e.g., Clinical Trial NCT02978716), this newly emergent subset of patients did not form part of the study analysis.
Accordingly, provided herein is a method of treating a patient in a second-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC comprising administering to the patient an effective amount of trilaciclib in a chemotherapeutic protocol, wherein the chemotherapeutic protocol further comprises the administration of gemcitabine and carboplatin (GC), and wherein the patient’s TNBC is PD-L1 status positive and the patient’s tumor has advanced or progressed following administration of an immune checkpoint inhibitor in a first- line setting. In particular embodiments, the method comprises administering to the patient in a 21- day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine. In some embodiments, the 21 -day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.
In alternative embodiments, the TNBC protocol incorporates a platinum-containing chemotherapeutic agent other than carboplatin, for example, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin. In some embodiments, the platinum containing chemotherapeutic used is cisplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is oxaliplatin.
In some embodiments, the patient being treated in the second-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC. In some embodiments, the patient being treated in the second-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC, defined as >1% tumor-infiltrating immune cells as confirmed by an in vitro diagnostic (IVD) assay, for example the Ventana SP-142 in vitro diagnostic (IVD) assay or use I IHC 22C3 pharmDx PDL1 assay. In some embodiments, the patient being treated in the second-line chemotherapeutic protocol has previously been treated with an ICI for a minimum duration of 8 weeks in the advanced/metastatic TNBC treatment setting. In some embodiments, the patient being treated in the second-line chemotherapeutic protocol has received the ICI as the most recent TNBC therapy. In some embodiments, the ICI is a PD-1 inhibitor. In some embodiments, the PD- 1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is cemiplimab. In some embodiments, the PD-1 inhibitor is CS1003. In some embodiments, the PD-1 inhibitor is tislelizumab. In some embodiments, the PD-1 inhibitor is toripalimab. In some embodiments, the PD-1 inhibitor is sintilimab. In some embodiments, the PD-1 inhibitor is camrelizumab. In some embodiments, the PD-1 inhibitor is pidilizumab. In some embodiments, the PD-1 inhibitor is retifanlimab. In some embodiments, the ICI is a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-L1 inhibitor is avelumab. In some embodiments, the PD-L1 inhibitor is durvalumab. In some embodiments, the PD-L1 inhibitor is sugemalimab. In some embodiments, the PD-L1 inhibitor is utomilumab. In some embodiments, the ICI is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, the CTLA-4 inhibitor is tremelimumab. In some embodiments, the ICI is a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is relatlimab. In some embodiments, the LAG-3 inhibitor is eftilagimod alpha. . In some embodiments, the ICI is a B7-H3 inhibitor. In some embodiments, the B7-H3 inhibitor is enoblituzumab. In some embodiments, the B7-H3 inhibitor is MGD009. In some embodiments, the ICI is a PD-l/Lag-3 specific DART molecule, for example, tebotelimab. In some embodiments, the ICI is a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is sabatolimab. In some embodiments, the ICI is a CD137 inhibitor. In some embodiments, the CD 137 inhibitor is urelumab. In some embodiments, the previous first-line chemotherapeutic protocol received by the patient for the treatment of TNBC did not include the administration of gemcitabine and carboplatin. In some embodiments, the patient has not received an ICI, for example a PD-1 inhibitor or PD-L1 inhibitor, within at least 14 days from the administration of trilaciclib. In some embodiments, the patient has a TNBC that is CDK4/6-positive. In some embodiments, the TNBC to be treated is CDK4/6-negative. In some embodiments, the TNBC to be treated has at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as i) homozygous deletion, ii) a frameshift mutation, or iii) a stop-gained mutation (i.e., a mutation that results in a premature termination codon (a stop codon was gained)). In alternative embodiments, the TNBC to be treated is CDK4/6-positive. In still other alternative embodiments, the TNBC is CDK4/6 indeterminate.
In an alternative aspect, provided herein is a method of treating a patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC comprising administering to the patient an effective amount of trilaciclib in a chemotherapeutic protocol, wherein the chemotherapeutic protocol further comprises the administration of gemcitabine and the platinum containing chemotherapeutic carboplatin (GC) (or alternatively, an alternative platinum containing chemotherapeutic agent including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), and wherein the patient is ICI treatment naive. Trilaciclib enhances immune activation and promotes anti-tumor immunity by differentially arresting cytotoxic and regulatory T-cell subsets followed by a faster recovery of cytotoxic T lymphocytes than regulatory T cells in tumors. This differential alteration of cell cycle kinetics between cytotoxic T lymphocytes and regulatory T cells results in a higher proportion of cytotoxic T lymphocytes than regulatory T cells, enhancement of T cell activation, and a decrease in regulatory T cell-mediated immunosuppressive functions. Together, these events promote the cytotoxic T lymphocyte-mediated clearance of tumor cells, which can be achieved without the administration of an ICI and the associated side-effects which accompany such ICI use. In some embodiments, the patient has a PD-L1 negative tumor. In some embodiments, the patient has a PD-L1 positive tumor.
In particular embodiments, the method comprises administering to the patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21 -day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting. In some embodiments, the 21-day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In an alternative embodiment, the platinum containing chemotherapeutic used is selected from cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is cisplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is oxaliplatin. In some embodiments, the patient has a PD-L1 negative tumor. In some embodiments, the patient has a PD-L1 positive tumor.
In some embodiments, the method comprises administering to the patient in a first-line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21 -day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting, and wherein the patient’s TNBC is classified as PD-L1 negative. In some embodiments, the 21-day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In an alternative embodiment, the platinum containing chemotherapeutic used is selected from cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is cisplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is oxaliplatin.
In alternative embodiments, the method comprises administering to the patient in a first- line chemotherapeutic protocol for the treatment of advanced/metastatic TNBC in a 21-day chemotherapeutic treatment cycle, an effective amount of trilaciclib on day 1 and day 8, an effective amount of gemcitabine on day 1 and day 8, and an effective amount of carboplatin on day 1 and day 8, wherein the trilaciclib is administered less than about four hours prior to the administration of carboplatin and gemcitabine, and wherein the patient is ICI treatment naive and has not received prior systemic therapy in the advanced/metastatic TNBC setting, and wherein the patient’s TNBC is classified as PD-L1 positive. In some embodiments, the 21-day chemotherapeutic treatment cycle is repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times. In an alternative embodiment, the platinum containing chemotherapeutic used is selected from cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is cisplatin. In an alternative embodiment, the platinum containing chemotherapeutic used is oxaliplatin.
In some embodiments, the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC. In some embodiments, the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status positive TNBC, defined as >1% tumor-infiltrating immune cells as confirmed by the Ventana SP-142 in vitro diagnostic (IVD) assay or I H4C 22C3 pharmDx PDL1 assay. In an alternative embodiment, the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status negative TNBC. In an alternative embodiment, the patient being treated in the first-line chemotherapeutic protocol has a documented PD-L1 status negative TNBC, defined as <1% tumor-infiltrating immune cells as confirmed, for example, by the Ventana SP-142 in vitro diagnostic (IVD) assay, I IHC 22C3 pharmDx PDL1 assay, or other suitable assay. In some embodiments, the patient has been disease- free for <12 months between the end of the last treatment with curative intent and disease progression. In some embodiments, the patient has been disease free for >12 months or de novo metastatic TNBC. In some embodiments, the TNBC to be treated is CDK4/6 -negative. In some embodiments, the TNBC to be treated has at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as i) homozygous deletion, ii) a frameshift mutation, or iii) a stop-gained mutation (i.e., a mutation that results in a premature termination codon (a stop codon was gained)). In alternative embodiments, the TNBC to be treated is CDK4/6-positive. In still other alternative embodiments, the TNBC is CDK4/6 indeterminate.
It has also been discovered that patients with threshold T-cell clonal diversity at or above the median T-cell clonal diversity of the group as a whole, for example all TNBC patients, or TNBC patients similarly situated, greatly benefit, for example, experience improved overall survival (OS), from the administration of trilaciclib in combination with gemcitabine and carboplatin (see, e.g., Example 2, Figs. 7 & 8). Accordingly, in one embodiment, the select patient subgroup with advanced/metastatic TNBC to be treated according to the methods described herein has a T-cell clonal peripheral diversity score at or above the median for the TNBC patient population. In some embodiments, the patient to be treated has a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones. In some embodiments, has a peripheral diversity score of at least about 12,000, 12,250, 12,500, 12,750, 13,000, 13,250, 13,500, 13,750, 14,000, 14,250, 14,500, 14,750, 15,000, or greater than 15,000.
Likewise, it has been discovered that patients with a lower or threshold Simpson clonality score, which measures the evenness of the T-cell repertoire, greatly benefit, for example experience improved overall survival (OS), with the administration of trilaciclib compared to those with higher Simpson clonality scores. In one aspect, the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC with a threshold Simpson clonality score. In one aspect, the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC with a threshold Simpson clonality score of less than about 0.08. In some embodiments, the patient has a Simpson clonality score of less than about 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, or 0.010, or less.
Improved Patient Outcomes
In some embodiments, the administration of a treatment regimen described herein to the patient subgroups described herein provides enhanced anti-tumor efficacy in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin or oxaliplatin, or nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin) without trilaciclib. In some embodiments, the administration of a treatment regimen described herein in the particular patient subgroups described above provides improved progression free survival (PFS) and/or overall survival (OS) in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative) without trilaciclib. In some embodiments, an improvement in PFS is based on per Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1).
In some embodiments, the administration of a treatment regimen described herein to a TNBC patient subgroups described above provides improved my el opreservation of hematopoietic stem and progenitor cells (HSPCs) and immune effector cells such as lymphocytes including T- lymphocytes. In some embodiments, the administration of a treatment regimen described herein to the TNBC patient subgroups described above provides reduced chemotherapy-induced myelosuppression (CIM). In some embodiments, the administration of a treatment regimen described herein provides myelopreservation of the neutrophil lineage in patients compared to those receiving the chemotherapy without trilaciclib. In some embodiments, the administration of a treatment regimen described herein provides a reduction in the duration of severe (Grade 4) neutropenia in patients compared to those receiving gemcitabine and carboplatin (or platinum containing drug alternative) without trilaciclib.
In some embodiments, the administration of a treatment regimen described herein provides a reduction in severe neutropenia events, a reduction in granulocyte-colony stimulating factor (G- CSF) treatment, or a reduction in febrile neutropenia (FN) adverse events (AEs). In some embodiments, the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 decreased hemoglobin laboratory values, red blood cell (RBC) transfusions, or erythropoiesis-stimulating agent (ESA) administration. In some embodiments, the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 decreased platelet count laboratory values and/or the number of platelet transfusions. In some embodiments, the administration of a treatment regimen described herein provides a reduction in Grade 3 or 4 hematologic laboratory values.
In some embodiments, the administration of a treatment regimen to the TNBC patient subgroup described herein provides a reduction in all-cause dose reductions or cycle delays and relative dose intensity of gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin or oxaliplatin). In some embodiments, the administration of a treatment regimen described herein provides a reduction in i) hospitalizations, including but not limited to those due to all causes, febrile neutropenia/neutropenia, anemia/RBC transfusion, thrombocytopenia/bleeding and infections) or ii) antibiotic use, including but not limited to intravenous (IV), oral, and oral and IV administered antibiotics.
In some embodiments, the administration of a treatment regimen described herein provides a reduction of chemotherapy-induced fatigue (CIF) in patients compared to those receiving chemotherapy without trilaciclib. In some embodiments, the reduction of CIF is a reduction in the time to first confirmed deterioration of fatigue (TTCD-fatigue), as measured by the Functional Assessment of Cancer Therapy -Fatigue (FACIT-F). In some embodiments, the administration of a treatment regimen described herein provides an improvement to one or more of: Functional Assessment of Cancer Therapy-General (FACT- G) domain scores (physical, social/family, emotional, and functional well-being); Functional Assessment of Cancer Therapy-Anemia (FACT-An); 5-level EQ-5D (EQ-5D-5L); Patient Global Impression of Change (PGIC) fatigue item; or Patient Global Impression of Severity (PGIS) fatigue item.
Brief Description of the Figures
Fig. 1 is a schematic of a human clinical trial assessing the effect of trilaciclib on overall survival (OS) compared with placebo in patients receiving first-line gemcitabine and carboplatin (GC) for advanced/metastatic triple negative breast cancer (Cohort 1) and patients receiving GC as second-line therapy after treatment with a PD-1/PD-L1 inhibitor in advanced/metastatic triple negative breast cancer (Cohort 2). The treatment phase consists of 21 -day cycles: Trilaciclib will be administered IV prior to GC infusions. Trilaciclib will be administered via IV prior to GC administration at 240mg/m2. Gemcitabine will be administered via IV at 1000mg/m2. Carboplatin will be administered IV at an AUC=2. The study will include 3 study phases: Screening Phase, Treatment Phase, and Survival Follow up Phase. The Treatment Phase begins on the day of the first dose with study treatment and completes at the Safety Follow-up Visit. The first Survival Follow-up assessment should occur 3 months after the Safety Follow-up Visit. GC= Gemcitabine and Carboplatin, DC=discontinuation PD=Progressive disease, PI=Principal Investigator, WD=W ithdrawal .
Fig. 2 is a bar graph showing the median treatment effect (25% and 75% quartiles) of Trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on Simpson clonality score. The x-axis represents the timing of the measurement. C1D1= cycle 1, day 1; C3D1= cycle 3, day 1; C5D1= cycle 5, day 1. The y-axis represents the Simpson clonality score ranging from 0-0.25. There was a significant decrease in Simpson clonality among patients who received Trilaciclib prior to GCb compared with GCb alone (P interactional.012).
Fig. 3 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median Simpson clonality score. The x-axis depicts months from randomization and number of patients at risk. The y-axis depicts the probability of survival. For Kaplan-Meier estimates of probability of survival, patients were stratified by high (equal or above median, solid lines) and low (below median, dashed lines). When patients were stratified above or below median Simpson clonality, there was a trend for improved overall survival among patients with decreased peripheral clonality, with a statistically significant improvement among patients receiving trilaciclib (P=0.02).
Fig. 4 is a bar graph showing the median treatment effect (25% and 75% quartiles) of Trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on Fraction of newly detected expanded clones. The x-axis represents response to treatment (Yes or No) and the y-axis represents the median value (25% and 75% quartiles) of the fraction of newly detected expanded clones. Responders receiving trilaciclib in groups 2 and 3 had more newly detected expanded clones compared with responders receiving GCb alone (P=0.09), with no difference between responders and non-responders in the Trilaciclib groups (P=0.79).
Fig. 5 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of newly detected expanded clones. The x-axis depicts months from randomization and number of patients at risk. The y-axis depicts the probability of survival. For Kaplan-Meier estimates of probability of survival, patients were stratified by high (equal or above median, solid lines) and low (below median, dashed lines). Although not statistically significant, when patients were stratified above or below median fraction of newly detected expanded clones, OS was improved among patients with a higher fraction of newly detected expanded clones who received trilaciclib.
Fig. 6 is a bar graph showing the median treatment effect (25% and 75% quartiles) of trilaciclib + gemcitabine/carboplatin (GCb) versus placebo + GCb on T-cell diversity. The x-axis represents the timing of the measurement. C1D1= cycle 1, day 1; C3D1= cycle 3, day 1; C5D1= cycle 5, day 1. The y-axis represents the number of T-cell clones (T-cell clonality). There was a significant increase in T-cell diversity among patients who received Trilaciclib prior to GCb compared with GCb alone (P interactional.007).
Fig. 7 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of peripheral diversity as measured by the number of T-cell clones (T-cell clonality). The x-axis depicts months from randomization and number of patients at risk. The y-axis depicts the probability of survival. For Kaplan-Meier estimates of probability of survival, patients were stratified by high (equal or above median) and low (below median).
Fig. 8 is a Kaplan-Meier plot of overall survival of triple negative breast cancer human patients based on stratification above or below the median fraction of peripheral diversity as measured by the number of T cell clones (T-cell clonality) across the different treatment group. The x-axis depicts months from randomization and number of patients at risk. For Kaplan-Meier estimates of probability of survival, patients were stratified by high (equal or above median, solid lines) and low (below median, dashed lines).
Fig. 9 is a table of ORR, PFS and OS by Prior Line of Therapy for Recurrent/Metastatic TNBC as assessed in a Phase 2 clinical trial, a. PFS and ORR based on data with cutoff date of 17May2019; OS results and duration of survival follow-up are updated based on final database lock on 17Jul2020. b The two-sided p-value is calculated using stratified exact CMH method with the term of liver involvement for by line analysis; for 0-2 group one more factor was controlled: the prior line of therapy, c The two-sided p-value was calculated using stratified log-rank test with the term of liver involvement for by line analysis; for 0-2 group one more factor was controlled: the prior line of therapy, d The hazard ratio (HR) was calculated from a Cox proportional hazard model with the stratification factor of liver involvement for by line analysis; for 0-2 group one more factor was controlled: the prior line of therapy, e Duration of survival follow up was calculated from first dose to the death date or the last contact date.
Figure imgf000017_0001
The 95% CI for ORR was calculated using the exact Clopper-Pearson method. CI = confidence interval; HR = hazard ratio; ITT = intent to treat analysis set; LOT = line of therapy; NA = not applicable; ORR = objective response rate; OS = overall survival; PFS = progression free survival.
Fig. 10 is table of ORR, PFS and OS by Prior Line of Therapy (pooled trilaciclib + G/C + SOC). a. PFS and ORR based on data with cutoff date of 17May2019, OS updated with final database lock on 17Jul2020. b. The two-sided p-value is calculated using stratified exact CMH method to account for liver involvement [Yes or No] as the stratification factor, c. The two-sided p-value was calculated using stratified log-rank test to account for liver involvement [Yes or No] as the stratification factor, d. The hazard ratio (HR) between the 2 treatments groups (trilaciclib versus GC only) was calculated from a Cox proportional hazard model in which treatment and the stratification factors (liver involvement [Yes or No] were included as fixed effects, e. Duration of survival follow up was calculated from first dose to the death date or the end of study date. IJThe 95% CI for ORR was calculated using the exact Clopper-Pearson method. { The stratification factors of number of prior lines of therapy [0 vs 1-2] and liver involvement were included for HR estimation and for generating two-sided p-value. CI = confidence interval; HR = hazard ratio; ITT = intent to treat analysis set; LOT = line of therapy; NA = not applicable; ORR = objective response rate; OS = overall survival; PFS = progression free survival.
Detailed Description of the Invention
Definitions
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In the specification, singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed application. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
In some embodiments of each compound described herein, the compound may be in the form of a racemate, enantiomer, mixture of enantiomers, diastereomer, mixture of diastereomers, tautomer, N-oxide, or isomer, such as a rotamer, as if each is specifically described unless specifically excluded by context. The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and/or”. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individual recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention unless otherwise claimed.
In some embodiments of each compound described herein, the compound may be in the form of a tautomer, N-oxide, or isomer, such as a rotamer, as if each is specifically described unless specifically excluded by context.
An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease, disorder, or side-effect experienced by a patient (i.e. palliative treatment) or to decrease a cause or effect of the disease, disorder (i.e. disease-modifying treatment), or side effect experienced by a patient as a result of the administration of a therapeutic agent.
Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the invention. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
As used herein, “pharmaceutical compositions” are compositions comprising at least one active agent, and at least one other substance, such as a carrier. “Pharmaceutical combinations” are combinations of at least two active agents which may be combined in a single dosage form or provided together in separate dosage forms with instructions that the active agents are to be used together to treat any disorder described herein.
As used herein, “pharmaceutically acceptable salt” is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.
Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n- COOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Wherein the methods described herein identify the administration of a particular compound, it is understood that administration of the compound’s pharmaceutically acceptable salt, if applicable, is encompassed as an embodiment. As used herein, the term "prodrug" means a compound which when administered to a host in vivo is converted into the parent drug. As used herein, the term "parent drug" means any of the presently described chemical compounds that are useful to treat any of the disorders described herein, or to control or improve the underlying cause or symptoms associated with any physiological or pathological disorder described herein in a host, typically a human. Prodrugs can be used to achieve any desired effect, including to enhance properties of the parent drug or to improve the pharmaceutic or pharmacokinetic properties of the parent. Prodrug strategies exist which provide choices in modulating the conditions for in vivo generation of the parent drug, all of which are deemed included herein. Nonlimiting examples of prodrug strategies include covalent attachment of removable groups, or removable portions of groups, for example, but not limited to acylation, phosphorylation, phosphonylation, phosphoramidate derivatives, amidation, reduction, oxidation, esterification, alkylation, other carboxy derivatives, sulfoxy or sulfone derivatives, carbonylation or anhydride, among others.
The term “carrier” applied to pharmaceutical compositions/combinations of the invention refers to a diluent, excipient, or vehicle with which an active compound is provided.
A “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition/combination that is generally safe, and neither biologically nor otherwise inappropriate for administration to a host, typically a human.
In non-limiting embodiments, trilaciclib can be used in a form that has at least one desired isotopic substitution of an atom, at an amount above the natural abundance of the isotope, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., the same number of protons but a different number of neutrons.
Examples of isotopes that can be incorporated into trilaciclib for use in the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine and iodine such as 2H, 3H, 11C, 13C, 14C, 15N, 18F 3 IP, 32P, 35S, 36CI, and 1251 respectively. In one non-limiting embodiment, isotopically labelled compounds can be used in metabolic studies (with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 18F labeled compound may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
By way of general example and without limitation, isotopes of hydrogen, for example, deuterium (2H) and tritium (3H) may be used anywhere in described structures that achieves the desired result. Alternatively, or in addition, isotopes of carbon, e.g., 13C and 14C, may be used.
Isotopic substitutions, for example deuterium substitutions, can be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted with deuterium. In certain embodiments, the isotope is 90, 95, or 99% or more enriched in an isotope at any location of interest. In one non-limiting embodiment, deuterium is 90, 95, or 99% enriched at a desired location.
Trilaciclib for use in the present invention may form a solvate with solvents (including water). Therefore, in one non-limiting embodiment, the invention includes a solvated form of trilaciclib. The term "solvate" refers to a molecular complex of trilaciclib (including a salt thereof) with one or more solvent molecules. Non-limiting examples of solvents are water, ethanol, dimethyl sulfoxide, acetone and other common organic solvents. The term "hydrate" refers to a molecular complex comprising a compound of the invention and water. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent may be isotopically substituted, e.g., D2O, d6-acetone, d6-DMSO. A solvate can be in a liquid or solid form.
The “patient” or “subject” treated is typically a human patient, although it is to be understood the methods described herein are effective with respect to other animals, such as mammals. More particularly, the term patient can include animals used in assays such as those used in preclinical testing including but not limited to mice, rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine (pigs and hogs), ruminants, equine, poultry, felines, bovines, murines, canines, and the like.
As generally contemplated herein, the term “hematopoietic stem and progenitor cells” (HSPCs) includes, but are not limited to, long term hematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells (ST-HSCs), hematopoietic progenitor cells (HPCs), multipotent progenitors (MPPs), oligodendrocyte pre-progenitors (OPPs), monocyte progenitors, granulocyte progenitors, common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), granulocyte progenitors, monocyte progenitors, and megakaryocyte-erythroid progenitors (MEPs), megakaryocyte progenitors, erythroid progenitors, HSC/MPPs (CD45dim/CD34+/CD38-), OPPs (CD45dim/CD34+/CD38+), monocyte progenitors (CD45+/CD14+/CDl lb+), granulocyte progenitors (CD45+/CD14-/CD1 lb+), erythroid progenitors (CD45-/CD71+), and megakaryocyte progenitors (CD45+/CD61+).
The term “immune effector cell” generally refers to an immune cell that performs one or more specific functions. Immune effector cells are known in the art and include for example, but are not limited to, T-cells, including Naive T-cells, Memory T-cells, Activated T-cells (Thelper (CD4+) and Cytotoxic T cells (CD8+)), TH1 activated T-cells, TH2 activated T-cells, TH17 activated T-cells, Naive B cells, Memory B cells, plasmablasts, dendritic cells, monocytes, and natural killer (NK) cells.
As used herein, the term “immune checkpoint inhibitor (ICI)” refers to inhibitory therapy targeting immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. ICIs block inhibitory checkpoints, and may act to restore immune system function. ICIs include those targeting immune checkpoint proteins such as programmed cell death- 1 protein (PD-1), PD-1 Ligand- 1 (PD-L1), PD-1 Ligand- 2 (PD-L2), CTLA-4, LAG-3, TIM-3, and V-domain Ig suppressor of T-cell activation (VISTA), B7-H3/CD276, indoleamine 2, 3 -dioxygenase (IDO), killer immunoglobulin-like receptors (KIRs), carcinoembryonic antigen cell adhesion molecules (CEACAM) such as CEACAM-1, CEACAM- 3, and CEACAM-5, sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and B and T lymphocyte attenuator (BTLA) protein, and the like. Immune checkpoint inhibitors are known in the art.
Triple Negative Breast Cancer
Triple-negative Breast Cancer (TNBC) is a highly aggressive breast cancer subtype that accounts for 15-20% of breast cancer cases annually and 25% of all breast cancer deaths. TNBC has been characterized by several aggressive clinicopathologic features, including onset at a younger age; large, high-grade tumors; and a propensity for visceral metastasis (Cheang et al., Basal-like breast cancer defined by five biomarkers has superior prognostic value than triplenegative phenotype. Clin Cancer Res. 2008;14(5): 1368-76.; Foulkes et al., Triple-negative breast cancer. N Engl J Med. 2010 Nov 11;363(20): 1938-48).
A breast cancer is generally classified as TNBC based on local ER-negative, progesterone receptor-negative, HER2-negative status, which can be determined through a histological or cytological hormone receptor immunohistochemistry (IHC) assessment for estrogen and progesterone (defined as <1% nuclei staining), and by IHC [0 or 1+] OR in situ hybridization [ratio <2.0] OR average gene copy number of <4 signals/nucleus) for HER2-negative, non- overexpression (per 2018 American Society of Clinical Oncology and the College of American Pathologists (ASCO CAP) criteria).
PD-L1 Status
In certain embodiments, the patient is receiving a second-line therapy in the advanced/metastatic TNBC setting following failure of or advancement on an ICI inhibitor and the TNBC to be treated is PD-L1 positive. In alternative embodiments, the patient is receiving a first- line therapy in the advanced/metastatic TNBC setting and the TNBC to be treated is PD-L1 positive. In still yet another alternative embodiment, the patient is receiving a first-line therapy in the advanced/metastatic TNBC setting and the TNBC to be treated is PD-L1 negative.
PD-L1 is a transmembrane protein that down-regulates immune responses through binding to its two inhibitory receptors, programmed death-1 (PD-1) and B7.1. PD-1 is an inhibitory receptor expressed on T cells following T-cell activation, which is sustained in states of chronic stimulation such as in chronic infection or cancer (Blank, C and Mackensen, A, Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunol. Immunother., 2007. 56(5): p. 739-745). Binding of PD-L1 with PD-1 inhibits T cell proliferation, cytokine production and cytolytic activity, leading to the functional inactivation or exhaustion of T cells. B7.1 is a molecule expressed on antigen presenting cells and activated T cells. PD-L1 binding to B7.1 on T cells and antigen presenting cells can mediate down-regulation of immune responses, including inhibition of T-cell activation and cytokine production (see Butte MJ, Keir ME, Phamduy TB, et al. Programmed death- 1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity. 2007;27(l): 111-122). PD-L1 expression has been observed in immune cells and tumor cells. See Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T- cell proliferation and interleukin- 10 secretion. Nat Med. 1999;5(12): 1365-1369; Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of response to the anti-PD-Ll antibody MPDL3280A in cancer patients. Nature. 2014;515(7528):563-567. Aberrant expression ofPD-Ll on tumor cells has been reported to impede anti-tumor immunity, resulting in immune evasion.
PD-L1 expression can be determined by methods known in the art. For example, PD-L1 expression can be detected using PD-L1 IHC 22C3 pharmDx, the FDA-approved in vitro diagnostic immunohistochemistry (IHC) test developed by Dako and Bristol -Meyers Squibb as a companion test for treatment with pembrolizumab. This is qualitative assay using Monoclonal Mouse Anti-PD-Ll, Clone 22C3 PD-L1 and EnVision FLEX visualization system on Autostainer Lin 48 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human cancer tissue. Expression levels can be measured using the tumor proportion score (TPS), which measures the percentage of viable tumor cells showing partial or complete membrane staining. Staining can show PD-Ll expression from l% to 100%.
PD-L1 expression can also be detected using PD-L1 IHC 28-8 pharmDx, the FDA- approved in vitro diagnostic immunohistochemistry (IHC) test developed by Dako and Merck as a companion test for treatment with nivolumab. This qualitative assay uses the Monoclonal rabbit anti-PD-Ll, Clone 28-8 and EnVision FLEX visualization system on Autostainer Lin 48 to detect PD-L1 in formalin-fixed, paraffin-embedded (FFPE) human cancer tissue.
Other commercially available tests for PD-L1 detection include the Ventana SP263 assay (developed by Ventana in collaboration with AstraZeneca) that utilizes monoclonal rabbit anti- PD-Ll, Clone SP263 and the Ventana SP142 Assay (developed by Ventana in collaboration with Genentech/Roche) that uses rabbit monoclonal anti-PD-Ll clone SP142. Determination of PD- L1 status is indication-specific, and evaluation is based on either the proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells (% IC) of any intensity or the percentage of PD-L1 expressing tumor cells (% TC) of any intensity. For example, PD-L1 positive status in TNBC is considered > 1% IC. In some embodiments, the TNBC has a PD-L1 positive status > 1% IC. In some embodiments, the TNBC has a PD-L1 positive status > 1% IC as determined by the Ventana SP142 Assay.
In some embodiments, the TNBC has a PD-L1 positive status < 1% IC. In some embodiments, the TNBC has a PD-L1 positive status < 1% IC as determined by the Ventana SP142 Assay.
CDK4/6 Status
In certain embodiments, the TNBC to be treated is CDK4/6 -negative. In alternative embodiments, the TNBC to be treated is CDK4/6-positive. In still other alternative embodiments, the TNBC is CDK4/6 indeterminate.
CDK4/6 replication independent cancers generally have a retinoblastoma gene (Rbl) aberration. The gene product of Rbl — Rb-protein — is a downstream target of CDK4/6. RBI is commonly dysregulated in cancer cells through deletion, mutation or epigenetic modification resulting in loss of RB expression, as well as by aberrant CDK kinase activity leading to excessive phosphorylation and inactivation of RB function (Chen et al. Novel RBI -Loss Transcriptomic Signature Is Associated with Poor Clinical Outcomes across Cancer Types. Clin Cancer Res. 2019;25(14); Sherr, C.J., and McCormick, F. The RB and p53 pathways in cancer. Cancer Cell, 2002;2: 103 12.). CCNE1/2 (cyclin E) is part of a parallel pathway that provides functional redundancy with CDK4/6 and helps to transition cells from the G1 to S phase. Overexpression will decrease the reliance on the CDK4/6 pathway leading to CDK4/6 independence (Turner et al., Cyclin El Expression and Palbociclib Efficacy in Previously Treated Hormone Receptor-Positive Metastatic Breast Cancer. J Clin Oncol. 2019;37(14): 1169-78.). Therefore, a tumor with either CCNE1/2 amplification or RB loss will generally be considered “CDK4/6 independent”.
Cancers that are CDK4/6 replication dependent require the activity of CDK4/6 for replication or proliferation. CDK 4/6 replication dependent TNBCs generally have an intact and functional Rb pathway and/ increased expression of CDK4/6 activators (cyclin D), and/or a d-type cyclin activating features (DCAF) — including CCND1 translocation, CCND1-3 3’UTR loss, and amplification of CCND2 or CCND3 (see Gong et al. Genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4 and CDK5 inhibitor abemaciclib. Cancer Cell. 2017;32(6):761-76). Tumors that are wildtype for RB and CCNE1/2 as well as have one of the DCAF described above are generally classified as “CDK4/6 dependent”.
Tumors that cannot be classified as either CDK4/6-replication dependent or CDK4/6- replication independent are generally classified as “CDK4/6 indeterminate” since they cannot be confirmed as CDK4/6 dependent or independent.
In some embodiments, the TNBC is classified as CDK4/6-replication dependent. In some embodiments, the TNBC is classified as CDK4/6-replication independent. In some embodiments, the TNBC is classified as CDK4/6 indeterminate.
Methods of determining CDK4/6 genetic signature analysis are known in the art and involve the utilization of tumor tissue collected from a patients’ biopsy (TNBC primary or metastatic site) and are described in Shapiro GI. Genomic biomarkers predicting response to selective CDK4/6 inhibition: Progress in an elusive search. Cancer Cell. 2017;32(6):721-3 and Gong et al. Genomic aberrations that activate D-type cyclins are associated with enhanced sensitivity to the CDK4 and CDK5 inhibitor abemaciclib. Cancer Cell. 2017;32(6):761-76. Alternative methods of determining CDL4/6 status includes the Prosigna Breast Cancer Prognostic Gene Signature Assay [PAM50] and Lehmann triple-negative breast cancer type (see, e.g., Prat et al. Research-based PAM50 subtype predictor identifies higher responses and improved survival outcomes in HER2-positive breast cancer in the NOAH study. Clin Cancer Res. 2014;20:511-21; Lehmann et al., Refinement of triple-negative breast cancer molecular subtypes: implications for neoadjuvant chemotherapy selection. PLoS One. 2016;l le0157368, and Asghar et al. Single-cell dynamics determines response to CDK4/6 inhibition in triple-negative breast cancer. Clin Cancer Res. 2017;23:5561-72).
In some embodiments, the patient receiving trilaciclib in combination with gemcitabine and carboplatin (or alternatively, a platinum containing compound), has a CDK4/6 independent TNBC having at least one of the following: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as i) homozygous deletion, ii) a frameshift mutation, or iii) a stop-gained mutation (i.e., a mutation that results in a premature termination codon (a stop codon was gained)). In some embodiments, the patient receiving trilaciclib in combination with gemcitabine and carboplatin (or alternatively, a platinum containing compound), has a CDK4/6 dependent TNBC which does not have 1) at least one of the following: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as i) homozygous deletion, ii) a frameshift mutation, or iii) a stop-gained mutation (i.e., a mutation that results in a premature termination codon ( a stop codon was gained));
2) does have a) wild type: i) CCNE1; ii) CCNE2; and iii) RBI; and
3) has at least one of the following D-cyclin activating features: i) CCND2 amplification; ii) CCND3 amplification; and iii) CCD 1-3 3’ UTR loss defined as a homozygous or heterozygous deletion of any of these UTRs.
Prior Exposure to Immune Checkpoint Inhibitor Treatment
In March 2019, the FDA granted accelerated approval to atezolizumab (TECENTRIQ, Genentech) plus nab-paclitaxel (ABRAXANE, Celgene) as first-line therapy for unresectable locally advanced or metastatic TNBC whose tumors express PD-L1 of 1% or greater. This was groundbreaking, as it represented the first approval of immunotherapy for breast cancer and the first biomarker-driven treatment for TNBC. Moreover, it is the first drug combination approved specifically for a group of patients with metastatic TNBC in desperate need of more effective targeted therapies. Median OS for metastatic TNBC is approximately 12 months.
Atezolizumab is a monoclonal antibody that targets and blocks recognition of PD-L1 on cancer cells by T cells. This prevents binding of PD-1 on the T-cell to the PD-L1 receptor on the cancer cell and, therefore, releases the brakes on the T cell to allow for attack and killing of the cancer cells.
The FDA based the approval on the randomized phase 3 IMpassionl30 trial, in which Schmid and colleagues compared atezolizumab plus nab-paclitaxel to placebo plus nab-paclitaxel. After median follow-up of 12.9 months, the results — published last year in The New England Journal of Medicine — showed a statistically significant improvement in median PFS (7.5 months vs. 5 months; HR = 0.62; P < .0001) in the PD-L1 -positive population. Researchers observed no statistically significant PFS improvement in the PD-L1 -negative subgroup.
After median follow-up of 18 months, a second interim analysis showed a compelling 7- month median OS advantage in the PD-L1 -positive population, (25 months vs. 18 months; HR = 0.71). Significance could not be concluded for OS as per the study design, which stated that statistical significance must be achieved in the intent-to-treat population (this was not significant) before it could be concluded in the PD-L1 -positive subgroup. Importantly, PD-L1 positivity was defined as PD-L1 expression on at least 1% of immune cells based on the Ventana PD-L1 (SP142) Assay. A higher percentage of patients with PD-L1 -positive disease achieved 24-month OS (51% vs. 37%), and a numerically superior ORR was observed in the PD-L1 -positive population (59% vs. 49%).
On November 13, 2020, the Food and Drug Administration granted accelerated approval to pembrolizumab (KEYTRUDA, Merck & Co.) in combination with chemotherapy for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS >10) as determined by an FDA approved test. FDA also approved the PD-L1 IHC 22C3 pharmDx (Dako North America, Inc.) as a companion diagnostic for selecting patients with TNBC for pembrolizumab.
Approval was based on KEYNOTE-355 (NCT02819518), a multicenter, double-blind, randomized, placebo-controlled trial in patients with locally recurrent unresectable or metastatic TNBC, who had not been previously treated with chemotherapy in the metastatic setting. Patients were randomized (2: 1) to receive pembrolizumab 200 mg on day 1 every 3 weeks or placebo in combination with different chemotherapy treatments (paclitaxel protein-bound, or paclitaxel, or gemcitabine plus carboplatin) via intravenous infusion.
The main efficacy outcome measure was progression-free survival (PFS) as assessed by blinded independent review according to RECIST 1.1, tested in the subgroup of patients with CPS >10. Median PFS was 9.7 months (95% CI: 7.6, 11.3) in the pembrolizumab plus chemotherapy arm and 5.6 months (95% CI:5.3, 7.5) in the placebo arm (HR 0.65; 95% CI: 0.49, 0.86; one-sided p-value=0.0012).
In certain aspects of the present invention, provided herein are methods of treating patients with advanced/metastatic triple negative breast cancer (TNBC) who have prior exposure to an immune checkpoint inhibitor, for example a programmed cell death protein-1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting in the case of TNBC, and who have developed therapeutic resistance to the immune checkpoint inhibitor leading to disease progression after an initial response. By administering the short acting, selective, and reversible cyclin dependent kinase (CDK) 4/6 inhibitor trilaciclib in a therapeutic protocol that further includes the administration of the chemotherapeutic agents gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), mechanisms of immune checkpoint inhibitor resistance resulting in the dysregulation of cytotoxic T-cell activity within the tumor microenvironment (TME) can be overcome, increasing the limited treatment options these patients have in the second-line and third-line setting, and increasing overall survival in a subset of difficult to treat patients.
Example of immune checkpoint inhibitors and immune modulating agents include, but are not limited to, a PD-1 inhibitor, PD-L1 inhibitor, PD-L2 inhibitor, CTLA-4 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, V-domain Ig suppressor of T-cell activation (VISTA) inhibitors, TIGIT inhibitor, Siglec-15 inhibitor, B7-H3 (CD272) inhibitor, BTLA inhibitor (CD272), small molecule, peptide, nucleotide, or other inhibitor. In certain aspects, the immune modulator is an antibody, such as a monoclonal antibody.
In some embodiments, the patient has previously been administered a PD-1 inhibitor. PD- 1 inhibitors include, for example, but are not limited to, nivolumab (Opdivo®), pembrolizumab (Keytruda®), pidilizumab (Medivation), AMP-224 (Amplimmune); sasanlimab (PF-06801591; Pfizer), spartalizumab (PDR001; Novartis), cemiplimab (Libtayo®; REGN2810; Regeneron), retifanlimab (MGA012; MacroGenics), tislelizumab (BGB-A317; BeiGene), camrelizumab (SHR-1210; Jiangsu Hengrui Medicine Company and Incyte Corporation), CS1003 (Cstone Pharmaceuticals), and dostarlimab (TSR-042;Tesaro).
In some embodiments, the patient has previously been administered a PD-L1 inhibitor. PD-L1 inhibitors include, for example, but are not limited to, atezolizumab (Tecentriq®, Genentech), durvalumab (Imfinzi®, AstraZeneca); avelumab (Bavencio®; Merck), envafolimab (KN035; Alphamab), BMS-936559 (Bristol-Myers Squibb), lodapolimab (LY3300054; Eli Lilly), cosibelimab (CK-301; Checkpoint Therapeutics), sugemalimab (CS-1001; Cstone Pharmaceuticals), adebrelimab (SHR-1316; Jiangsu HengRui Medicine), CBT-502 (CBT Pharma), and BGB-A333 (BeiGene).
In some embodiments, the patient has previously been administered a dual PD-L1/PD-1 inhibitor.
In some embodiments, the patient has previously been administered a PD-L1/VISTA inhibitor. PD-L1 -VISTA inhibitors include, but are not limited to, CA-170 (Curis Inc.). In some embodiments, the immune checkpoint inhibitor is a VISTA immune checkpoint inhibitor. VISTA inhibitors include, but are not limited to, JNJ-61610588 (Johnson & Johnson).
In some embodiments, the patient has previously been administered a CTLA-4 immune checkpoint inhibitor. CTLA-4 inhibitors include, but are not limited to, ipilimumab (Yervoy®, Bristol Myers Squibb); tremelimumab (AstraZeneca/Medlmmune), zalifrelimab (AGEN1884; Agenus) and AGEN2041 (Agenus).
In some embodiments, the patient has previously been administered a LAG-3 immune checkpoint inhibitor. Examples of LAG-3 immune checkpoint inhibitors include, but are not limited to, relatlimab (BMS-986016; Bristol-Myers Squibb), GSK2831781 (GlaxoSmithKline), eftilagimod alpha (IMP321; Prima BioMed), leramilimab (LAG525; Novartis), MK-4280 (Merck), REGN3767 (Regeneron), TSR-033 (Tesaro), BI754111 (Bohringer Ingelheim), Sym022 (Symphogen). the dual PD-1 and LAG-3 inhibitor tebotelimab (MGD013; MacroGenics), and the dual PD-L1 and LAG-3 inhibitor FS118 (F-Star).
In some embodiments, the patient has previously been administered a TIM-3 immune checkpoint inhibitor. TIM-3 inhibitors include, but are not limited to, TSR-022 (Tesaro), MBG453 (Novartis), Sym023 (Symphogen), INCAGN2390 (Incyte), LY3321367 (Eli Lilly and Company), BMS-986258 (BMS), SHR-1702 (Jiangsu HengRui), and RO7121661 (Roche).
In some embodiments, the patient has previously been administered a TIGIT (T cell immunoreceptor with Ig and ITIM domains) immune checkpoint inhibitor. TIGIT immune checkpoint inhibitors include, but are not limited to, MK-7684 (Merck), Etigilimab /OMP- 313 M32 (OncoMed), Tiragolumab/MTIG7192A/RG-6058 (Genentech), BMS-986207 (BMS), AB-154 (Arcus Biosciences), and ASP-8374 (Potenza).
In some embodiments, the patient has previously been administered an immune checkpoint inhibitor including, for example, but not limited to, a B7-H3/CD276 immune checkpoint inhibitor such as enoblituzumab (MGA217, Macrogenics) MGD009 (Macrogenics), 13 H-8H9/omburtamab (Y-mabs), and I-8H9/omburtamab (Y-mabs), an indoleamine 2,3-dioxygenase (IDO) immune checkpoint inhibitor such as Indoximod and INCB024360, a killer immunoglobulin-like receptors (KIRs) immune checkpoint inhibitor such as Lirilumab (BMS-986015), a carcinoembryonic antigen cell adhesion molecule (CEACAM) inhibitor (e.g., CEACAM-1, -3 and/or -5). Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 and WO 2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al. PLoS One. 2010 September 2; 5(9). pii: el2529 (DOI: 10: 1371/journal. pone.0021146), or crossreacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.
In some embodiments, the patient has previously been administered an immune checkpoint inhibitor directed to B and T lymphocyte attenuator molecule (BTLA), for example as described in Zhang et al., Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus, Clin Exp Immunol. 2011 Jan; 163(1): 77-87, and TAB004/JS004 (Junshi Biosciences).
In some embodiments, the patient has previously been administered a sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) inhibitor, including, but not limited to, NC318 (an anti- Siglec-15 mAb).
T-cell Diversity Status
As provided herein, in some embodiments, the patient to be treated is selected based on the presence of sufficient peripheral T-cell clone diversity. Without wishing to be bound by any one theory, it is believed that TNBC patients, with sufficiently high levels of T-cell clone diversity can further benefit from the inclusion of trilaciclib in combination with the described chemotherapeutic agents, even when such patients may have clinically progressed on other immunotherapies, such immune checkpoint inhibitors, for example a PD-1 and/or PD-L1 inhibitor. T-cell receptors (TCR) are respectively found on the surfaces of T-cells. These receptors are heterodimers consisting of a/p or y/8 chains. TCRs usually recognize antigenic peptides in complex with major histocompatibility complex (MHC) molecules. The receptor of each mature T-cell forms via a specific gene recombination mechanism (V(D)J recombination), in which, one from each of the multiple variable (V), diversity (D) and joining (J) genomically encoded segments is selected and joined together. Recombination occurs in two consecutive stages, wherein D and J segment joining is followed by the addition of a V segment. During this process, exonucleases can remove several nucleotides from the ends of each segment and random nucleotides may then also be added to the segment junctions to form the hypervariable complementarity determining region 3 (CDR3) that is primarily responsible for the antigen recognition. It should be noted that D segment is only present in the TCRP and TCRS; for the other chains only V and J segments are involved in this recombination process. This mechanism enables the generation of a large number of different receptor variants, and thus ensures the possibility to recognize virtually any potential alien or self-antigen. The set of T-cell receptors in a single individual or in a given sample is called the TCR repertoire or T-cell clonal diversity. The formation of each repertoire is the result of multiple processes including: (i) the initial recombination event, which occurs during immune cell maturation, (ii) positive and negative selection of T-cells in the thymus, and (iii) expansion of particular sequences as a result of interaction with the environment, including the tumor microenvironment.
Immuno-sequencing can provide the information about counts and receptor chain sequences for the TCR clonotypes present in a repertoire. This information can then be used to calculate summary statistics of clone size distribution, which in turn gives insight in repertoire diversity and clonality. Methods of determining T-cell repertoire of T-cell clonal diversity are described in, for example, Minervina et al., T-cell receptor and B-cell receptor repertoire profiling in adaptive immunity. Transplant International Nov. 2019, Vol. 32 (11); pg. 1111-1123.
It has been discovered that patients with threshold T-cell clonal diversity at or above the median T-cell clonal diversity of the group as a whole, greatly benefit, for example, experience improved overall survival (OS) from the administration of trilaciclib in combination with the described chemotherapeutics (see, e.g., Example 2, Figs. 7 & 8). Accordingly, in one embodiment, the select patient subgroup with advanced/metastatic TNBC, to be treated according to the methods described herein has a T-cell clonal peripheral diversity score at or above the median for the TNBC patient population. In some embodiments, the patient to be treated has a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones. In some embodiments, has a peripheral diversity score of at least about 12,000, 12,250, 12,500, 12,750, 13,000, 13,250, 13,500, 13,750, 14,000, 14,250, 14,500, 14,750, 15,000, or greater than 15,000.
In one aspect, the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC, with a threshold Simpson clonality score. Simpson clonality is a T-cell clonal diversity single number metric that describes the characteristic shape of a sample repertoire. Simpson clonality measures the evenness of the repertoire, that is the extent to which one or a few clones dominate the sample repertoire. Simpson Clonality is calculated as follows:
Figure imgf000034_0001
, wherein R = the total number of rearrangements; i = each rearrangement; Pi = productive frequency of rearrangement i. Simpson Clonality is the square root of Simpson’s Index. Simpson’s Index is 1 — Simpson’s Diversity Index. Simpson clonality is further described in, for example, Wong, et al. J Immunol. 2016; 197(5): 1642-9; Schneider-Hohendorf, et al. Nat Commun. 2016;7: 11153 ; Weir, et al. J Immunother Cancer. 2016;4:68; Nunes-Alves, et al. PLoS Pathog. 2015;l l(5):el004849; Suessmuth, et al. Blood. 2015;125(25):3835-50; Mahalingam, et al. Clin Can Res. 2020;26(l):71-81; Morris, et al. Sci Transl Med. 2015;7(272):272ral0; Roh, et al. Sci Transl Med. 2017;9(379); Zhu, et al. Oncolmmunology. 2015;4(12):el051922; Tumeh, et al. Nature. 2014;515(7528):568-71; Keane, et al. Clin Cancer Res. 2017;23(7): 1820-1828; Kirsch, et al. Sci Transl Med. 2015;7(308):308ral58; Hershberg, et al. Phil. Trans. R. Soc. B. 2015;370(1676)20140239; Wu, et al. Sci Transl Med. 2012;4(134) L 134ra63; Carey, et al. J Immunol. 2016;196(6):2602-13; Seay, et al. JCI Insight. 2016;l(20):e88242; Emerson, et al. Nat Genet. 2017;49(5):659-665; Lindau et al. J Immunol. 2018; j i 1800217, each of which is incorporated herein by reference. 0 represents a completely even sample, while 1 represents a monoclonal sample. In one aspect, the improved methods of treatment are administered to a select patient subgroup with advanced/metastatic TNBC, with a threshold Simpson clonality score of less than about 0.08. In some embodiments, the patient has a Simpson clonality score of less than about 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, or 0.010, or less.
Improved Chemotherapeutic Protocols
Trilaciclib
Trilaciclib (2'-((5-(4-methylpiperazin-l -yljpyri din-2 -yl)amino)-7',8'-dihydro-6'H- spiro(cyclohexane-l,9'-pyrazino(r,2':l,5)pyrrolo(2,3-d)pyrimidin)-6'-one) is a highly selective CDK4/6 inhibitor having the structure:
Figure imgf000035_0001
As provided herein, trilaciclib or its pharmaceutically acceptable salt, composition, isotopic analog, or prodrug thereof is administered in a suitable carrier. Trilaciclib is described in US 2013-0237544, incorporated herein by reference in its entirety. Trilaciclib can be synthesized as described in US 2019-0135820, incorporated herein by reference in its entirety. Trilaciclib can be administered in any manner that achieves the desired outcome, including systemically, parenterally, intravenously, intramuscularly, subcutaneously, or intradermally. For injection, trilaciclib may be provided, in some embodiments, for example, as a 300 mg/vial as a sterile, lyophilized, yellow cake providing 300 mg of trilaciclib (equivalent to 349 mg of trilaciclib dihydrochloride, dihydrate). The product, for example, may be supplied in single-use 20-mL clear glass vials which does not contain a preservative. For example, prior to administration, trilaciclib for injection, 300 mg/vial may be reconstituted with 19.5 ml of 0.9% sodium chloride injection or 5% dextrose injection. This reconstituted solution has a trilaciclib concentration of 15 mg/mL and would typically be subsequently diluted prior to intravenous administration. Trilaciclib can be administered intravenously as described herein. In certain embodiments, trilaciclib is in the form of a dihydrochloride optionally as a hydrate. For example, trilaciclib can be used in the present invention as a dihydrochloride, dihydrate or as a pharmaceutical composition formed from trilaciclib dihydrochloride, dihydrate.
In some embodiments, trilaciclib is administered at between about 180 mg/m2 and 300 mg/m2. In some embodiments, trilaciclib is administered at about 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or about 280 mg/m2. In some embodiments, trilaciclib is administered at least 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 mg/m2. In some embodiments, trilaciclib is administered at about 240 mg/m2, prior to administration of for example, gemcitabine and carboplatin (or platinum containing drug alternative including, but not limited to, cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin), for example prior to about 4 hours or less, for example about 4 hours or less, 3 hours or less, 2 hours or less, about 1 hour or less, or about 30 minutes prior to administration of gemcitabine and/or carboplatin. In some embodiments, trilaciclib is administered intravenously over a period of about 30 minutes. In some embodiments, trilaciclib is completely administered prior to administration of gemcitabine and carboplatin (or alternative) or alternatively docetaxel (or alternative).
As provided herein, for the treatment of advanced/metastatic TNBC, trilaciclib is administered on day 1 and day 8 of each 21 -day cycle, or as otherwise provided herein. Trilaciclib is administered prior to the initiation of administration of gemcitabine and carboplatin, generally via intravenous injection/infusion over about 30 minutes about 4 hours or less, for example about 3 hours or less, 2 hours or less, 1 hour or less, or about 30 minutes prior to the initiation of the administration of gemcitabine and carboplatin in the protocol. In some embodiments, the trilaciclib is completely administered on day 1 and day 8 no more than 4 hours prior to initiation of the administration of the gemcitabine and carboplatin.
In an alternative embodiment, a different CDK4/6 inhibitor is administered. For example, in alternative embodiments, the CDK4/6 inhibitor used instead of trilaciclib in the protocols described herein is ribociclib (Novartis), palbociclib (Pfizer), or abemaciclib (Eli Lily), or a pharmaceutically acceptable salt thereof. In an additional alternative embodiment, the CDK4/6 inhibitor is lerociclib, which has the structure:
Figure imgf000037_0001
or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, which is described in US 2013-0237544, incorporated herein by reference in its entirety, and can be synthesized as described in US 2019-0135820, incorporated herein by reference in its entirety. In some embodiments, lerociclib is administered as a pharmaceutically acceptable salt, for example, the dihydrocloride salt.
In an additional alternative embodiment, the CDK4/6 inhibitor has the structure:
Figure imgf000037_0002
or a pharmaceutically acceptable composition, salt, isotopic analog, or prodrug thereof, which is described in US 2013-0237544, incorporated herein by reference in its entirety, and can be synthesized as described in US 2019-0135820, incorporated herein by reference in its entirety.
Gemcitabine
Gemcitabine is a nucleoside metabolic inhibitor. Descriptions of formulations of commercially available gemcitabine can be found in current available prescribing information.
Gemcitabine hydrochloride is 2’-deoxy-2’,2’-difluorocytidine monohydrochloride (P-isomer) with the following molecular structure:
Figure imgf000038_0001
Gemcitabine kills cells undergoing DNA synthesis and blocks the progression of cells through the Gl/S-phase boundary. Gemcitabine is metabolized by nucleoside kinases to diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleosides. Gemcitabine diphosphate inhibits ribonucleotide reductase, an enzyme responsible for catalyzing the reactions that generate deoxynucleoside triphosphates for DNA synthesis, resulting in reductions in deoxynucleotide concentrations, including dCTP. Gemcitabine triphosphate competes with dCTP for incorporation into DNA. The reduction in the intracellular concentration of dCTP by the action of the diphosphate enhances the incorporation of gemcitabine triphosphate into DNA (self-potentiation). After the gemcitabine nucleotide is incorporated into DNA, only one additional nucleotide is added to the growing DNA strands, which eventually results in the initiation of apoptotic cell death.
Gemcitabine is generally administered by intravenous injection on day 1 and day 8 of each 21-day cycle after administration of trilaciclib. As provided herein, administration of gemcitabine should not be longer than 4 hours. In the methods provided herein, gemcitabine can be administered according to institutional guidelines. In some embodiments, gemcitabine can be administered at its standard of care dose. In some embodiments, gemcitabine is administered at a dose of between about 600 mg/m2 and 1250 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 600 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 800 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 1000 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 1200 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 1250 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 600 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 800 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 1000 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 1200 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 1250 mg/m2.
In some embodiments, gemcitabine is administered at a starting dose of about 1000 mg/m2. In some embodiments, gemcitabine is administered at about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose. In some embodiments, gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 m2 for at least one dose.
The use of gemcitabine is associated with bone marrow suppression: neutropenia, thrombocytopenia, and anemia, including Grade 3 or 4 hematologic toxicity. Additional risks associated with the use of gemcitabine include: capillary leak syndrome; hemolytic uremic syndrome: may lead to renal failure and dialysis (including fatalities); hepatotoxicity; hypersensitivity: anaphylaxis and allergic reactions (including bronchospasm and anaphylactoid reactions) have been observed; posterior reversible encephalopathy syndrome (PRES): may manifest with blindness, confusion, headache, hypertension, lethargy, seizure, and other visual and neurologic disturbances; and, pulmonary toxicity: including adult respiratory distress syndrome, interstitial pneumonitis, pulmonary edema, and pulmonary fibrosis.
Carboplatin
Carboplatin is a platinum coordination compound. Descriptions of formulations of commercially available carboplatin can be found in current available prescribing information. The chemical name for carboplatin is platinum, diamine [l,l-cyclobutane-dicarboxylato(2-)-0,0']-,(SP- 4-2), and carboplatin has the following structural formula:
Figure imgf000039_0001
Carboplatin, like cisplatin, produces predominantly inter-strand DNA cross-links rather than DNA-protein cross-links. This effect is apparently cell-cycle nonspecific. The aquation of carboplatin, which is thought to produce the active species, occurs at a slower rate than in the case of cisplatin. Despite this difference, it appears that both carboplatin and cisplatin induce equal numbers of drug-DNA cross-links, causing equivalent lesions and biological effects. The differences in potencies for carboplatin and cisplatin appear to be directly related to the difference in aquation rates.
Carboplatin is generally administered by intravenous injection on day 1 and day 8 of each 21-day cycle after administration of trilaciclib. As provided herein, administration of carboplatin should not be longer than 4 hours. In the methods provided herein, carboplatin can be administered according to institutional guidelines. In some embodiments, carboplatin is administered at its standard of care dose. In some embodiments, carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2 (maximum of 300 mg), which is known in the art. In some embodiments, in patients with abnormally low serum creatinine, a minimum of 0.7 mg/dL should be used for the calculation of dose. In some embodiments, carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2. In some embodiments, carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2. In some embodiments, carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
The use of carboplatin is associated with: bone marrow suppression: leukopenia, neutropenia, and thrombocytopenia; nephrotoxic potential: concomitant treatment with aminoglycosides has resulted in increased renal and/or audiologic toxicity; emesis; peripheral neurotoxicity: observed infrequently, but its incidence is increased in patients older than 65 years and in patients previously treated with cisplatin; Loss of vision; allergic reactions, including anaphylaxis; abnormal liver function tests; and, fetal harm.
Cisplatin
Cisplatin is a metallic (platinum) coordination compound with a square planar geometry that is delivered intravenously. Cisplatin for injection is indicated for the treatment of advanced testicular cancer, advanced ovarian cancer, and advanced bladder cancer. It is also used off-label for indications including TNBC. Descriptions of formulations of commercially available cisplatin can be found in current available prescribing information. The chemical name for cisplatin is cisplatinum, or c/.s-diamminedichloroplatinum (II) and cisplatin has the following structural formula:
Figure imgf000041_0001
The mode of action of cisplatin has been linked to its ability to crosslink with the purine bases on the DNA; interfering with DNA repair mechanisms, causing DNA damage, and subsequently inducing apoptosis in cancer cells. The use of cisplatin is associated with: nausea, vomiting, nephrotoxicity, low blood counts, blood test abnormalities, thrombocytopenia, neutropenia, ototoxicity (especially in children), myelosuppression, anaphylaxis and alopecia.
In alternative embodiments, cisplatin is used in combination with gemcitabine.
Oxaliplatin
Oxaliplatin is a platinum-based chemotherapy drug in the same family as cisplatin and carboplatin. Compared to cisplatin the two amine groups are replaced by cyclohexyldiamine for improved antitumour activity. The chlorine ligands are replaced by the oxalato bidentate derived from oxalic acid in order to improve water solubility. It is also used off-label for indications including TNBC. Descriptions of formulations of commercially available oxaliplatin can be found in current available prescribing information. Oxaliplatin is an antineoplastic agent with the molecular formula CsHi4N2O4Pt and the chemical name of cis-[(l R,2 R)-l,2- cyclohexanediamine-N,N'] [oxalato(2)-O,O'] platinum. Oxaliplatin is an organoplatinum complex in which the platinum atom is complexed with 1,2-diaminocyclohexane (DACH) and with an oxalate ligand as a leaving group with a structural formula of:
Figure imgf000042_0001
The use of oxaliplatin is associated with peripheral neuropathy, nausea and vomiting, diarrhea, mouth sores, low blood counts, fatigue, loss of appetite, constipation, fever, generalized pain, headache, cough, temporary increases in blood tests measuring liver function, and anaphylaxis. In alternative embodiments, oxaliplatin is used in combination with gemcitabine.
In certain embodiments, an alternative platin is used in combination with gemcitabine, for example, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, or satraplatin.
Advanced/Metastatic Treatment of TNBC Using Trilaciclib + Gemcitabine/Carboplatin As provided herein, the defined subpopulations of patients with advanced/metastatic
TNBC as described herein are administered trilaciclib, or a pharmaceutically acceptable salt thereof, in combination with the chemotherapeutic agents gemcitabine and carboplatin in a specifically timed administrative protocol. Accordingly, provided herein is a method of treating a human patient with advanced/metastatic TNBC comprising: i) administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000042_0002
or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii) administering to the patient an effective amount of gemcitabine; and, iii) administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered prior to the initiation of administration of the gemcitabine and carboplatin, and wherein the patient is receiving either 1) first-line treatment for advanced/metastatic TNBC and is immune-checkpoint inhibitor treatment-naive or, alternatively^) second-line treatment for advanced/metastatic TNBC and has had prior exposure to an immune checkpoint inhibitor, for example, a programmed cell death protein- 1 (PD-1) and/or programmed death-ligand- 1 (PD-L1) inhibitor, in a first-line chemotherapeutic setting and who have developed therapeutic resistance to the immune checkpoint inhibitor leading to disease progression after an initial response.
In some embodiments, trilaciclib is administered less than 4 hours or prior to the administration of gemcitabine and carboplatin. In some embodiments, trilaciclib is administered about one hour or less, for example, about 45 minutes, about 40 minutes, about 35 minutes, or about 30 minutes, prior to administration of gemcitabine and carboplatin.
In some embodiments, trilaciclib is administered to the patient intravenously between about 190 and 280 mg/m2. In some embodiments, the trilaciclib is administered at about 240 mg/m2.
In some embodiments, gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 600 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 800 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 1000 mg/m2. In some embodiments, gemcitabine is administered at a dose of about 1200 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 600 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 800 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 1000 mg/m2. In some embodiments, gemcitabine is administered at a dose of at least about 1200 mg/m2. In some embodiments, the gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours. In some embodiments, gemcitabine is administered at a starting dose of about 1000 mg/m2. In some embodiments, gemcitabine is administered at about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose. In some embodiments, gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
In some embodiments, carboplatin is administered at its standard of care dose. In some embodiments, carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2 (maximum of 300 mg), which is known in the art. In some embodiments, in patients with abnormally low serum creatinine, a minimum of 0.7 mg/dL should be used for the calculation of dose. In some embodiments, carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2. In some embodiments, carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2. In some embodiments, carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2. In some embodiments, carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
In some embodiments, the trilaciclib/GC chemotherapy regimen is administered in 1 or more cycles, 2 or more cycles, 3 or more cycles, 4 or more cycles, 5 or more cycles, 6 or more cycles, 7 or more cycles, 8 or more cycles, 9 or more cycles, 10 or more cycles, or 11 or more cycles. In some embodiments, the trilaciclib/GC chemotherapy is administered up to 12 times.
In some embodiments, the protocol comprises one or more 21 -day therapeutic cycles, wherein trilaciclib, carboplatin, and gemcitabine are administered on days 1 and 8 of each 21 -day cycle, wherein trilaciclib is administered no more than 4 hours prior to the administration of the gemcitabine and carboplatin, and wherein the trilaciclib is completely administered before the start of the administration of gemcitabine and/or carboplatin.
In an alternative embodiment, cisplatin is used instead of carboplatin. In an alternative embodiment, oxaliplatin is used instead of carboplatin.
Anti-tumor Efficacy Assessment
In some embodiments, the inclusion of trilaciclib in a gemcitabine/carboplatin (GC) chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased anti-tumor efficacy compared to those patients receiving a GC chemotherapy protocol without trilaciclib. Methods of accessing tumor response are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased or extended progression free survival (PFS) compared to those patients not receiving trilaciclib. PFS is generally defined as the time (number of months) from date of protocol administration until the date of documented radiologic disease progression or death from any cause. Methods of accessing increased PFS are well known in the art and include, for example RECIST vl. l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an increased or extended overall survival (OS) compared to those patients not receiving trilaciclib. OS is generally calculated as the time (months) from the date of the onset of protocol administration to the date of death due to any cause.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an improved objective response rate (ORR) compared to those patients not receiving trilaciclib. ORR is generally defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Examples of an objective response (OR) includes a complete response (CR), which is the disappearance of all signs of the tumor in response to treatment and a partial response (PR), which is a decrease in the size of a tumor in response to treatment. In some embodiments, the objective response (OR) is a complete response (CR). In some embodiments, the objective response (OR) is a partial response (PR). The ORR is an important parameter to demonstrate the efficacy of a treatment and it serves as a primary or secondary end-point in clinical trials. Methods of assessing ORR are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247) and World Health Organization (WHO) (World Health Organization. WHO Handbook for Reporting Results of Cancer Treatment. World Health Organization Offset Publication No. 48; Geneva (Switzerland), 1979). Statistical methods of measuring objective response rate are well known in the art and include, for example, the Clopper-Pearson Method (Clopper, C.; Pearson, E. S. (1934). "The use of confidence or fiducial limits illustrated in the case of the binomial". Biometrika. 26 (4): 404-413. doi: 10.1093/biomet/26.4.404).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for an improved Duration of Objective Response (DOR) compared to those patients not receiving trilaciclib. DOR is generally defined as the time between first objective response of CR or PR and the first date that progressive disease is objectively documented or death, whichever comes first. Methods of assessing improved DOR are well known in the art and include, for example RECIST vl.l (Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45: 228-247).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC provides for T-cell immune activation against the tumor. In some embodiments, the T-cell immune activation results in T-cell receptor (TCR) modulation. In some embodiments, the T-cell activation results in increased interferon gamma (IFNy) expression. In some embodiments, the T-cell activation results in increased activation-induced expression of CD137. In some embodiments, the T-cell activation results in increased TCR diversity. In some embodiments, the T-cell activation results in a decrease in the baseline Simpson clonality score.
Reduction of Toxicity
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in improved myelopreservation of hematopoietic stem and progenitor cells (HSPCs) and immune effector cells such as lymphocytes including T-lymphocytes, as well as enhanced anti -turn or efficacy in patients compared to those receiving a chemotherapy protocol without trilaciclib. Improvements in myelopreservation of hematopoietic stem and progenitor cells (HSPCs) and immune effector cells such as lymphocytes including T-lymphocytes are measured with increases in hematology assessments (complete blood count (CBC), red blood cell count (RBC), platelet count, white blood cell count (WBC) and absolute neutrophil count (ANC)), reduction in severe adverse events (AEs), reduction in supportive care interventions (including transfusions and G-CSF administration), reduction in dose modifications, and improved patient recorded outcomes (PROs).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the incidence of chemotherapeutic induced-myelosuppression (CIM). Patients who develop myelosuppression while receiving a chemotherapeutic agent such as gemcitabine and carboplatin, or alternatively docetaxel, are more likely to experience infections, sepsis, bleeding, and fatigue, often leading to the need for hospitalizations, hematopoietic growth factor support, transfusions (red blood cells [RBCs] and/or platelets), and even death (see, e.g., Gustinetti et al., Bloodstream infections in neutropenic cancer patients: A practical update. Virulence. 2016; 7(3): 280-97; Li et al., Relationship between severity and duration of chemotherapy-induced neutropenia and risk of infection among patients with nonmyeloid malignancies. Support Care Cancer 2016; 24(10): 4377-83; Caggiano et al., Incidence, cost, and mortality of neutropenia hospitalization associated with chemotherapy. Cancer. 2005; 103(9): 1916-24). Moreover, CIM commonly leads to dose reductions and delays, which limit therapeutic dose intensity and can compromise the anti-tumor efficacy benefits of chemotherapy. Attempts at developing and implementing clinical algorithms to guide chemotherapy dose reductions and treatment delays in patients with neutropenia and/or thrombocytopenia during treatments have been examined (see, for example, Clinical Trial of a Novel Dose Adjustment Algorithm for Preventing Cytopenia-Related Delays During FOLFOX Chemotherapy, ClinicalTrials.gov Identifier: NCT04526886). Nonetheless, chemotherapy- induced cellular damage to the immune system may also limit anti-tumor efficacy due to an inability of the host immune system to effectively mount a response against the cancer. Prolonged exposure to myelosuppressive agents can lead to cumulative bone marrow toxicity and myelosuppression that can limit the ability to deliver subsequent lines of therapy at the standard of care doses and schedule.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in myelopreservation of the neutrophil lineage in patients compared to those receiving the chemotherapy protocol without trilaciclib. Endpoints to measure myelopreservation of the neutrophil lineage include a reduction in duration of severe neutropenia, for example after cycle 1, and the reduction in the occurrence of severe neutropenia. Neutropenia is generally defined as a condition that results when the body does not have enough neutrophils, an important white blood cell that fights infections. The lower the neutrophil count, the more vulnerable one is to infectious diseases. Neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 1500 per microliter (1500/pL). Severe neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 500 per microliter (500/ pL). Methods of calculating absolute neutrophil count (ANC) are well known in the art and include multiplying the WBC count times the percent of neutrophils in the differential WBC count. The percent of neutrophils consists of the segmented (fully mature) neutrophils + the bands (almost mature neutrophils).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the duration of severe (Grade 4) neutropenia (DSN) in patients compared to those receiving the chemotherapy protocol without trilaciclib. The duration of SN (DSN) is generally defined as the number of days from the date of the first ANC value of <0.5 x 109/L to the date of the first ANC value >0.5 x 109/L where no additional ANC values <0.5 x 109/L are observed for the remainder of that cycle. Severe neutropenia is generally quantified as an absolute neutrophil count (ANC) of less than 500 per microliter (500/ pL). Methods of calculating absolute neutrophil count (ANC) are well known in the art and include multiplying the WBC count times the percent of neutrophils in the differential WBC count. The percent of neutrophils consists of the segmented (fully mature) neutrophils + the bands (almost mature neutrophils).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction of chemotherapy- induced fatigue (CIF) in patients compared to those receiving the chemotherapy protocol without trilaciclib. In some embodiments, the reduction of CIF is a reduction in the time to first confirmed deterioration of fatigue (TTCD-fatigue), as measured by the Functional Assessment of Cancer Therapy -Fatigue (FACIT-F). FACIT-F is a 13-item subscale that measures fatigue severity and the impact of fatigue on functioning and is described in Yellen et al., Measuring fatigue and other anemia-related symptoms with the Functional Assessment of Cancer Therapy (FACT) measurement system. J Pain Symptom Manage. 1997; 13: 63-74. In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in severe neutropenia events, a reduction in granulocyte-colony stimulating factor (G-CSF) treatment, or a reduction in febrile neutropenia (FN) adverse events (AEs) in patients compared to those receiving the chemotherapy protocol without trilaciclib. G-CSF treatment will be utilized according to the treatment guidelines outlined in Aapro et al. 2010 update of EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours. Eur J Cancer. 2011; 47:8-32.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 decreased hemoglobin laboratory values, red blood cell (RBC) transfusions, or erythropoiesisstimulating agent (ESA) administration.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 decreased platelet count laboratory values and/or the number of platelet transfusions. As described in (Kaufman, 2015; Schiffer, 2017), platelets are generally transfused at a threshold of < 10,000/pL. Platelets are also generally transfused in any patient who is bleeding with a platelet count < 50,000/pL (100,000/pL for central nervous system or ocular bleeding).
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in Grade 3 or 4 hematologic laboratory values. In some embodiments, the use of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in all-cause dose reductions or cycle delays and relative dose intensity of a chemotherapy protocol described herein.
In some embodiments, the inclusion of trilaciclib a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in i) hospitalizations, including but not limited to those due to all causes, febrile neutropenia/neutropenia, anemia/RBC transfusion, thrombocytopenia/bleeding and infections) or ii) antibiotic use, including but not limited to intravenous (IV), oral, and oral and IV administered antibiotics. In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in an improvement to one or more of: Functional Assessment of Cancer Therapy -General (FACT-G) domain scores (physical, social/family, emotional, and functional well-being); Functional Assessment of Cancer Therapy- Anemia (FACT-An); 5-level EQ-5D (EQ-5D-5L); Patient Global Impression of Change (PGIC) fatigue item; or Patient Global Impression of Severity (PGIS) fatigue item.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the number of severe diarrhea episodes (Grade 3 or greater) experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the development of, severity of, or episodes of mucositis experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
In some embodiments, the inclusion of trilaciclib in a chemotherapy protocol described herein for the treatment of advanced or metastatic TNBC results in a reduction in the development of, severity of, or episodes of stomatitis experienced by a patient compared to those receiving the chemotherapy protocol described herein without trilaciclib.
Pharmaceutical Compositions
The selected compounds of the protocols described herein or their pharmaceutically acceptable salts can be administered as the neat chemical, but is more typically administered as a pharmaceutical composition, that includes an effective amount for a patient, typically a human, in need of such treatment in a pharmaceutically acceptable carrier. The pharmaceutical composition may contain a compound or salt thereof as the only active agent, or, in an alternative embodiment, the compound or its salt and at least one additional active agent for the disease to be treated.
The pharmaceutical compositions may be administered in a therapeutically effective amount by any desired mode of administration, but is typically administered as an intravenous injection or infusion. In alternative embodiments, the compounds or pharmaceutically acceptable salts are delivered in an effective amount with a pharmaceutically acceptable carrier for oral delivery. As more general non-limiting examples, the pharmaceutical composition one suitable for oral (including buccal and sub-lingual), rectal, nasal, topical, transdermal, pulmonary, vaginal or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous), injections, inhalation or spray, intra-aortal, intracranial, subdermal, intraperitioneal, subcutaneous, or by other means of administration containing conventional pharmaceutically acceptable carriers.
Suitable dosage ranges depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the patient, the potency of the compound used, the route and form of administration, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this application, to ascertain a therapeutically effective amount of the compositions of the disclosure for a given disease.
In certain embodiments the pharmaceutical composition is in a dosage form that contains from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 750 mg, from about 1 mg to about 500 mg, or from about 5, 10, 15, or 20 mg to about 250 mg of the active compound or its pharmaceutically acceptable salt. Examples are dosage forms are those delivering at least 0.01, 0.05, 0.1, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 500, 600, 700, or 750 mg of active compound, or its salt. When the weight is used herein, it can refer to either the compound alone or the compound in combination with its pharmaceutically acceptable salt.
An effective amount of the disclosed compound or its salt may be administered based on the weight, size or age of the patient. For example, a therapeutic amount may for example be in the range of about 0.01 mg/kg to about 250 mg/kg body weight, or about 0.1 mg/kg to about 10 mg/kg, in at least one dose. The patient can be administered as many doses as are required to reduce and/or alleviate and/or cure the disorder in question. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient.
In certain embodiments the dose ranges from about 0.01-100 mg/kg of patient body weight, for example about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packed tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
In certain embodiments the compound is administered as a pharmaceutically acceptable salt. Non-limiting examples of pharmaceutically acceptable salts include: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.
Depending on the intended mode of administration, the pharmaceutical compositions can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, syrup, suspensions, creams, ointments, lotions, paste, gel, spray, aerosol, foam, or oil, injection or infusion solution, a transdermal patch, a subcutaneous patch, an inhalation formulation, in a medical device, suppository, buccal, or sublingual formulation, parenteral formulation, or an ophthalmic solution, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose. The compositions will include an effective amount of the selected drug in combination with a pharmaceutically acceptable carrier and, in addition, can include other pharmaceutical agents, adjuvants, diluents, buffers, and the like.
Carriers include excipients and diluents and should be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert, or it can possess pharmaceutical benefits of its own. The amount of carrier employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.
Classes of carriers include, but are not limited to adjuvants, binders, buffering agents, coloring agents, diluents, disintegrants, excipients, emulsifiers, flavorants, gels, glidents, lubricants, preservatives, stabilizers, surfactants, solubilizer, tableting agents, wetting agents or solidifying material.
Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others.
Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.
Some excipients include, but are not limited, to liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, and the like. The compound can be provided, for example, in the form of a solid, a liquid, spray dried material, a microparticle, nanoparticle, controlled release system, etc., as desired according to the goal of the therapy. Suitable excipients for non-liquid formulations are also known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients and salts is available in Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990).
Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, can be present in such vehicles. A biological buffer can be any solution which is pharmacologically acceptable, and which provides the formulation with the desired pH, i.e., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank’ s buffered saline, and the like.
For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, and the like, an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered can also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington’s Pharmaceutical Sciences, referenced above.
In yet another embodiment provided is the use of permeation enhancer excipients including polymers such as: poly cations (chitosan and its quaternary ammonium derivatives, poly-L- arginine, aminated gelatin); polyanions (N-carboxymethyl chitosan, poly-acrylic acid); and thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil-cysteine, chitosanthiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).
In certain embodiments the excipient is selected from butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. The pharmaceutical compositions containing the active agents can be formulated for oral administration. For oral administration, the composition may take the form of a tablet, capsule, a softgel capsule or can be an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules are typical oral administration forms. Tablets and capsules for oral use can include one or more commonly used carriers such as lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. Typically, the compositions of the disclosure can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
When liquid suspensions are used, the active agent can be combined with any oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like and with emulsifying and suspending agents. If desired, flavoring, coloring and/or sweetening agents can be added as well. Other optional components for incorporation into an oral formulation herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.
Parenteral formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or as emulsions. Typically, sterile injectable suspensions are formulated according to techniques known in the art using suitable carriers, dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a sterile injectable solution or a suspension in a acceptably nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are conventionally employed as solvents or suspending media. In addition, parenteral administration can involve the use of a slow release or sustained release system such that a constant level of dosage is maintained.
Parenteral administration includes intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Administration via certain parenteral routes can involve introducing the formulations of the disclosure into the body of a patient through a needle or a catheter, propelled by a sterile syringe or some other mechanical device such as a continuous infusion system. A formulation provided by the disclosure can be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.
The claimed invention is further described by way of the following non-limiting examples. Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures.
Examples
Example 1. Evaluation of Trilaciclib + Gemcitabine/Carboplatin versus placebo + Gemcitabine/Carboplatin on measures of T cell activation.
Clonality measures how evenly receptor sequences (rearrangements) are distributed amongst a set of T cells. This can quantify how focused the immune repertoire is on a particular set of antigens. Diversity metrics, like clonality, can provide insight into how disease or intervention mechanisms affect the immune system in various applications. Such metrics also have the potential to be a predictive or prognostic biomarker in research settings such as oncology. A diversity metric is a single number that describes the characteristic shape of a sample repertoire. When applied to the immune repertoire it is a powerful overall indicator of the state of the adaptive immune system. It can demonstrate an immune response to a specific threat or signify whether the system is healthy enough to respond to a variety of pathogens. Simpson clonality measures range from 0 to 1, where values approaching 0 represents a completely even sample and values approaching 1 represents a monoclonal sample.
Simpson clonality was measured in human patients with metastatic/advanced breast cancer receiving trilaciclib in combination with gemcitabine and carboplatin in a phase 2 clinical trial (Clinicaltrials.gov identifier: NCT02978716 - described further in Example 3). To assess the effect of trilaciclib addition to the GC chemotherapeutic regimen on the composition of lymphocyte subsets and clonal expansion, T-cell receptor (TCR) B CDR3 regions were amplified and sequenced from purified genomic DNA in peripheral blood mononuclear cells isolated from whole blood samples collected on Day 1 of cycles 1 (baseline), 3, and 5.
As shown in Figure 2, Simpson clonality significantly decreased over time in patients that received trilaciclib in addition to GC when compared to GC alone (Pinteraction=0.012). Furthermore, as shown in Figure 3, when patients were stratified above or below median Simpson clonality at baseline, there was a statistically significant improvement in overall survival (OS) among patients receiving trilaciclib (P=0.02). In addition to a decrease in Simpson clonality, responders receiving trilaciclib in groups 2 and 3 had more newly detected expanded clones compared with responders receiving GC alone (P=0.09), with no difference between responders and non-responders in the trilaciclib groups (P=0.79), which is shown in Figure 4. Although not statistically significant, when patients were stratified above or below median fraction of newly detected expanded clones, overall survival was improved among patients with a higher fraction of newly detected expanded clones who received trilaciclib, which is shown in Figure 5.
Furthermore, by showing a reduction in Simpson Clonality through the addition of trilaciclib to the GC chemotherapy treatment protocol resulted in greater heterogeneity of T cells relative to GC chemotherapy alone, which correlated with an increase in overall survival.
These data suggest trilaciclib enhances anti-tumor immunity through T cell activation leading to an anti-tumor response and may be particularly effective in reversing disease progression due to immunosuppressive tumor microenvironments in patients who have been treated with, and whose tumors developed resistance to, immune checkpoint inhibitors, or who otherwise have an immunosuppressive tumor microenvironment.
Furthermore, the induction of reduced Simpson clonality with the use of trilaciclib, without the use of an ICI, provides advantageous and alternative immunotherapeutic approaches for hard to treat TNBC patients, including metastatic/advanced TNBC patients receiving first line therapy who are ICI naive.
Example 2. Evaluation of Trilaciclib + Gemcitabine/Carboplatin versus placebo + Gemcitabine/Carboplatin on measures of T cell diversity.
To evaluate the trilaciclib treatment effect on peripheral diversity, peripheral samples from patients participating in a Phase 2 clinical trial (Clinicaltrials.gov identifier: NCT02978716 - described further in Example 3) that were used to examine peripheral Simpson clonality across visits (described in Examplel) were computationally downsampled to a common number of T cells clones (n= 17,890). As shown in Figure 6, diversity, as measured by an increase in number of diverse clones, increases across visits in patients treated with Trilaciclib + gemcitabine/carboplatin (Pinteraction=0.007). In patients treated only with gemcitabine/carboplatin, diversity remained constant. When stratified by treatment arm, diversity increases over time in patients treated with trilaciclib + gemcitabine/carboplatin (Pimm=>0.001) while patients treated only with gemcitabine/carboplatin did not (Pimm=0.92). Furthermore, as shown in Figure 7, all treated patients that were above the median for T cell diversity showed a trend for better overall survival than treated patients that were below the median (P=0.22). When stratified by treatment arm, patients stratified above or below median diversity, overall survival was improved among patients above median diversity who received trilaciclib (P=0.15), which is shown in Figure 8.
Example 3: A Phase 2 study of trilaciclib administered prior to gemcitabine plus carboplatin (GCb) in patients with metastatic triple-negative breast cancer (NCT02978716).
A Phase 2 study of trilaciclib, an intravenous cyclin-dependent kinase 4/6 inhibitor, administered prior to gemcitabine plus carboplatin (GCb) in patients with metastatic triplenegative breast cancer was performed (NCT02978716). Patients receiving a prior treatment with an ICI such as a PD-1 or PD-L1 inhibitor were excluded from the study. Patients were randomized (1 : 1 : 1) to group 1 (GCb [days 1, 8]; n = 34), group 2 (trilaciclib prior to GCb [days 1, 8]; n = 33), or group 3 (trilaciclib [days 1, 8] and trilaciclib prior to GCb [days 2, 9]; n = 35). Subgroup analyses of antitumor efficacy were performed according to CDK4/6 dependence, level of PD-L1 expression, and RNA-based immune signatures using proportional hazards regression. T-cell receptor (TCR) P CDR3 regions were amplified and sequenced to identify, quantify, and compare the abundance of each unique TCR P CDR3 at baseline and on treatment (described in Example 1 and Example 2, above).
Patients were randomized (1 : 1 : 1) to one of three treatments, given in 21-day cycles: group 1 received GCb alone on days 1 and 8; group 2 received trilaciclib prior to GCb on days 1 and 8; and group 3 received trilaciclib alone on days 1 and 8, and trilaciclib before GCb on days 2 and 9. Gemcitabine was administered at 1000 mg/m2 and carboplatin at AUC 2 (both IV administration). IV trilaciclib 240 mg/m2 was administered within 4 hours prior to GCb. Treatment was continued until disease progression, unacceptable toxicity, withdrawal of consent, or discontinuation by the investigator.
PD-L1 expression was assessed in archival tumor tissue samples from each patient using the Ventana SP142 PD-L1 assay (Ventana Medical Systems, Inc., Tuscon, AZ, USA; ref. (25)). Consistent with the standard approach for evaluating PD-L1 in TNBC, expression was scored as negative or positive if <1% or >1% of the total tumor area contained PD-Ll-labelled immune cells, respectively.
OS was analyzed following the final database lock on July 17, 2020; other endpoints (ORR, PFS) were based on a data cut-off of May 15, 2020. PFS and OS were assessed in the intention- to-treat population, and ORR in response-evaluable patients (patients in the intention-to-treat population who received at least one dose of study drug, had measurable disease at baseline, and either had at least one post-baseline tumor assessment, investigator-determined clinical progression before the first post-baseline scan, or died due to disease progression before the first post-baseline scan).
Kaplan-Meier methodology was used to estimate median PFS and OS. Treatment group differences in PFS and OS were evaluated using a stratified log-rank test, with hazard ratios and their 95% confidence intervals (trilaciclib prior to GCb versus GCb alone) generated using a Cox proportional hazard model that included number of lines of prior therapy (0 versus 1 or 2) and liver involvement (yes versus no) as stratification factors. Stratification factors were not included in any of the models for the subgroup analyses. Association of PD-L1 expression with antitumor efficacy was assessed using proportional hazards regression, with data restricted to only those patients in the relevant strata. The initial results of the study based on PD-L1 status are provided in Table 1. Table 1 : ORR; PFS; and OS by PD-L1 Status
Figure imgf000060_0001
HR and P values are for comparisons between group 2 and group 1, group 3 and group 1, and between groups 2 and 3 combined and group 1. HR, hazard ratio; NR, not reached; ORR, objective response rate; OS, overall survival; PD-L1, programmed death ligand-1; PFS, progression -free survival.
Association of chemotherapeutic line expression with antitumor efficacy was also assessed using proportional hazards regression, with data restricted to only those patients in the relevant strata. The initial results of the study based on prior treatment status are provided in Figures 9 and 10. Analysis of results by PD-L1 status and line of treatment is ongoing.
Example 4. A Phase 3, Randomized, Double-Blind Study of Trilaciclib or Placebo in Patients Receiving First- or Second-Line Gemcitabine and Carboplatin Chemotherapy for Advanced or Metastatic Triple-Negative Breast Cancer (PRESERVE 2).
Overview
A Phase 3, multicenter, randomized, double-blind, placebo-controlled study evaluating the safety and efficacy of trilaciclib versus placebo administered prior to gemcitabine-carboplatin (GC) for patients receiving first- or second-line treatment for advanced/metastatic triple negative breast cancer (TNBC) in two separate cohorts is underway (see ClinicalTrials.gov Identifier: NCT04799249: Trilaciclib, a CDK 4/6 Inhibitor, in Patients Receiving Gemcitabine and Carboplatin for Metastatic Triple-Negative Breast Cancer (TNBC) (PRESERVE 2)). A general schematic describing the clinical trial is provided in Fig. 1.
A total of 250 patients will be enrolled in this study with 170 in Cohort 1 and 80 in Cohort 2. Within each cohort, patients meeting entry criteria will be randomly assigned (1 : 1) to receive either trilaciclib prior to GC therapy or placebo prior to GC therapy. Study drugs will be administered intravenously (IV) on Days 1 and 8 in 21 -day cycles.
Study drugs will be administered as follows:
• Gemcitabine 1000 mg/m2 and carboplatin using Calvert formula with a target area under the curve (AUC)=2 - administered IV on Days 1 and 8 of each cycle.
• Trilaciclib 240 mg/m2 or placebo - administered IV prior to GC over approximately 30 minutes and no more than 4 hours prior to GC on Days 1 and 8 of each cycle.
Diagnosis and Main Eligibility Criteria (Cohorts 1 and 2)
Patients targeted for treatment in the clinical trial include patients >18 years of age at the time of signing the informed consent with evaluable locally advanced, unresectable, or metastatic TNBC (defined as <1% estrogen receptor [ER] and progesterone receptor by immunohistochemistry [IHC], human epidermal growth factor receptor 2 [HER2] -negative by immunohistochemistry (IHC) or in situ hybridization [ISH]) and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
For Cohort 1, patients receiving prior systemic therapy in the advanced/metastatic setting, and patients with rapid progression within 6 months from the end of last treatment with curative intent are excluded. In addition, prior PD-1/PD-L1 inhibitor treatment is not permitted in any setting, including in the neoadjuvant setting.
Cohort 2 comprises patients with a documented PD-L1 positive TNBC tumor and treatment with a PD 1/PD-L1 inhibitor for a minimum duration of 8 weeks as the most recent therapy in the advanced/metastatic setting.
Dose, Dosing Regimen, and Route
Trilaciclib is supplied as a sterile, preservative-free, yellow, lyophilized cake in a singledose vial (300 mg/20 mL). Trilaciclib must be reconstituted and further diluted prior to IV infusion. Upon reconstitution, the solution must then be diluted to the calculated dose (240 mg/m2) based on the body surface area (BSA) of the patient. Actual body weight (not ideal body weight) should be utilized for dose calculations.
Trilaciclib solution is administered as a 30-minute IV infusion no more than 4 hours prior to chemotherapy and is always administered first. Gemcitabine/carboplatin (GC) may be administered immediately following trilaciclib, but not until the completion of the trilaciclib infusion. The order of administration of gemcitabine and carboplatin is per institutional standards. The interval between trilaciclib administration and the first dose of chemotherapy (gemcitabine or carboplatin) administration should not be greater than 4 hours. If administration of trilaciclib is discontinued, GC therapy will also be discontinued.
Carboplatin is administered using Calvert formula with a target area under the curve (AUC)=2. In patients with abnormally low serum creatinine, a minimum of 0.7 mg/dL should be used. “Adjusted” rather than actual body weight should be used for patients who are overweight (those with body mass index [BMI] > 25 kg/m2). Actual weight should be used for patients with BMI < 25 kg/m2. Patients who have > 10% weight change from baseline or who experience CTCAE > Grade 2 renal toxicity (serum creatinine > 1.5 ULN) will require recalculation of the carboplatin dose for subsequent cycles (this would not be considered a dose reduction). In patients who require carboplatin dose modification, if the creatinine at the time of the dose modification is lower than the baseline creatinine that was used, the prior (higher) creatinine value should be used. If the creatinine at the time of dose modification is higher than the baseline creatinine value, the current (higher) value should be used. This is to ensure that patients receive the intended dose reduction. The maximum carboplatin dose based on target area under the curve (AUC) will be capped at 300 mg. The dose reductions for carboplatin following a hematologic toxicity are as follows: starting dose: AUC=2; first dose reduction: 20% from original dose; and second dose reduction: 40% from original dose.
Gemcitabine is administered at 1000 mg/m2 according to institutional standards. The dose reductions for gemcitabine following a hematologic or non-hematologic toxicity are as follows: starting dose: 1000 mg/m2; first dose reduction: 800 mg/m2; and second dose reduction: 600 mg/m2. Criteria for Starting Cycle 2 and Each Subsequent Cycle
In both groups, study drug administration will continue until progressive disease per RECIST vl. l or clinical progression as determined clinically by the Investigator, unacceptable toxicity, withdrawal of consent, Investigator decision, or the end of the trial. There should be no more than 4 weeks between doses of chemotherapy in all groups. Dosing delays greater than 4 weeks may be permitted on a case by case basis with the approval of the Investigator and Medical Monitor.
Criteria for Day 1 of Each Cycle
Patients must meet all of the following criteria to receive the Day 1 dose of study drug in each cycle: ANC > 1 x 109/L; Platelet count > 100 x 109/L; Nonhematologic toxi cities (except alopecia) must be < Grade 2 or have returned to baseline.
Criteria for Day 8 of Each Cycle
To receive the Day 8 dose of each cycle, patients must meet all the following criteria: ANC > 1 x 109/L and platelet count > 100 x 109/L.
If patients do not meet the prior criteria but meet the criteria of: ANC > 0.75 x 109/L to <1 x 109/L OR platelet count > 75 x 109/L to <100 x 109/L, then the patient may still receive the Day 8 dose, but doses of gemcitabine and carboplatin should be adjusted as described above. Dose reductions of trilaciclib are not permitted. Trilaciclib/placebo should be administered only on days that chemotherapy is administered.
If patients do not meet the prior criteria but meet the criteria of: ANC is <0.75 x 109/L OR platelets count <75 x 109/L, then all study drugs (GC and trilaciclib/placebo) will be skipped and doses of gemcitabine and carboplatin should be adjusted per Table XX. The patient will return at the next planned visit (approximately 7 days later) which will be Day 1 of the next cycle to resume dosing. Note that the criteria for starting Day 1 outlined above will now apply to resumption of dosing. Dose reductions of trilaciclib are not permitted. Trilaciclib/placebo should be administered only on days that chemotherapy is administered.
This specification has been described with reference to embodiments of the invention. However, one of ordinary skill in the art appreciates that various modification and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.

Claims

Claims
1. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient has previously been administered one or more immune checkpoint inhibitors (ICI) in a first-line chemotherapeutic setting and has developed therapeutic resistance to the ICI leading to disease progression, comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000065_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin; and, wherein the patient’s cancer is PD-L1 positive.
2. The method of claim 1, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
3. The method of claims 1 or 2, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
4. The method of claims 1-3, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
5. The method of claim 1-4, wherein trilaciclib is administered to the patient intravenously.
6. The method of claim 1-5, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
7. The method of claims 1-6, wherein trilaciclib is administered at about 240 mg/m2.
8. The method of claims 1-7, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
9. The method of claims 1-8, wherein gemcitabine is administered at a dose of about 600 mg/m2.
10. The method of claims 1-8, wherein gemcitabine is administered at a dose of about 800 mg/m2.
11. The method of claims 1-8, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
12. The method of claims 1-8, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
13. The method of claims 1-8, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
14. The method of claims 1-8, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
15. The method of claims 1-8, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
16. The method of claims 1-15, wherein gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
17. The method of claims 1-8, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
18. The method of claims 1-8, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
19. The method of claims 1-8, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
20. The method of claims 1-19, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
21. The method of claims 1-20, wherein carboplatin is administered at a maximum dose of 300 mg.
22. The method of claims 1-19, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
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23. The method of claims 1-19, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
24. The method of claims 1-19, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
25. The method of claims 1-24, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
26. The method of claims 1-25, wherein the method comprises one or more 21 -day therapeutic cycles, wherein trilaciclib, carboplatin, and gemcitabine are administered on days 1 and 8 of each 21 -day cycle.
27. The method of claim 26, wherein the 21-day cycle is administered 2 to 12 times.
28. The method of claim 26, wherein the 21-day cycle is administered up to 12 times.
29. The method of claims 1-28, wherein the previously administered ICI is a PD-1 inhibitor.
30. The method of claim 29, wherein the previously administered ICI is pembrolizumab.
31. The method of claims 1-28, wherein the previously administered ICI is a PD-L1 inhibitor.
32. The method of claim 31, wherein the previously administered ICI is atezolizumab.
33. The method of claims 1-32, wherein the patient has not previously received gemcitabine and carboplatin.
34. The method of claims 1-33, wherein the patient’s TNBC is CDK4/6-status positive.
35. The method of claims 1-33, wherein the patient’s TNBC is CDK4/6-status negative.
36. The method of claims 1-33, wherein the patient’s TNBC is CDK4/6-status indeterminate.
37. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient has previously been administered one or more immune checkpoint inhibitors (ICI) in a first-line chemotherapeutic setting and has developed therapeutic resistance to the ICI leading to disease progression, the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
65
Figure imgf000068_0001
or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin, and, wherein the patient’s cancer is PD-L1 -positive.
38. The method of claim 37, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
39. The method of claims 37 or 38, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
40. The method of claims 37-39, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
41. The method of claim 37-40, wherein trilaciclib is administered to the patient intravenously.
42. The method of claim 37-41, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
43. The method of claims 37-42, wherein trilaciclib is administered at about 240 mg/m2.
44. The method of claims 37-43, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
45. The method of claims 37-43, wherein gemcitabine is administered at a dose of about 600 mg/m2.
46. The method of claims 37-43, wherein gemcitabine is administered at a dose of about 800 mg/m2.
47. The method of claims 37-43, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
48. The method of claims 37-43, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
49. The method of claims 37-43, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
50. The method of claims 37-43, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
51. The method of claims 37-43, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
52. The method of claims 37-51, wherein gemcitabine is administered as a continuous infusion
(CI) over a period of between about 30 minutes to 2 hours.
53. The method of claims 37-43, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
54. The method of claims 37-43, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
55. The method of claims 37-43, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
56. The method of claims 37-55, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
57. The method of claims 37-56, wherein carboplatin is administered at a maximum dose of 300 mg.
58. The method of claims 37-55, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
59. The method of claims 37-55, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
60. The method of claims 37-55, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
61. The method of claims 37-60, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
67
62. The method of claim 61, wherein the 21-day cycle is administered 2 to 12 times.
63. The method of claim 61, wherein the 21-day cycle is administered up to 12 times.
64. The method of claims 37-63, wherein the previously administered ICI is a PD-1 inhibitor.
65. The method of claim 64, wherein the previously administered ICI is pembrolizumab.
66. The method of claims 37-63, wherein the previously administered ICI is a PD-L1 inhibitor.
67. The method of claim 66, wherein the previously administered ICI is atezolizumab.
68. The method of claims 37-67, wherein the patient has not previously received gemcitabine and carboplatin.
69. The method of claims 37-68, wherein the patient’s TNBC is CDK4/6-status positive.
70. The method of claims 37-68, wherein the patient’s TNBC is CDK4/6-status negative.
71. The method of claims 37-68, wherein the patient’s TNBC is CDK4/6-status indeterminate.
72. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000070_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin.
73. The method of claim 72, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
74. The method of claims 72 or 73, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
75. The method of claims 72-74, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
76. The method of claim 72-75, wherein trilaciclib is administered to the patient intravenously.
77. The method of claim 72-76, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
78. The method of claims 72-77, wherein trilaciclib is administered at about 240 mg/m2.
79. The method of claims 72-78, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
80. The method of claims 72-79, wherein gemcitabine is administered at a dose of about 600 mg/m2.
81. The method of claims 72-79, wherein gemcitabine is administered at a dose of about 800 mg/m2.
82. The method of claims 72-79, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
83. The method of claims 72-79, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
84. The method of claims 72-79, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
85. The method of claims 72-79, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
86. The method of claims 72-79, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
87. The method of claims 72-86, wherein gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
88. The method of claims 72-79, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
89. The method of claims 72-79, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
90. The method of claims 72-79, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
91. The method of claims 72-81, wherein the carboplatin is administered at a standard of care dose.
92. The method of claims 72-90, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
93. The method of claims 72-92, wherein carboplatin is administered at a maximum dose of 300 mg.
94. The method of claims 72-92, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
95. The method of claims 72-92, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
96. The method of claims 72-92, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
97. The method of claims 72-96, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
98. The method of claims 72-97, wherein the method comprises one or more 21-day therapeutic cycles, wherein trilaciclib, carboplatin, and gemcitabine are administered on days 1 and 8 of each 21-day cycle.
99. The method of claim 98, wherein the 21-day cycle is administered 2 to 12 times.
100. The method of claim 98, wherein the 21-day cycle is administered up to 12 times.
101. The method of claims 72-100, wherein the patent’s TNBC is PD-L1 status positive.
102. The method of claims 72-100, wherein the patent’s TNBC is PD-L1 status negative.
103. The method of claims 72-102, wherein the patient has not previously received gemcitabine and carboplatin.
104. The method of claims 72-103, wherein the patient’s TNBC is CDK4/6-status positive.
105. The method of claims 72-103, wherein the patient’s TNBC is CDK4/6-status negative.
106. The method of claims 72-103, wherein the patient’s TNBC is CDK4/6-status indeterminate.
107. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000073_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin.
108. The method of claim 107, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
109. The method of claims 107 or 108, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
110. The method of claims 107-109, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
111. The method of claim 107-110, wherein trilaciclib is administered to the patient intravenously.
112. The method of claim 107-111, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
113. The method of claims 107-112, wherein trilaciclib is administered at about 240 mg/m2.
71
114. The method of claims 107-113, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
115. The method of claims 107-113, wherein gemcitabine is administered at a dose of about 600 mg/m2.
116. The method of claims 107-113, wherein gemcitabine is administered at a dose of about 800 mg/m2.
117. The method of claims 107-113, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
118. The method of claims 107-113, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
119. The method of claims 107-113, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
120. The method of claims 107-113, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
121. The method of claims 107-113, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
122. The method of claims 107-112, wherein gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
123. The method of claims 107-113, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
124. The method of claims 107-113, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
125. The method of claims 107-113, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
126. The method of claims 107-125, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
127. The method of claims 107-126, wherein carboplatin is administered at a maximum dose of 300 mg.
128. The method of claims 107-125, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
72
129. The method of claims 107-125, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
130. The method of claims 107-125, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
131. The method of claims 107-130, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
132. The method of claim 107-131, wherein the 21-day cycle is administered 2 to 12 times.
133. The method of claim 107-131, wherein the 21-day cycle is administered up to 12 times.
134. The method of claims 107-133, wherein the patient has not previously received gemcitabine and carboplatin.
135. The method of claims 107-134, wherein the patient’s TNBC is CDK4/6-status positive.
136. The method of claims 107-134, wherein the patient’s TNBC is CDK4/6-status negative.
137. The method of claims 107-134, wherein the patient’s TNBC is CDK4/6-status indeterminate.
138. The method of claims 107-134, wherein the patent’s TNBC is PD-L1 status positive.
139. The method of claims 107-134, wherein the patent’s TNBC is PD-L1 status negative.
140. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), and wherein the patient has a TNBC that is PD-L1 status negative, comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000075_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin.
141. The method of claim 140, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
142. The method of claims 140 or 141, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
143. The method of claims 140-142, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
144. The method of claim 140-143, wherein trilaciclib is administered to the patient intravenously.
145. The method of claim 140-144, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
146. The method of claims 140-145, wherein trilaciclib is administered at about 240 mg/m2.
147. The method of claims 140-146, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
148. The method of claims 140-147, wherein gemcitabine is administered at a dose of about 600 mg/m2.
149. The method of claims 140-147, wherein gemcitabine is administered at a dose of about 800 mg/m2.
150. The method of claims 140-147, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
151. The method of claims 140-147, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
152. The method of claims 140-147, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
153. The method of claims 140-147, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
154. The method of claims 140-147, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
74
155. The method of claims 140-154, wherein gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
156. The method of claims 140-147, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
157. The method of claims 140-147, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
158. The method of claims 140-147, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
159. The method of claims 140-158, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
160. The method of claims 140-159, wherein carboplatin is administered at a maximum dose of 300 mg.
161. The method of claims 140-158, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
162. The method of claims 140-158, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
163. The method of claims 140-158, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
164. The method of claims 140-163, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
165. The method of claims 140-164, wherein the method comprises one or more 21-day therapeutic cycles, wherein trilaciclib, carboplatin, and gemcitabine are administered on days 1 and 8 of each 21-day cycle.
166. The method of claim 165, wherein the 21-day cycle is administered 2 to 12 times.
167. The method of claim 165, wherein the 21-day cycle is administered up to 12 times.
168. The method of claims 140-167, wherein the patient has not previously received gemcitabine and carboplatin.
169. The method of claims 140-168, wherein the patient’s TNBC is CDK4/6-status positive.
170. The method of claims 140-168, wherein the patient’s TNBC is CDK4/6-status negative.
75
171. The method of claims 140-168, wherein the patient’s TNBC is CDK4/6-status indeterminate.
172. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), and wherein the patient has a TNBC that is PD-L1 status negative, the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000078_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and/or carboplatin.
173. The method of claim 172, wherein trilaciclib is fully administered to the patient before the administration of the gemcitabine and carboplatin.
174. The method of claims 172 or 173, wherein trilaciclib is administered about one hour or less prior to administration of gemcitabine and carboplatin.
175. The method of claims 172-174, wherein trilaciclib is administered about 30 minutes or less prior to administration of gemcitabine and carboplatin.
176. The method of claim 172-175, wherein trilaciclib is administered to the patient intravenously.
177. The method of claim 172-176, wherein trilaciclib is administered to at a dosage of between about 190 and 280 mg/m2.
178. The method of claims 172-177, wherein trilaciclib is administered at about 240 mg/m2.
179. The method of claims 172-178, wherein gemcitabine is administered at a dose of between about 600 mg/m2 and 1200 mg/m2.
180. The method of claims 172-178, wherein gemcitabine is administered at a dose of about 600 mg/m2.
181. The method of claims 172-178, wherein gemcitabine is administered at a dose of about 800 mg/m2.
182. The method of claims 172-178, wherein gemcitabine is administered at a dose of about 1000 mg/m2.
183. The method of claims 172-178, wherein gemcitabine is administered at a dose of about 1200 mg/m2.
184. The method of claims 172-178, wherein gemcitabine is administered at a dose of at least about 600 mg/m2.
185. The method of claims 172-178, wherein gemcitabine is administered at a dose of at least about 800 mg/m2.
186. The method of claims 172-178, wherein gemcitabine is administered at a dose of at least about 1000 mg/m2.
187. The method of claims 172-186, wherein gemcitabine is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
188. The method of claims 172-178, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2.
189. The method of claims 172-178, wherein gemcitabine is administered at a starting dose of about 1000 mg/m2 for one or more doses, and at about 800 mg/m2 for at least one dose.
190. The method of claims 172-178, wherein gemcitabine is administered at 1000 mg/m2 in one or more doses, at 800 mg/m2 for one or more doses, and about 600 mg/m2 for at least one dose.
191. The method of claims 172-190, wherein carboplatin is administered using Calvert formula with a target area under the curve (AUC) = 2.
77
192. The method of claims 172-191, wherein carboplatin is administered at a maximum dose of
300 mg.
193. The method of claims 172-190, wherein carboplatin is administered at a 20% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
194. The method of claims 172-190, wherein carboplatin is administered at a 30% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
195. The method of claims 172-189, wherein carboplatin is administered at a 40% reduced level compared to the calculated Calvert formula with a target area under the curve (AUC) = 2.
196. The method of claims 172-195, wherein the carboplatin is administered as a continuous infusion (CI) over a period of between about 30 minutes to 2 hours.
197. The method of claim 172-196, wherein the 21-day cycle is administered 2 to 12 times.
198. The method of claim 172-196, wherein the 21-day cycle is administered up to 12 times.
199. The method of claims 172-198, wherein the patient has not previously received gemcitabine and carboplatin.
200. The method of claims 172-199, wherein the patient’s TNBC is CDK4/6-status positive.
201. The method of claims 172-199, wherein the patient’s TNBC is CDK4/6-status negative.
202. The method of claims 172-199, wherein the patient’s TNBC is CDK4/6-status indeterminate.
203. The method of claims 1-202, wherein the treatment does not include the administration of an immune checkpoint inhibitor.
204. The method of any of claims 1-203, wherein the treatment results in a reduction and/or prevention in one or more carboplatin and/or gemcitabine chemotherapy-induced myelosuppression (CIM).
205. The method of any of claims 1-204, wherein the treatment results in an improvement in progression free survival (PFS) in comparison to the predicted PFS based on administration of gemcitabine and carboplatin chemotherapy without trilaciclib.
206. The method of any of claims 1-205, wherein the treatment results in an improvement in overall survival (OS) in comparison to the predicted OS based on administration of gemcitabine and carboplatin chemotherapy without trilaciclib.
78
207. The method of any of claims 1-206, wherein the treatment results in my el opreservation of hematopoietic stem and progenitor cells (HSPCs).
208. The method of any of claims 1-207, wherein the treatment results in protection of immune effector cells.
209. The method of any of claims 1-208, wherein the treatment results in enhanced anti -tumor efficacy in patients compared to those receiving gemcitabine and carboplatin chemotherapy without trilaciclib.
210. The method of any of claims 1-209, wherein the treatment results in my el opreservation of the neutrophil lineage in patients compared to those receiving gemcitabine and carboplatin chemotherapy without trilaciclib.
211. The method of any of claims 1-210, wherein the treatment results in a reduction in the duration of severe (Grade 4) neutropenia in patients compared to those receiving gemcitabine and carboplatin chemotherapy without trilaciclib.
212. The method of any of claims 1-211, wherein the treatment results in a reduction of chemotherapy-induced fatigue (CIF) in patients compared to those receiving gemcitabine and carboplatin chemotherapy without trilaciclib.
213. The method of any of claims 1-212, wherein the treatment results in reduction of CIF is a reduction in the time to first confirmed deterioration of fatigue (TTCD-fatigue), as measured by the Functional Assessment of Cancer Therapy -Fatigue (FACIT-F).
214. The method of any of claims 1-213, wherein the treatment results in improved progression free survival (PFS) and/or overall survival (OS) in patients compared to those receiving gemcitabine and carboplatin chemotherapy without trilaciclib.
215. The method of any of claims 1-214, wherein the treatment results in an improvement in PFS is based on per Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1).
216. The method of any of claims 1-215, wherein the treatment results in a reduction in severe neutropenia events, a reduction in granulocyte-colony stimulating factor (G-CSF) treatment, or a reduction in febrile neutropenia (FN) adverse events (AEs).
217. The method of any of claims 1-216, wherein the treatment results in a reduction in Grade 3 or 4 decreased hemoglobin laboratory values, red blood cell (RBC) transfusions, or erythropoiesis-stimulating agent (ESA) administration.
79
218. The method of any of claims 1-217, wherein the treatment results in a reduction in Grade 3 or 4 decreased platelet count laboratory values and/or the number of platelet transfusions.
219. The method of any of claims 1-218, wherein the treatment results in a reduction in Grade 3 or 4 hematologic laboratory values.
220. The method of any of claims 1-219, wherein the treatment provides a reduction in all-cause dose reductions or cycle delays and relative dose intensity for gemcitabine and carboplatin chemotherapy without trilaciclib.
221. The method of any of claims 1-220, wherein the treatment results in a reduction in i) hospitalizations, including but not limited to those due to all causes, febrile neutropenia/neutropenia, anemia/RBC transfusion, thrombocytopenia/bleeding and infections) or ii) antibiotic use, including but not limited to intravenous (IV), oral, and oral and IV administered antibiotics.
222. The method of any of claims 1-221, wherein the treatment results in an improvement to one or more of: Functional Assessment of Cancer Therapy-General (FACT-G) domain scores (physical, social/family, emotional, and functional well-being); Functional Assessment of Cancer Therapy- Anemia (FACT-An): Anemia; Functional Assessment of Cancer Therapy (FACT-C); 5- level EQ-5D (EQ-5D-5L); Patient Global Impression of Change (PGIC) fatigue item; or Patient Global Impression of Severity (PGIS) fatigue item.
223. The method of any of claims 1-222, wherein the patient maintains an absolute neutrophil count (ANC) of greater than 1.0 x 109/L when measured prior to the beginning of each trilaciclib/gemcitabine and carboplatin chemotherapy treatment.
224. The method of any of claims 1-223, wherein the patient maintains a platelet count of greater than 100 x 109/L when measured prior to the beginning of each trilaciclib/gemcitabine and carboplatin chemotherapy treatment.
225. The method of any of claims 1-224, wherein the administration of the trilaciclib/gemcitabine and carboplatin regimen results in a decrease in Simpson clonality.
226. The method of any of claims 35, 70, 105, 136, 170 and 201, wherein the TNBC to be treated has at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or
80 c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation.
227. The method of any of claims 1-226, wherein the patient has a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones.
228. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 12,500.
229. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 13,000.
230. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 13,500.
231. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 14,000.
232. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 14,500.
233. The method of claim 227, wherein the patient has a T-cell clonal peripheral diversity score of at least 15,000.
234. The method of any of claims 1-233, wherein the patient has a Simpson clonality score of less than about 0.08.
235. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.070.
236. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.060.
237. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.050.
238. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.040.
239. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.030.
240. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.020.
241. The method of claim 234, wherein the patient has a Simpson clonality score of less than about 0.010.
242. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient has previously been administered one or more immune checkpoint inhibitors (ICI) in a first-line chemotherapeutic setting and has developed therapeutic resistance to the ICI leading to disease progression, comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000084_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
243. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient has previously been administered one or more immune checkpoint inhibitors (ICI) in a first-line chemotherapeutic setting and has developed therapeutic resistance to the ICI leading to disease progression, the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000085_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
244. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000086_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
245. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000087_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
246. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), and wherein the patient has a TNBC that is PD-L1 status negative, comprising i. administering to the patient an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000088_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient an effective amount of gemcitabine; and, iii. administering to the patient an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
247. A method of treating a patient having advanced/metastatic triple negative breast cancer, wherein the patient is receiving first-line treatment for advanced/metastatic TNBC, wherein the patient has not previously been administered one or more immune checkpoint inhibitors (ICI), and wherein the patient has a TNBC that is PD-L1 status negative, the treatment comprising one or more 21-day cycles, wherein each cycle comprises: i. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of a CDK 4/6 inhibitor having the structure:
Figure imgf000089_0001
(trilaciclib), or a pharmaceutically acceptable salt, composition, isotope, or prodrug thereof; ii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of gemcitabine; and, iii. administering to the patient on day 1 and day 8 of the 21-day cycle an effective amount of carboplatin; wherein trilaciclib is administered no more than about 4 hours prior to the initiation of administration of the gemcitabine and carboplatin; wherein the patient has a TNBC with at least one of the following characteristics: a. a CCNE1 amplification; b. a CCNE2 amplification; or c. a Retinoblastoma protein 1 (Rbl) loss, which is defined as: i) a homozygous deletion; ii) a frameshift mutation; or iii) a stop-gained mutation; and, wherein the patient has either i) a T-cell clonal peripheral diversity score of at least about 12,000 T cell clones, ii) a Simpson clonality score of less than about 0.08, or iii) a T-cell clonal
87 peripheral diversity score of at least about 12,000 T cell clones and a Simpson clonality score of less than about 0.08.
248. The method of any of claims 1-19, 25-55, 61-91, 97-125, 131-158, 164-190, and 196-247, wherein an alternative platinum containing drug is administered in place of carboplatin.
249. The method of claim 248, wherein the alternative platinum containing drug administered in place of carboplatin is cisplatin.
250. The method of claim 248, wherein the alternative platinum containing drug administered in place of carboplatin is oxaliplatin.
251. The method of claims 1-250, wherein administration of the protocol induces T-cell immune activation against the tumor.
252. The method of claims 1-251, wherein administration of the protocol induces T-cell receptor (TCR) modulation.
253. The method of claims 1-252, wherein the T-cell activation results in increased interferon gamma (IFNy) expression.
254. The method of claims 1-253, wherein T-cell activation results in increased activation- induced expression of CD137.
255. The method of claims 1-254, wherein the T-cell activation results in increased TCR diversity.
256. The method of claims 1-255, wherein the T-cell activation results in a decrease in the baseline Simpson clonality score.
88
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