Methods of treating cancer using B-RAF inhibitors and immune checkpoint inhibitors

Abstract

The present invention relates to therapies, including dosage regimens, for the treatment of cancer using B-RAF and an immune checkpoint inhibitor in combination with and / or without a MEK inhibitor.

Description

[0001] This application is a divisional application of the patent application with the application date of November 18, 2016, the Chinese application number of 201680051333.4, and the invention name of "Methods of treating cancer using B-RAF inhibitors and immune checkpoint inhibitors". TECHNICAL FIELD

[0002] Provided herein are therapies for treating cancer using B-RAF and immune checkpoint inhibitors in combination with and / or without a MEK inhibitor, including dosage regimens. BACKGROUND

[0003] In 2014, the American Cancer Society estimated 76,100 new cases of melanoma and approximately 9,710 patients would die of the disease (American Cancer Society 2013). In 2008, the age-standardized melanoma incidence and mortality rates were 9.5 and 1.8 per 100,000 in men and 8.6 and 1.1 per 100,000 in women, respectively. Melanoma incidence rates are particularly high in Australia (42.4 per 100,000) and Western Europe (10.6 per 100,000) in Caucasian populations. In addition, melanoma incidence rates continue to rise; it is increasing more rapidly than any other malignancy in men and more rapidly than any other malignancy except lung cancer in women. The lifetime risk of developing melanoma in 2005 was approximately one in 55, with a median age at diagnosis of 59 years. Melanoma ranks second to adult leukemia in terms of years of life lost per death. Metastatic melanoma accounts for approximately 4% of all newly diagnosed melanoma cases. However, survival in this patient group remains extremely poor, with a 5-year relative survival rate of 15.3%.

[0004] Until recently, there were only a limited number of therapies available for metastatic melanoma. Dacarbazine is considered the standard first-line therapy, with response rates of 5%-12%, a median progression-free survival (PFS) of less than 2 months, and a median overall survival (OS) of 6.4 to 9.1 months. Combination chemotherapy and chemotherapy combined with interferon (IFN)-alpha (IFN-a) or interleukin-2 (IL-2) showed improved response rates while not leading to improved OS. More recently, newer agents with the potential to improve outcomes in patients with metastatic melanoma have been investigated. Ipilimumab, a humanized IgGl monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), was approved in 2011 for the treatment of patients with unresectable stage III or IV melanoma based on a 3.4-month improvement in median OS and a 1.3-month improvement in median PFS compared to chemotherapy. approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of unresectable or metastatic melanoma based on two Phase III studies showing OS improvement compared to glycoprotein 100 (gp100) peptide vaccination in previously untreated patients with advanced melanoma and compared to dacarbazine plus placebo in combination with dacarbazine. In these studies, the overall response rate (10.9%) and median PFS (2.8 months) were within the range historically observed for dacarbazine. However, the 3-year survival rate for ipilimumab plus dacarbazine was 20.8%, indicating that a subset of patients experienced durable clinical.

[0005] Vemurafenib, a specific inhibitor of the V600 mutant BRAF prevalent in approximately 50% of cutaneous melanomas approved by the FDA in 2011 for the treatment of BRAF V600 mutation-positive, unresectable, or metastatic melanoma. In patients with previously untreated metastatic melanoma with the BRAF V600E mutation, vemurafenib demonstrated a 63% reduction in the risk of death and a 74% improvement in PFS compared to dacarbazine. The response rate for vemurafenib was 48% versus 5% for dacarbazine. Updated OS and PFS results continue to demonstrate the clinical benefit of vemurafenib treatment. With a median follow-up of 12.5 months, vemurafenib treatment was associated with a nearly 4-month improvement in median OS (13.6 versus 9.7 months). The hazard ratio for death in this analysis was 0.70 (95% CI: 0.57, 0.87) in favor of vemurafenib.

[0006] Despite the PFS and OS prolongation, disease recurrence occurs in nearly all patients treated with BRAF inhibitors. MEK inhibitors have emerged as an additional treatment option for BRAF mutant melanoma. MEK is a downstream component of the BRAF / MEK / ERK signaling pathway, which is thought to be upregulated in response to BRAF inhibition. Additional data show that combining BRAF and MEK inhibition can provide greater clinical benefit compared to BRAF inhibitor monotherapy and potentially reduce side effects associated with BRAF inhibition. Cobimetinib (GDC-0973, RO5514041, XL518) is a MEK inhibitor in active clinical investigation and recently approved by the FDA.

[0007] Recently, cancer immunotherapy has made therapeutic inroads in metastatic melanoma. The programmed death-1 (PD-1) receptor blocking antibodies Nivolumab and Pembrolizumab have both received accelerated approval in the United States for the treatment of patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor, contingent upon verification of clinical benefit in confirmatory trials (see [Pembrolizumab] and [Nivolumab] U.S. package insert). Atezolizumab (also known as MPDL3280A) is a humanized IgGl monoclonal antibody that targets human programmed death-ligand 1 (PD-L1) and inhibits its interaction with its receptor, PD-1. Atezolizumab also blocks the binding of PD-L1 to B7-1, an interaction that is reported to provide an additional inhibitory signal to T cells.

[0008] Among the goals of new therapies is the challenge of combining the high response rates observed with B-RAF and MEK inhibitors in B-RAF V600 mutant patients with the potential for prolonged duration of response that can be elicited by immune modulation. Inhibition of BRAF V600 by vemurafenib and dabrafenib exhibits relatively little impact on other kinases, and in vitro / in vivo modeling suggests that BRAF inhibition improves T lymphocyte recognition of melanoma antigens and increases the number of CD4+ and CD8+ tumor infiltrating lymphocytes observed in melanoma tumors. In contrast to BRAF V600E inhibition, MEK inhibition can have a negative impact on T cells. See Luke, Journal American Journal of Hematology / Oncology 11(2):34-38 (February 2015). Also, PD-L1 expression is a mechanism of resistance to BRAF inhibitors, as BRAF resistant cell lines express high PD-L1, and the addition of a MEK inhibitor has a suppressive effect on PD-L1 expression. See Kim et al., Cancer Biol. Med 11(4):237-246 (2014). Continued development of new therapies, and the pursuit of the optimal treatment regimen (sequential combination) that will provide the greatest therapeutic benefit to patients, is ongoing. As such, clinical trials remain a viable option for initial treatment of advanced melanoma. SUMMARY

[0009] Provided herein are combinations comprising a B-RAF inhibitor and an immune checkpoint inhibitor with or without a MEK inhibitor. Provided herein are methods of treating cancer in an individual, the method comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor.

[0010] Also provided herein are methods of increasing the efficacy of a cancer treatment, the method comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor.

[0011] Also provided herein are methods of treating cancer in an individual, wherein the cancer treatment comprises first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor, wherein the cancer treatment has increased efficacy compared to administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of the immune checkpoint inhibitor alone.

[0012] In some embodiments of any of the methods, the B-RAF inhibitor is propane-1- sulfonic acid {3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro- phenyl}-amide or a pharmaceutically acceptable salt thereof. In some embodiments, the B-RAF is pembrolizumab, encorafenib, or vemurafenib. In some embodiments, the B-RAF inhibitor is vemurafenib. In some embodiments of any of the methods, the first administration and the second administration of the B-RAF inhibitor is at a dose of about 960 mg twice a day, about 720 mg twice a day, and / or about 480 mg twice a day. In some embodiments of any of the methods, the first administration of the B-RAF inhibitor is at a greater dose than the second administration of the B-RAF inhibitor. In some embodiments of any of the methods, the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor and the first dose is greater than the second dose. In some embodiments of any of the methods, the first administration of the B-RAF inhibitor is about 28 days or about 56 days. In some embodiments of any of the methods, the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor, the first dose is greater than the second dose, and the first dose is administered for 21 days and the second dose is administered for 7 days. In some embodiments of any of the methods, the B-RAF inhibitor is administered orally.

[0013] In some embodiments of any of the methods, the immune checkpoint inhibitor is a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is atezolizumab. In some embodiments, atezolizumab is administered at a dose of about 15 mg / kg q3w, about 20 mg / kg q3w, about 800 mg q2w, or about 1200 mg q3w. In some embodiments, the immune checkpoint inhibitor is administered intravenously.

[0014] In some embodiments of any of the methods, the first administration and the second administration further comprise an effective amount of a MEK inhibitor. In some embodiments, the MEK inhibitor is Trametinib, Binimetinib, or Cobimetinib. In some embodiments, the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane, hemifumarate. In some embodiments, the MEK inhibitor is Cobimetinib. In some embodiments of any of the methods, the first administration and the second administration of the MEK inhibitor is at a dose of about 60 mg per day on a 21 days on / 7 days off schedule or about 40 mg per day on a 21 days on / 7 days off schedule. In some embodiments of any of the methods, the first administration of the MEK inhibitor is about a 28 day schedule. In some embodiments of any of the methods, the MEK inhibitor is administered orally.

[0015] In some embodiments of any of the methods, the cancer is melanoma. In some embodiments of any of the methods, the melanoma is unresectable or metastatic melanoma. In some embodiments of any of the methods, the melanoma is a B-RAF V600 mutant melanoma. In some embodiments of any of the methods, the B-RAF V600E mutant melanoma or B-RAF V600K mutant melanoma.

[0016] Further provided herein is a method of treating melanoma in an individual, the method comprising first administering to the individual vemurafenib on a 28-day schedule, wherein vemurafenib is administered at a dose of 960 mg twice a day for 21 days on the 28-day schedule, followed by 720 mg twice a day for 7 days, and second administering to the individual vemurafenib and atezolizumab, wherein vemurafenib is administered at a dose of 720 mg twice a day and atezolizumab is administered at a dose of 1200 mg q3w. In some embodiments, vemurafenib is administered orally as a tablet. In some embodiments, atezolizumab is administered intravenously. In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is unresectable or metastatic melanoma. In some embodiments, the melanoma is B-RAF V600 mutant melanoma. In some embodiments, the B-RAF V600E mutant melanoma or B-RAF V600K mutant melanoma.

[0017] Further provided herein is a method of treating melanoma in an individual, the method comprising first administering to the individual vemurafenib and cobimetinib on a 28-day schedule, wherein vemurafenib is administered at a dose of 960 mg twice a day for 21 days on the 28-day schedule, followed by 720 mg twice a day for 7 days, and cobimetinib is administered at a dose of 60 mg daily for 21 days on the 28-day schedule and 7 days off, and second administering to the individual vemurafenib, cobimetinib, and atezolizumab, wherein vemurafenib is administered at a dose of 720 mg twice a day, cobimetinib is administered at a dose of 60 mg daily for 21 days and 7 days off, and atezolizumab is administered at a dose of 800 mg q2w. In some embodiments, vemurafenib is administered orally as a tablet. In some embodiments, cobimetinib is administered orally as a tablet. In some embodiments, atezolizumab is administered intravenously. In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is unresectable or metastatic melanoma. In some embodiments, the melanoma is B-RAF V600 mutant melanoma. In some embodiments, the B-RAF V600 mutant melanoma is B-RAF V600E mutant melanoma or B-RAF V600K mutant melanoma. DETAILED DESCRIPTION

[0018] I. DEFINITIONS

[0019] The terms "RAF" and "RAF kinase" refer to a family of three RAF kinases, which are serine / threonine-specific protein kinases. The RAF kinases are A-Raf, BRAF, and CRAF. RAF kinases are involved in retroviral oncogenes. RAF is an acronym for Rapidly Accelerated Fibrosarcoma.

[0020] Unless otherwise indicated, the term "B-RAF" as used herein refers to any native or variant (whether natural or synthetic) B-RAF polypeptide. The term "wild-type B-RAF" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring B-RAF protein. See, e.g., Uni. Prot No. P15056, accessed 11.16.15. The term "V600 mutant B-RAF" or "V600 mutant B-RAF" generally refers to a b-Raf polypeptide comprising a missense mutation at amino acid V600, e.g., B-RAF V600E or B-RAF V600K.

[0021] An "antagonist" (interchangeably referred to as an "inhibitor") of a polypeptide of interest is a molecule that interferes with the activation or function of the polypeptide of interest, e.g., partially or totally blocks, inhibits, or neutralizes a biological activity mediated by the polypeptide of interest. For example, an antagonist of polypeptide X can refer to any molecule that partially or totally blocks, inhibits, or neutralizes a biological activity mediated by polypeptide X. Preferably, the inhibitor is a small molecule that binds to the polypeptide of interest. In a particular embodiment, the inhibitor has a binding affinity (dissociation constant) for the polypeptide of interest of about 1,000 nM or less. In another embodiment, the inhibitor has a binding affinity for the polypeptide of interest of about 100 nM or less. In another embodiment, the inhibitor has a binding affinity for the polypeptide of interest of about 50 nM or less. In a particular embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 1,000 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 500 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 50 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 10 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 1 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 0.1 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 0.01 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 0.001 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 0.0001 nM or less. In another embodiment, the inhibitor inhibits the polypeptide of interest with an IC50of 0.00001 nM or less. 50 inhibits signaling of the polypeptide of interest. In another embodiment, the inhibitor inhibits signaling of the polypeptide of interest with an IC50of 500 nM or less. 50 inhibits signaling of the polypeptide of interest. In another embodiment, the inhibitor inhibits signaling of the polypeptide of interest with an IC50of 500 nM or less. 50 inhibits signaling of the polypeptide of interest. In another embodiment, the inhibitor inhibits signaling of the polypeptide of interest with an IC50of 500 nM or less.

[0022] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis - a result being the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, and / or target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0023] The term "PD-1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with the signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with the signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1 binding antagonist decreases the negative co-stimulatory signal mediated by or through a cell surface protein expressed on T lymphocytes (mediating signaling through PD-1), rendering a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-1 binding antagonist is an anti-PD-1 antibody. In a particular embodiment, a PD-1 binding antagonist is MDX-1106 (nivolumab) described herein. In another particular embodiment, a PD-1 binding antagonist is MK-3475 (pembrolizumab) described herein. In another particular embodiment, a PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another particular aspect, a PD-1 binding antagonist is MEDI-0680 (AMP-514) described herein. In another particular aspect, a PD-1 binding antagonist is PDR001 described herein. In another particular aspect, a PD-1 binding antagonist is REGN2810 described herein. In another particular aspect, a PD-1 binding antagonist is BGB-108 described herein.

[0024] The term "PD-L1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with the signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist includes anti-PD-Ll antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with the signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one embodiment, a PD-L1 binding antagonist decreases the negative co-stimulatory signal mediated by or through a cell surface protein expressed on T lymphocytes (mediating signaling through PD-L1), rendering a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L1 binding antagonist is an anti-PD-Ll antibody. In a specific aspect, an anti-PD-Ll antibody is MPDL3280A (atezolizumab) described herein. In another specific aspect, an anti-PD-Ll antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD-Ll antibody is YW243.55.S70 described herein. In still another specific aspect, an anti-PD-Ll antibody is MEDI4736 (durvalumab) described herein. In still another specific aspect, an anti-PD-Ll antibody is MSB0010718C (avelumab) described herein.

[0025] The term "PD-L2 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a particular aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist decreases the negative co-stimulatory signal mediated by or through a cell surface protein expressed on T lymphocytes (mediating signaling through PD-L2), thereby making a dysfunctional T cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.

[0026] The term "small molecule" refers to any molecule having a molecular weight of about 2000 daltons or less, preferably about 500 daltons or less.

[0027] "Individual response" or "response" can be assessed using any endpoint indicative of benefit to the individual, including, but not limited to: (1) some degree of inhibition of progression of the disease (e.g., cancer progression), including slowing down and complete arrest; (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or complete termination) of cancer cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing down or complete termination) of metastasis; (5) some degree of alleviation of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase in length of progression-free survival; and / or (8) reduction in mortality at a given time point post-treatment.

[0028] An "effective amount" of a substance / molecule (e.g., a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0029] A "therapeutically effective amount" of a substance / molecule can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactically effective amount will be lower than a therapeutically effective amount since a prophylactic dose is used before or at the earliest signs of disease, as opposed to a therapeutic dose, which is used after disease is established.

[0030] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0031] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than an active ingredient that does not cause unacceptable toxicity in a subject to which the formulation would be administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizing agents, or preservatives.

[0032] As used herein, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound.

[0033] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a subject's pathology, and can be performed either for prophylaxis or during the course of pathology. Desirable effects of treatment include, but are not limited to, preventing or delaying the onset of disease, alleviating symptoms, diminishment of any direct or indirect pathological consequences, preventing or delaying a metastasis, decreasing the rate of disease progression, ameliorating or eliminating symptoms, and improving prognosis. In some embodiments, the antibodies of the application are used to delay development of a disease or to slow the progression of a disease.

[0034] "Subject" or "individual" refers to a mammal. Mammals include, but are not limited to, domestic and farm animals (e.g., cows, sheep, pigs, goats, horses, dogs, and cats), primates (e.g., humans, apes, monkeys and lemurs), rabbits, and rodents (e.g., mice, rats, and hamsters). In certain embodiments, the subject or individual is a human.

[0035] The term "concomitantly" is used herein to refer to administration of two or more therapeutic agents in sufficient proximity in time wherein the effects of the various therapeutic agents overlap in time. In some embodiments, concomitant administration is concurrent, sequential, and / or simultaneous. In some embodiments, concurrent administration includes dosing regimens where administration of one or more agents is continued after administration of one or more other agents is discontinued.

[0036] "Decreasing or inhibiting" refers to the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Decreasing or inhibiting can refer to symptoms of the disorder being treated, the presence or size of metastases, or the size of a primary tumor.

[0037] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0038] An "article of manufacture" is a manufacture (e.g., package or container) or kit including at least one reagent, such as a drug for treating a disease or disorder (e.g., cancer) or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is marketed, distributed, or sold as a unit for practicing the methods described herein.

[0039] As those skilled in the art understand, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X."

[0040] It is to be understood that aspects and embodiments of the application described herein include that which "comprises" and / or "consists of the stated aspects and embodiments. As used herein, the singular forms "a", "an" and "the" include plural referents unless otherwise indicated.

[0041] II. Methods and Uses

[0042] Provided herein are methods of treating cancer with a B-RAF inhibitor (e.g., vemurafenib) and a cancer immune checkpoint inhibitor (e.g., an anti-PD1 / anti-PD-L1 antibody). In addition, provided herein are methods of treating cancer with a B-RAF inhibitor (e.g., vemurafenib), a MEK inhibitor (e.g., cobimetinib), and an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor).

[0043] Provided herein are methods of treating cancer in an individual comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor). For example, provided herein are methods of treating cancer in an individual comprising first administering to the individual an effective amount of vemurafenib and second administering to the individual an effective amount of vemurafenib and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab).

[0044] Also provided herein are methods of treating cancer in an individual comprising first administering to the individual an effective amount of a B-RAF inhibitor and an effective amount of a MEK inhibitor, and second administering to the individual an effective amount of the B-RAF inhibitor, an effective amount of the MEK inhibitor, and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor). For example, provided herein are methods of treating cancer in an individual comprising first administering to the individual an effective amount of vemurafenib and an effective amount of cobimetinib and second administering to the individual an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab).

[0045] Provided herein are methods of improving the efficacy of a cancer treatment comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor). For example, provided herein are methods of improving the efficacy of a cancer treatment comprising first administering to the individual an effective amount of vemurafenib and second administering to the individual an effective amount of vemurafenib and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab). In some embodiments, the methods improve immune tumor CD8+ T cell infiltration. In some embodiments, the methods improve PD-L1 expression, e.g., as determined by IHC.

[0046] Further provided herein are methods of improving the efficacy of a cancer treatment comprising first administering to the individual an effective amount of a B-RAF inhibitor and an effective amount of a MEK inhibitor, and second administering to the individual an effective amount of the B-RAF inhibitor, an effective amount of the MEK inhibitor, and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor). For example, provided herein are methods of improving the efficacy of a cancer treatment comprising first administering to the individual an effective amount of vemurafenib and an effective amount of cobimetinib and second administering to the individual an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab).

[0047] Also provided herein are methods of treating a cancer in an individual, wherein the cancer treatment comprises first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor), wherein the cancer treatment has improved efficacy compared to administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of the immune checkpoint inhibitor (e.g., a PD1 axis inhibitor) alone (administered concurrently by itself). For example, provided herein are methods of treating a cancer in an individual, wherein the cancer treatment comprises first administering to the individual an effective amount of vemurafenib and second administering to the individual an effective amount of vemurafenib and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab), wherein the cancer treatment has improved efficacy compared to administering to the individual an effective amount of vemurafenib and an effective amount of the anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) alone (administered concurrently by itself).

[0048] Provided herein are methods of treating cancer in an individual, wherein the cancer treatment comprises a first administration of an effective amount of a B-RAF inhibitor and an effective amount of a MEK inhibitor to the individual, and a second administration of an effective amount of the B-RAF inhibitor, an effective amount of the MEK inhibitor, and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor) to the individual, wherein the cancer treatment has improved efficacy compared to administration of an effective amount of the B-RAF inhibitor, an effective amount of the MEK inhibitor, and an effective amount of an immune checkpoint inhibitor (e.g., a PD1 axis inhibitor) to the individual alone (administered concurrently by itself). For example, provided herein are methods of treating cancer in an individual, wherein the cancer treatment comprises a first administration of an effective amount of vemurafenib and an effective amount of cobimetinib to the individual and a second administration of an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual, wherein the cancer treatment has improved efficacy compared to administration of an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of the anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual alone (administered concurrently by itself).

[0049] In some embodiments of any of the methods, the first administration is about any of 7, 14, 21, 28, 35, 42, 49, or 56 days. In some embodiments, the first administration is between about 21 and 35 days. In some embodiments, the first administration is about 28 days. In some embodiments, the first administration is about 56 days. For example, the method of treating cancer in an individual comprises a first administration of an effective amount of vemurafenib to the individual for 28 days and a second administration of an effective amount of vemurafenib and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual. Also, for example, the method of treating cancer in an individual comprises a first administration of an effective amount of vemurafenib and an effective amount of cobimetinib to the individual for 28 days and a second administration of an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual.

[0050] In some embodiments of any of the methods, the individual according to any of the above embodiments can be a human.

[0051] In some embodiments of any of the methods, the first administration comprises administration of a single dose of the B-RAF inhibitor and / or the MEK inhibitor. In some embodiments of any of the methods, the first administration comprises administration of more than one dose of the B-RAF inhibitor and / or the MEK inhibitor. In some embodiments, the first administration comprises administration of a first dose for about any of 7, 14, 21, 28, 35, 42, or 49 days, followed by a second dose for about any of 7, 14, 21, 28, 35, 42, or 49 days. In some embodiments, the first administration comprises administration of a first dose for about 49 days, followed by a second dose for about any of 7 days. In some embodiments, the first administration comprises administration of a first dose for about 21 days, followed by a second dose for about any of 7 days. In some embodiments of any of the methods, the first administration comprises a first dose of the B-RAF inhibitor and / or the MEK inhibitor that is greater than a second dose. For example, a method of treating cancer in an individual comprises a first administration of an effective amount of vemurafenib to the individual, wherein vemurafenib is administered in a first dose for 21 days, followed by a second dose for 7 days, and a second administration of an effective amount of vemurafenib and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual. Also, for example, a method of treating cancer in an individual comprises a first administration of an effective amount of vemurafenib and an effective amount of cobimetinib to the individual, wherein vemurafenib is administered in a first dose for 21 days, followed by a second dose for 7 days, and a second administration of an effective amount of vemurafenib, an effective amount of cobimetinib, and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual.

[0052] In some embodiments of any of the methods, the combination therapies described above encompass co-administration (where two or more therapeutic agents are comprised in the same or separate formulations), and separate administration (in which case the antagonists of the application can be administered prior to, simultaneously with, sequentially with, concurrently with, and / or following the administration of the other therapeutic agents and / or adjuvants). In some embodiments, the combination therapies further comprise radiation therapy and / or additional therapeutic agents.

[0053] The B-RAF inhibitors, MEK inhibitors and / or immune checkpoint inhibitors described herein can be administered by any appropriate means, including oral, parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending on whether administration is short term or long term, the dosing can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0054] In some embodiments of any of the methods, administration of the effective amount of the B-RAF, the effective amount of the MEK inhibitor, and / or the effective amount of the immune checkpoint inhibitor can occur via administration of different routes and / or administration at different times. For example, the B-RAF inhibitor can be administered orally, e.g., orally twice a day. The MEK inhibitor can be administered orally, e.g., orally once a day. In some embodiments, when the first administration comprises a B-RAF inhibitor and a MEK inhibitor, the B-RAF inhibitor and the MEK inhibitor act concomitantly. In some embodiments, when the second administration comprises a B-RAF inhibitor and an immune checkpoint inhibitor, the B-RAF inhibitor and the immune checkpoint inhibitor act concomitantly. In some embodiments, when the second administration comprises a B-RAF inhibitor, a MEK inhibitor, and an immune checkpoint inhibitor, the B-RAF inhibitor, the MEK inhibitor, and the immune checkpoint inhibitor act concomitantly. In some embodiments, the first and second administrations act concomitantly. In some embodiments, the first administration and the second administration are sequential.

[0055] The B-RAF inhibitors, MEK inhibitors, and / or immune checkpoint inhibitors described herein can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the part of the body to be treated, the method of administration, the scheduling of the administration, and other factors known to medical practitioners. The B-RAF inhibitors, MEK inhibitors, and / or immune checkpoint inhibitors need not be, but are optionally formulated with one or more agents currently used in the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of B-RAF inhibitors, MEK inhibitors, and / or immune checkpoint inhibitors present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes and schedules as are employed with the B-RAF inhibitors, MEK inhibitors, and / or immune checkpoint inhibitors, or as are standard for the other agents being administered.

[0056] For the prevention or treatment of disease, the appropriate dosage of the B-RAF inhibitor, MEK inhibitor, and / or immune checkpoint inhibitor described herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the B-RAF inhibitor, MEK inhibitor, and / or immune checkpoint inhibitor is being used for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the B-RAF inhibitor, MEK inhibitor, and / or immune checkpoint inhibitor, and the judgment of the treating physician. The B-RAF inhibitor, MEK inhibitor, and / or immune checkpoint inhibitor is suitably administered to the patient at one time or over a series of treatments. For repeated administrations over several days or longer, depending on the condition, the treatment is generally continued until desired suppression of disease symptoms occurs.

[0057] In some embodiments, the B-RAF inhibitor (e.g., vemurafenib) is administered daily or twice a day. In some embodiments, the B-RAF inhibitor (e.g., vemurafenib) is administered at a dose of about 960 mg, 720 mg, 480 mg, or 240 mg twice a day. In some embodiments, the first administration comprises administering the B-RAF inhibitor (e.g., vemurafenib) at a first dose of 960 mg twice a day, followed by a second dose of 720 mg twice a day. In some embodiments, the first administration comprises administering the B-RAF inhibitor (e.g., vemurafenib) at a first dose of 720 mg twice a day, followed by a second dose of 480 mg twice a day. In some embodiments, the second administration comprises administering the B-RAF inhibitor (e.g., vemurafenib) at a dose of 960 mg twice a day. In some embodiments, the second administration comprises administering the B-RAF inhibitor (e.g., vemurafenib) at a dose of 720 mg twice a day. For example, in some embodiments, the method of treating cancer in an individual comprises a first administration of a B-RAF inhibitor (e.g., vemurafenib) to the individual for 28 days, wherein the B-RAF inhibitor (e.g., vemurafenib) is administered at 960 mg twice a day for 21 days and 720 mg twice a day for 7 days and a second administration of a B-RAF inhibitor (e.g., vemurafenib) at a dose of 720 mg twice a day and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual.

[0058] In some embodiments, the MEK inhibitor (e.g., cobimetinib) is administered daily or twice a day. In some embodiments, the MEK inhibitor (e.g., cobimetinib) is administered daily at a dose of about 60 mg or 20 mg either. In some embodiments, the first administration comprises administering the MEK inhibitor (e.g., cobimetinib) at a first dose of 60 mg daily, 21 days on and 7 days off (21 / 7). In some embodiments, the first administration comprises administering the MEK inhibitor (e.g., cobimetinib) at a first dose of 40 mg daily, 21 days on and 7 days off (21 / 7). In some embodiments, the second administration comprises administering the MEK inhibitor (e.g., cobimetinib) at a dose of 60 mg daily, 21 days on and 7 days off (21 / 7). In some embodiments, the second administration comprises administering the MEK inhibitor (e.g., cobimetinib) at a dose of 40 mg daily, 21 days on and 7 days off (21 / 7). For example, in some embodiments, the method of treating cancer in an individual comprises a first administration of a B-RAF inhibitor (e.g., vemurafenib) and a MEK inhibitor (e.g., cobimetinib) to the individual for 28 days, wherein the B-RAF inhibitor (e.g., vemurafenib) is administered at 960 mg twice a day for 21 days and at 720 mg twice a day for 7 days and the MEK inhibitor (e.g., cobimetinib) is administered at a dose of 60 mg daily, 21 days on and 7 days off and a second administration of a B-RAF inhibitor (e.g., vemurafenib) at a dose of 720 mg twice a day, a MEK inhibitor (e.g., cobimetinib) at a dose of 60 mg daily, 21 days on and 7 days off and an effective amount of an anti-PD1 / anti-PD-L1 antibody (e.g., atezolizumab) to the individual.

[0059] In some embodiments, the anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) is administered every 3 weeks (q3w) or every 2 weeks (q2w). In some embodiments, the anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) is administered at a dose of about 15 mg / kg q3w, about 20 mg / kg q3w, about 800 mg q2w, or about 1200 mg q3w. In some embodiments, the second administration comprises administering the anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) at a dose of 800 mg q2w. In some embodiments, the second administration comprises administering the anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) at a dose of 1200 mg q3w. For example, in some embodiments, the method of treating cancer in an individual comprises a first administration of a B-RAF inhibitor (e.g., vemurafenib) to the individual for 28 days, wherein the B-RAF inhibitor (e.g., vemurafenib) is administered at 960 mg twice a day for 21 days and 720 mg twice a day for 7 days, and a second administration of a B-RAF inhibitor (e.g., vemurafenib) at a dose of 720 mg twice a day and an anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) at a dose of 1200 mg q3w to the individual. For example, in some embodiments, the method of treating cancer in an individual comprises a first administration of a B-RAF inhibitor (e.g., vemurafenib) and a MEK inhibitor (e.g., cobimetinib) to the individual for 28 days, wherein the B-RAF inhibitor (e.g., vemurafenib) is administered at 960 mg twice a day for 21 days and 720 mg twice a day for 7 days and the MEK inhibitor (e.g., cobimetinib) is administered at a dose of 60 mg per day, 21 days on and 7 days off, and a second administration of a B-RAF inhibitor (e.g., vemurafenib) at a dose of 720 mg twice a day, a MEK inhibitor (e.g., cobimetinib) at a dose of 60 mg per day, 21 days on and 7 days off, and an anti-PD1 / anti-PD-Ll antibody (e.g., atezolizumab) at a dose of 800 mg q2w to the individual.

[0060] In some embodiments of any of the methods, the cancer is melanoma. In some embodiments, the melanoma is unresectable or metastatic melanoma. In some embodiments, the melanoma is a B-RAF V600 mutant melanoma. In some embodiments, the B-RAF V600 mutant melanoma is a B-RAF V600E mutant melanoma. In some embodiments, the B-RAF V600 mutant melanoma is a B-RAF V600K mutant melanoma.

[0061] III. Therapeutic Compositions

[0062] Provided herein are combinations comprising a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor. In certain embodiments, the combinations improve efficacy compared to treatment comprising administration of a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor alone or co-administration (concurrent administration) of a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor. In certain embodiments, the combinations comprising a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor as described herein improve T cell infiltration and / or PD-L1 expression levels compared to treatment comprising administration of a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor alone or co-administration (concurrent administration) of a B-RAF inhibitor, a MEK inhibitor, or an immune checkpoint inhibitor.

[0063] In some embodiments of any of the methods, the methods described herein can use a B-RAF inhibitor. Exemplary BRAF inhibitors are known in the art and include, for example, sorafenib, PLX4720, PLX-3603, dabrafenib (GSK2118436), encorafenib (LGX818), GDC-0879, RAF265 (Novartis), XL281, ARQ736, BAY73-4506, vemurafenib, and those described in WO2007 / 002325, WO2007 / 002433, WO2009111278, WO2009111279, WO2009111277, WO2009111280, and U.S. Patent No. 7,491,829. In some embodiments, the BRAF inhibitor is a selective BRAF inhibitor. In some embodiments, the BRAF inhibitor is a selective inhibitor of BRAF V600. In some embodiments, BRAF V600 is BRAF V600E, BRAF V600K, and / or V600D. In some embodiments, BRAF V600 is BRAF V600R. In some embodiments, the BRAF inhibitor is vemurafenib. In some embodiments, the BRAF inhibitor is vemurafenib. In some embodiments, the B-RAF inhibitor is propane-1 -sulfonic acid {3-[5-(4-chlorophenyl)-1 H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide or a pharmaceutically acceptable salt thereof. In some embodiments, the B-RAF inhibitor is vemurafenib. In some embodiments, the B-RAF inhibitor is dabrafenib. In some embodiments, the B-RAF inhibitor is encorafenib.

[0064] Vemurafenib (RG7204, PLX-4032, CAS Registry Number 1029872-55-5) has been shown to cause programmed cell death in a variety of cancer cell lines, such as melanoma cell lines. Vemurafenib interrupts the BRAF / MEK step on the BRAF / MEK / ERK pathway if BRAF has the common V600E mutation. Vemurafenib works in patients whose cancer has the V600E BRAF mutation (i.e., the normal valine is replaced by glutamic acid at amino acid position number 600 of the BRAF protein) such as FDA-approved melanoma patients. About 60% of melanomas have the V600E BRAF mutation. The V600E mutation is present in a variety of other cancers, including lymphoma, colon cancer, melanoma, thyroid cancer and lung cancer. Vemurafenib has the following structure:

[0065]

[0066] ZELBORAF® (vemurafenib) (Genentech, Inc.) is a drug product approved in the United States and indicated for the treatment of patients with unresectable or metastatic melanoma with a BRAF V600E mutation (as detected by a FDA-approved test). ZELBORAF® (vemurafenib) is not recommended for melanoma patients who lack the BRAF V600E mutation (wild-type BRAF melanoma). ZELBORAF® (vemurafenib) is a kinase inhibitor available as a 240 mg tablet for oral use.

[0067] In some embodiments of any of the combination therapy methods described herein, the targeted therapeutic agent is a MEK inhibitor. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, and / or a MEK1 / 2 inhibitor. Exemplary MEK inhibitors include, but are not limited to, trametinib (GSK 1120212), MEK162, selumetinib (AZD 6244, ARRY-142886), pimasertib (MSC1936369B, AS-703026, AS703026), refametinib, cobimetinib (GDC-0973), BI-847325, GDC-0623, PD-325901, CI-1040, and PD035901. In some embodiments, the MEK inhibitor is selumetinib, pimasertib, cobimetinib (GDC-0973), GDC-0623, binimetinib, or trametinib. In certain embodiments, the MEK inhibitor is cobimetinib (GDC-0973).

[0068] Cobimetinib (GDC-0973 or XL518) is a selective inhibitor of MEK, also known as mitogen-activated protein kinase kinase (MAPKK), a key component of the RAS / RAF / MEK / ERK pathway frequently activated in human tumors. GDC-0973 can be prepared as described in International Patent Application Publication No. WO2007044515 (Al). GDC-0973 is named (S)-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl)(3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl)methanone and has the following structure:

[0069]

[0070] In some embodiments, the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4- iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane, hemifumarate. In some embodiments, the MEK inhibitor is cobimetinib.

[0071] Cobimetinib is a fumarate salt that appears as a white to off-white solid and exhibits pH-dependent solubility. COTELLIC (cobimetinib) tablets are supplied as white, round, film-coated 20 mg tablets for oral administration, one side is engraved with "COB". Each 20 mg tablet contains 22 mg of cobimetinib fumarate, which corresponds to 20 mg of cobimetinib free base. The inactive ingredients of COTELLIC are: Core: microcrystalline cellulose, lactose monohydrate, croscarmellose sodium, magnesium stearate. Coating: polyvinyl alcohol, titanium dioxide, polyethylene glycol 3350, talc.

[0072] Trametinib (GSK 1120212, CAS Registry Number 871700-17-3) is named N-(3-{3- cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxa-3,4,6,7- tetrahydropyrido[4,3-d]pyrimidin-l(2H)-yl}phenyl)acetamide and has the following structure:

[0073]

[0074] In some embodiments of any of the methods, the methods described herein can use an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. PD-1 (programmed death 1) is also known in the art as "programmed cell death 1", "PDCD1", "CD279", and "SLEB2". An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot Accession No. Q15116. PD-L1 (programmed death-ligand 1) is also known in the art as "programmed cell death 1 ligand 1", "PDCD1LG1", "CD274", "B7-H", and "PDL1". An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1. PD-L2 (programmed death-ligand 2) is also known in the art as "programmed cell death 1 ligand 2", "PDCD1LG2", "CD273", "B7-DC", "Btdc", and "PDL2". An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot Accession No. Q9BQ51. In some embodiments, the PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0075] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0076] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of MPDL3280A, YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 described in WO 2010 / 077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO 2011 / 066389 and US 2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO 2006 / 121168. MK-3475, also known as lambrolizumab, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0077] In some embodiments, the PD-1 axis binding antagonist is an anti-PD-Ll antibody. In some embodiments, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-Ll and PD-1 and / or the binding between PD-Ll and B7-1. In some embodiments, the anti-PD-Ll antibody is a monoclonal antibody. In some embodiments, the anti-PD-Ll antibody is an antibody fragment selected from the group consisting of a Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragment. In some embodiments, the anti-PD-Ll antibody is a humanized antibody. In some embodiments, the anti-PD-Ll antibody is a human antibody.

[0078] Examples of anti-PD-Ll antibodies useful in the methods herein are described in PCT Patent Application WO 2010 / 077634, WO 2007 / 005874, WO 2011 / 066389, and US2013 / 034559, which are incorporated herein by reference.

[0079] Anti-PD-1 Antibodies

[0080] In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In still another embodiment, there is provided an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and / or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 2. In still another embodiment, there is provided an isolated anti-PD-1 antibody comprising a heavy chain and / or light chain sequence, wherein:

[0081] (a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the following heavy chain sequence:

[0082] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWV

[0083] AVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT

[0084] NDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE

[0085] PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD

[0086] HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE

[0087] VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL

[0088] TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE

[0089] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1), and

[0090] (b) The light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the following light chain sequences:

[0091] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS

[0092] NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKV

[0093] EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

[0094] QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2).

[0095] Anti-PD-L1 antibody

[0096] In some embodiments, the antibody in the formulation comprises at least one (e.g., at least two, at least three, or at least four) tryptophan in the heavy and / or light chain sequence. In some embodiments, the amino acid tryptophan is in the HVR regions, framework regions, and / or constant regions of the antibody. In some embodiments, the antibody comprises two or three tryptophan residues in the HVR regions. In some embodiments, the antibody in the formulation is an anti-PD-Ll antibody. PD-L1 (programmed death-ligand 1), also known as PDL1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein and its interaction with PD-1 inhibits T cell activation and cytokine production. In some embodiments, the anti-PD-Ll antibody described herein binds to human PD-L1. Examples of anti-PD-Ll antibodies useful in the methods described herein are described in PCT Patent Application WO 2010 / 077634 Al and U.S. Patent No. 8,217,149, which are incorporated herein by reference in their entirety.

[0097] In some embodiments, the anti-PD-Ll antibody is capable of inhibiting the binding between PD-L1 and PD-1 and / or PD-L1 and B7-1. In some embodiments, the anti-PD-Ll antibody is a monoclonal antibody. In some embodiments, the anti-PD-Ll antibody is an antibody fragment selected from the group consisting of a Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragment. In some embodiments, the anti-PD-Ll antibody is a humanized antibody. In some embodiments, the anti-PD-Ll antibody is a human antibody.

[0098] The anti-PD-Ll antibodies described in WO 2010 / 077634 Al and US 8,217,149 can be used in the methods described herein. In some embodiments, the anti-PD-Ll antibody comprises the heavy chain variable region sequence of SEQ ID NO: 3 and / or the light chain variable region sequence of SEQ ID NO: 4. In still another embodiment, an isolated anti-PD-Ll antibody is provided, comprising a heavy chain variable region and / or a light chain variable region sequence, wherein:

[0099] (a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the following heavy chain sequence:

[0100] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO: 3), and

[0101] (b) the light chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following light chain sequence:

[0102] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0103] In one embodiment, the anti-PD-Ll antibody comprises a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

[0104] (a) the HVR-H1 sequence is GFTFSXiSWIH (SEQ ID NO: 5);

[0105] (b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6);

[0106] (c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7);

[0107] Further wherein: Xi is D or G; X2 is S or L; X3 is T or S. In a specific aspect, Xi is D; X2 is S and X3 is T.

[0108] In another aspect, the polypeptide further comprises an inter-HVR juxtaposed variable region heavy chain framework sequence according to the following formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequences are derived from a human consensus framework sequence. In another aspect, the framework sequences are VH subgroup III consensus frameworks. In yet another aspect, at least one of the framework sequences is as follows:

[0109] HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 8)

[0110] HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 9)

[0111] HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10)

[0112] In yet another aspect, the heavy chain polypeptide is further combined with a variable region light chain comprising HVR-L1, HVR-L2 and HVR-L3, wherein:

[0113] (a) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12);

[0114] (b) the HVR-L2 sequence is SASX9LX 10 S (SEQ ID NO: 13);

[0115] (c) the HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T (SEQ ID NO: 14);

[0116] wherein: X4is D or V; X5is V or I; X6is S or N; X7is A or F; X8is V or L; X9is F or T; X 10 is Y or A; X 11 is Y, G, F, or S; X 12 is L, Y, F or W; X 13 is Y, N, A, T, G, F or I; X 14 is H, V, P, T or I; X 15 is A, W, R, P or T. In yet another aspect, X4is D; X5is V; X6is S; X7is A; X8is V; X9is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 is H; X 15 is A.

[0117] In yet another aspect, the light chain further comprises inter-HVR juxtaposed variable region light chain framework sequences according to the following formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the framework sequences are VL kappa I consensus framework. In yet another aspect, at least one framework sequence is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)

[0118] LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16)

[0119] LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17) LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 18).

[0120] In another embodiment, an isolated anti-PD-Ll antibody or antigen binding fragment is provided, comprising heavy and light chain variable region sequences, wherein:

[0121] (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein further:

[0122] (i) the HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO: 5),

[0123] (ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6)

[0124] (iii) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 7), and

[0125] (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein further:

[0126] (i) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12),

[0127] (ii) the HVR-L2 sequence is SASX9LX 10 S (SEQ ID NO: 13), and

[0128] (iii) the HVR-L3 sequence is QQX 11 X 12 X 13X 14 PX 15 T (SEQ ID NO: 14)

[0129] wherein: X1is D or G; X2is S or L; X3is T or S; X4is D or V; X5is V or I; X6is S or N; X7is A or F; X8is V or L; X9is F or T; X 10 is Y or A; X 11 is Y, G, F, or S; X 12 is L, Y, F or W; X 13 is Y, N, A, T, G, F or I; X 14 is H, V, P, T or I; X 15 is A, W, R, P or T. In one particular aspect, X1is D; X2is S and X3is T. In another aspect, X4is D; X5is V; X6is S; X7is A; X8is V; X9is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 is H; X 15 is A. In yet another aspect, X1is D; X2is S and X3is T, X4is D; X5is V; X6is S; X7is A; X8is V; X9is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 is H and X 15 is A.

[0130] In another aspect, the heavy chain variable region comprises one or more framework sequences interposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences interposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a further aspect, the framework sequences are derived from human consensus framework sequences. In a further aspect, the heavy chain framework sequences are derived from Kabat Subgroup I, II, or III sequences. In a further aspect, the heavy chain framework sequences are VH Subgroup III consensus framework. In a further aspect, the one or more heavy chain framework sequences are as set forth in SEQ ID NOs: 8, 9, 10, and 11. In a further aspect, the light chain framework sequences are derived from Kabat kappa I, II, II, or IV subgroup sequences. In a further aspect, the light chain framework sequences are VL kappa I consensus framework. In a further aspect, the one or more light chain framework sequences are as set forth in SEQ ID NOs: 15, 16, 17, and 18.

[0131] In a further specific aspect, the antibody further comprises a human or murine constant region. In a further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a further specific aspect, the human constant region is IgGl. In a further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a further aspect, the murine constant region is IgG2A. In a further specific aspect, the antibody has reduced or minimal effector function. In a further specific aspect, the minimal effector function is derived from an "effector-less Fc mutation" or aglycosylation. In a further embodiment, the effector-less Fc mutation is N297A or D265A / N297A substitution in the constant region.

[0132] In a further embodiment, an anti-PD-Ll antibody is provided, which comprises heavy and light chain variable region sequences, wherein:

[0133] (a) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively, or

[0134] (b) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively.

[0135] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0136] In another aspect, the heavy chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a further aspect, the framework sequences are derived from human consensus framework sequences. In a further aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In a further aspect, the heavy chain framework sequences are VH subgroup III consensus framework. In a further aspect, the one or more heavy chain framework sequences are set forth in SEQ ID NOs: 8, 9, 10, and 11. In a further aspect, the light chain framework sequences are derived from Kabat kappa I, II, II, or IV subgroup sequences. In a further aspect, the light chain framework sequences are VL kappa I consensus framework. In a further aspect, the one or more light chain framework sequences are set forth in SEQ ID NOs: 15, 16, 17, and 18.

[0137] In a further specific aspect, the antibody further comprises a human or murine constant region. In a further aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In a further specific aspect, the human constant region is IgGl. In a further aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In a further aspect, the murine constant region is IgG2A. In a further specific aspect, the antibody has reduced or minimal effector function. In a further specific aspect, the minimal effector function is derived from an "effector-less Fc mutation" or aglycosylation. In a further embodiment, the effector-less Fc mutation is N297A or D265A / N297A substitution in the constant region.

[0138] In yet another embodiment, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain variable region sequences, wherein:

[0139] (a) the heavy chain sequence has at least 85% sequence identity to the following heavy chain sequence:

[0140] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 25), and / or

[0141] (b) the light chain sequence has at least 85% sequence identity to the following light chain sequence:

[0142] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0143] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat Subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH Subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is as set forth in SEQ ID NO: 8, 9, 10, and WGQGTLVTVSS (SEQ ID NO: 27).

[0144] In yet another aspect, the light chain framework sequences are derived from Kabat subgroup I, II, III, or IV sequences. In yet another aspect, the light chain framework sequences are VL subgroup I consensus framework sequences. In yet another aspect, one or more of the light chain framework sequences are as set forth in SEQ ID NOs: 15, 16, 17, and 18.

[0145] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In yet another specific aspect, the human constant region is IgGl. In yet another aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from production in a prokaryotic cell. In yet another specific aspect, the minimal effector function results from an "effector less Fc mutation" or aglycosylation. In yet another embodiment, the effector less Fc mutation is N297A or D265A / N297A substitution in the constant region.

[0146] In yet another aspect, the heavy chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus framework sequences. In yet another aspect, one or more of the heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 29) HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO: 30) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 10) HC-FR4 WGQGTLVTVSS (SEQ ID NO: 27).

[0147] In yet another aspect, the light chain framework sequences are derived from Kabat Kl, Kl, KII or KlV subgroup sequences. In yet another aspect, the light chain framework sequences are VL Kl consensus framework. In yet another aspect, one or more of the light chain framework sequences are as follows:

[0148] LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)

[0149] LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 16)

[0150] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17)

[0151] LC-FR4 FGQGTKVEIK (SEQ ID NO: 28).

[0152] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In yet another specific aspect, the human constant region is IgGl. In yet another aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "effector less Fc mutation" or aglycosylation. In yet another embodiment, the effector less Fc mutation is N297A or D265A / N297A substitution in the constant region.

[0153] In yet another embodiment, an anti-PD-Ll antibody is provided comprising heavy and light chain variable region sequences, wherein:

[0154] (c) the heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), respectively, and / or

[0155] (d) the light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively.

[0156] In one specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0157] In another aspect, the heavy chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more framework sequences interposed between the HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from a human consensus framework sequence. In another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequences are VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences are as set forth in SEQ ID NO: 8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO: 31).

[0158] In yet another aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are as set forth in SEQ ID NO: 15, 16, 17, and 18. In still yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgGl, IgG2, IgG2, IgG3, IgG4. In yet another specific aspect, the human constant region is IgGl. In yet another aspect, the murine constant region is selected from the group consisting of IgGl, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function is derived from an "effector-less Fc mutation" or aglycosylation. In yet another aspect, the effector-less Fc mutation is N297A or D265A / N297A substitution in the constant region.

[0159] In still yet another embodiment, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain variable region sequences, wherein:

[0160] (a) the heavy chain sequence has at least 85% sequence identity to the following heavy chain sequence:

[0161] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO: 26), or

[0162] (b) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence of:

[0163] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 4).

[0164] In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain variable region sequences, wherein the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26. In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain variable region sequences, wherein the light chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4, and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 26. In some embodiments, one, two, three, four or five amino acid residues can be deleted, substituted or modified at the N-terminus of the heavy and / or light chain.

[0165] In still another embodiment, an isolated anti-PD-Ll antibody is provided, comprising heavy and light chain sequences, wherein:

[0166] (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32), and / or

[0167] (b) the light chain sequence has at least 85% sequence identity to the light chain sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0168] In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33. In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32. In some embodiments, an isolated anti-PD-Ll antibody is provided, comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 33 and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0169] In some embodiments, the isolated anti-PD-Ll antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to carbohydrate moieties attached to the side chain of an asparagine residue. The tripeptide sequences asparagine-X- serine and asparagine-X-threonine where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. As such, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylga lactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxy lysine can also be used. Glycosylation sites can be conveniently removed from an antibody by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration can be made by substituting the asparagine, serine or threonine residue in the glycosylation site with another amino acid residue (e.g., glycine, alanine or a conservative substitution).

[0170] In any of the embodiments herein, the isolated anti-PD-Ll antibody can bind to human PD-L1 (e.g., human PD-L1 as set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1) or a variant thereof.

[0171] In still another embodiment, an isolated nucleic acid is provided that encodes any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expression of the nucleic acid that encodes any of the anti-PD-Ll antibodies previously described. In a further particular aspect, the vector is in a host cell suitable for expression of the nucleic acid. In a further particular aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In a further particular aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0172] The antibodies or antigen-binding fragments thereof can be generated using methods known in the art, for example, by a method comprising culturing a host cell containing a nucleic acid that encodes any of the anti-PD-Ll antibodies or antigen-binding fragments previously described in a form suitable for expression, under conditions suitable for the production of such antibodies or fragments, and recovering the antibodies or fragments.

[0173] V. Pharmaceutical Formulations

[0174] Pharmaceutical formulations of B-RAF antagonists (e.g., vemurafenib), MEK antagonists (e.g., cobimetinib), and / or PD1 axis inhibitors (e.g., atezolizumab) as described herein can be prepared in lyophilized or aqueous solution form by co-formulation with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Generally, pharmaceutically acceptable carriers are non-toxic to recipients at the doses and concentrations used, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexanediamine chloride; benzalkonium chloride, benzyl chloride; phenols, butanol, or benzyl alcohol; p-hydroxybenzoic acid hydrocarbon esters, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) multi-component... Peptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming opposite ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polysorbates (e.g., TWEEN). TM ), poloxams (e.g., PLURONICS) TM Or polyethylene glycol (PEG). The active pharmaceutical ingredient may also be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse-drop emulsions. The illustrative pharmaceutically acceptable carriers described herein further comprise interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (… Baxter International, Inc. Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Publications Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glycosaminoglycans such as chondroitinase.

[0175] In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents into the composition. The compounds can be formulated as solid compositions, lyophilized compositions or as aqueous solutions.

[0176] Pharmaceutical formulations comprising an antagonist of B-RAF described herein (e.g. vemurafenib), an antagonist of MEK (e.g. cobimetinib) and / or a PD1 axis inhibitor (e.g. atezolizumab) can be formulated, dosed, and administered in a form considered

[0177] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an antagonist of B-RAF described herein (e.g. vemurafenib), an antagonist of MEK (e.g. cobimetinib) and / or a PD1 axis inhibitor (e.g. atezolizumab) which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic-glycolic acid copolymers, injectable microspheres of LUPRON®-Leuprolide acetate (LUPRON DEPOT® ) and poly-D-(-)-3-hydroxybutyric acid.

[0178] Formulations of the antagonists of B-RAF (e.g., vemurafenib) or the antagonists of MEK (e.g., cobimetinib) described herein can be suitable for oral administration, e.g., can be prepared as discrete units such as pills, capsules, cachets or tablets each containing a predetermined amount of the antagonist of B-RAF (e.g., vemurafenib) or the antagonist of MEK (e.g., cobimetinib). For the B-RAF antagonist vemurafenib, the formulation can comprise a tablet core comprising one or more hypromellose acetate succinate, sodium croscarmellose, colloidal silicon dioxide, magnesium stearate, and hydroxypropyl cellulose. In some embodiments, the tablet core can be coated with a coating comprising one or more polyvinyl alcohol, titanium dioxide, polyethylene glycol 3350, talc, and red ferric oxide. In some embodiments, the B-RAF antagonist vemurafenib can be formulated as a 240 mg tablet. Vemurafenib has the chemical name propane-1 -sulfonic acid {3-[5-(4-chlorophenyl)-1 H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide. For the antagonists of MEK (e.g., cobimetinib), the formulation can comprise a tablet core comprising one or more microcrystalline cellulose, lactose monohydrate, sodium croscarmellose, magnesium stearate. In some embodiments, the tablet core can be coated with a coating comprising one or more polyvinyl alcohol, titanium dioxide, polyethylene. In some embodiments, the MEK antagonist cobimetinib can be formulated as a 20 mg tablet. In some embodiments, the 20 mg tablet contains 22 mg of cobimetinib fumarate, which corresponds to 20 mg of cobimetinib free base. Formulations of the PD1 axis inhibitors (e.g., atezolizumab) described herein can be suitable for IV administration.

[0179] Formulations can be packaged in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, water, prior to use. Solutions and suspensions for immediate injection can be prepared from sterile powders, granules, and tablets of the type previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above described, or an appropriate fraction thereof, in an amount suitable for the subject being treated. Accordingly, in some embodiments, the formulations described herein are unit-dose formulations.

[0180] VI. Articles of Manufacture

[0181] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, bubble- packed bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antagonist of B-RAF (e.g., vemurafenib), an antagonist of MEK (e.g., cobimetinib), and / or a PD1 axis inhibitor (e.g., atezolizumab) as described herein. The label or package insert indicates that the compositions are used for treating the condition of choice. Moreover, the article of manufacture can comprise (a) a first container wherein said composition is contained within said first container and wherein said composition comprises an antagonist of B-RAF (e.g., vemurafenib); and (b) a second container wherein said composition is contained within said second container and wherein said composition comprises a PD1 axis inhibitor (e.g., atezolizumab). Furthermore, the article of manufacture can comprise (a) a first container wherein said composition is contained within said first container and wherein said composition comprises an antagonist of B-RAF (e.g., vemurafenib); (b) a second container wherein said composition is contained within said second container and wherein said composition comprises an antagonist of MEK (e.g., cobimetinib); and (c) a third container wherein said composition is contained within said third container and wherein said composition comprises a PD1 axis inhibitor (e.g., atezolizumab).

[0182] The article of manufacture in this embodiment of the application can further include a package insert indicating that the compositions can be used to treat a particular condition, such as cancer or melanoma, e.g., V600 mutant unresectable or metastatic melanoma. Package inserts can refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosages, administration, contraindications and / or warnings, concerning the use of such therapeutic products. Alternatively or additionally, the article of manufacture can further include a second (or third or fourth) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0183] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that certain changes and modifications can be practiced within the scope of the appended claims. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

[0184] EXAMPLE

[0185] The following are examples of the methods and compositions of the present application. It is to be understood that various other embodiments can be practiced in light of the general description provided herein.

[0186] Example 1

[0187] Materials and Methods

[0188] Patient Inclusion Criteria: Patients must meet the following criteria to be eligible for entry into the study: signed informed consent form, age > 18 years, and histologic or cytologic documentation of metastatic or Stage IIIc unresectable melanoma with a BRAF V600 mutation (of any missense type) as assessed by a BRAF V600 mutation test performed at a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory or equivalent. The primary tumor can be of cutaneous, mucosal, or acral location but not of uveal origin. Patients with unknown primary tumor can be eligible if uveal melanoma can be ruled out and at the discretion of the medical monitor. ECOG performance status of 0 or 1, adequate hematologic and end-organ function, defined by the following laboratory results obtained within 14 days prior to Day 1: neutrophils (ANC > 1500 cells / μL), WBC count > 2500 cells / μL and < 15,000 cells / μL, lymphocyte count > 500 cells / μL (or within the local laboratory normal limits), platelet count > 100,000 cells / μL, hemoglobin > 9.0 g / dL, total bilirubin < 1.5 x ULN (with the following exception: patients with known Gilbert’s disease with serum bilirubin levels < 3 x ULN can be enrolled), AST and ALT < 2.0 x ULN, ALP < 2.5 x ULN (with the following exception: patients with documented hepatic or bone metastases: ALP < 5 x ULN within 14 days prior to Day 1), creatinine clearance > 30 mL / min, INR and aPTT < 1.5 x ULN, and measurable disease per RECIST vl. l.

[0189] Patient Exclusion Criteria: Patients who meet any of the following criteria will be excluded from the study and will not be enrolled: Received prior systemic anticancer therapy (e.g., biologic or other targeted therapy, chemotherapy, investigational anticancer agent, or hormonal therapy) for unresectable, locally advanced, or metastatic melanoma except: IFN therapy in the adjuvant setting that was discontinued for > 28 days prior to Day 1, IL-2 therapy that was discontinued for > 28 days prior to Day 1, vaccine therapy that was discontinued for > 28 days prior to Day 1, herbal therapy intended as an anticancer therapy must be discontinued for > 7 days prior to Day 1, prior immunomodulatory agents, including PD-1 or PD-L1 targeted therapies or CTLA-4 targeted therapies, including ipilimumab, the above exceptions noted in the previous exclusion criteria apply, prior MAPK pathway agents, including MEK kinase inhibitors and BRAF kinase inhibitors, major surgical procedure within 28 days prior to Day 1 or requiring major surgical procedure during the study period, radiation therapy within < 7 days prior to Day 1, unresolved adverse events from prior anticancer therapy that are > Grade 1, except alopecia, current serious, uncontrolled systemic disease (including, but not limited to, clinically significant cardiovascular, pulmonary, or renal disease), except cancer, known clinically significant liver disease, including active viral, alcoholic, or other hepatitis, cirrhosis, fatty liver, and inherited liver disease, active or untreated CNS metastases as determined by computed tomography (CT) or magnetic resonance imaging (MRI) assessment during screening and prior assessment of CNS metastases.Note: Patients with a history of treated asymptomatic CNS metastases are eligible provided they meet all of the following criteria: no metastases within 10 mm of the brainstem, midbrain, pons, medulla, or visual apparatus (optic nerve and chiasm), leptomeningeal disease is also excluded, improved radiography documentation after completion of CNS-directed therapy and no evidence of interim progression between completion of CNS-directed therapy and screening radiographic study, no history of intracranial hemorrhage, no current requirement for dexamethasone as therapy for CNS disease; stable dose of anticonvulsant medication, no stereotactic radiation or whole brain radiation within 28 days prior to Day 1, screening CNS radiographic study > 4 weeks from completion of radiation therapy and > 2 weeks from discontinuation of corticosteroids, patients with active malignancy (except BRAF-mutant melanoma) or prior malignancy within the past 3 years are excluded, patients with resected melanoma, resected BCC, resected cuSCC, resected melanoma in situ, resected cervical carcinoma in situ, resected breast carcinoma in situ, or other malignancies with similar outcomes (dependent upon discussion with medical monitor) are excluded, prior allogeneic bone marrow transplant or prior solid organ transplant, history of autoimmune disease, including but not limited to myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, vascular thrombosis associated with antiphospholipid syndrome, Wegener's granulomatosis, Sjogren's syndrome, Guillain-Barre syndrome, multiple sclerosis, vasculitis, or glomerulonephritis (patients with a history of autoimmune-related hypothyroidism on a stable dose of thyroid replacement hormone can be eligible for this study, and patients with controlled type 1 diabetes mellitus on a stable insulin regimen can be eligible for this study), history of idiopathic pulmonary fibrosis (including pneumonitis), risk of pulmonary toxicity, or evidence of active pneumonia on screening chest CT scan, history of HIV infection, patients with active hepatitis B (defined as having a positive hepatitis B surface antigen [HBsAg] test at screening), active hepatitis C, tuberculosis, etc.

[0190] Study design: Open-label, multicenter, Phase lb study of atezolizumab in combination with two targeted therapies (vemurafenib monotherapy and vemurafenib + cobimetinib combination therapy) in previously untreated patients with BRAFV600 mutation-positive metastatic melanoma. Dose levels of atezolizumab are fixed at 15 or 20 mg / kg q3w either, or fixed doses of 1200 mg q3w or 800 mg q2w either, depending on the arm / armlet. For the targeted therapy, all vemurafenib doses will be PO BID, while all cobimetinib doses will be PO QD on a 21 days on / 7 days off schedule. The schedule can contain a run-in period consisting of targeted therapy with a starting dose of 960 mg vemurafenib with or without 60 mg cobimetinib prior to initiation of combination therapy with atezolizumab. The initial armlet starting dose level of targeted therapy during the combination therapy period with the targeted agent will be 720 mg vemurafenib and 60 mg cobimetinib (based on results from ongoing cobimetinib + atezolizumab GP28363 study).

[0191] Abbreviations: A = atezolizumab; BID = twice daily; C = cobimetinib; MTD = maximum tolerated dose; q2w = every 2 weeks; q3w = every 3 weeks; QD = once daily; V = vemurafenib. Note: all V dosing is BID; all C dosing is QD on a 21 days on / 7 days off schedule; A dosing is 15 mg / kg q3w or 1200 mg q3w with V only and 800 mg q2w with V + C. Note: C and V are given via oral administration while A is administered by IV.

[0192] Arm 1 : V at 720 mg BID + A at 20 mg / kg q3w are initiated concurrently. No run-in period is initially recommended; however, a run-in period was introduced after toxicities were observed in the concurrent V + A initiation in Arm 1 (although no DLTs were observed).

[0193] Arm 2: 56-day run-in, V at 960 mg BID for 49 days, then V at 720 mg BID for 7 days, followed by: V at 720 mg BID + A at 15 mg / kg q3w. The length of the run-in period in Arm 4 (28 days) was selected based on tolerability of the regimens tested in Arm 2 (56-day run-in) and Arm 3 (28-day run-in).

[0194] Arm 3: 28-day run-in, V at 960 mg BID for 21 days, then V at 720 mg BID for 7 days, followed by: V at 720 mg BID + A at 1200 mg q3w.

[0195] Cohort 4: 28-day run-in of V+C (V 21 days at 960 mg BID and 7 days V at 720 mg BID + C at 60 mg QD 21 / 7), followed by: V at 720 mg BID + C at 60 mg QD 21 / 7 + A at 800 mg q2w, 28-day cycle. The length of the run-in period in Cohort 4 (28 days) was selected based on tolerability of the regimens tested in Cohort 2 (56-day run-in) and Cohort 3 (28-day run-in).

[0196] Expansion Cohort A: V+A. Enroll patients into an expansion of the Cohort 3 regimen: 28-day vemurafenib run-in (21 days at 960 mg BID and 7 days at 720 mg BID), followed by a 21-day cycle of treatment including V at 720 mg BID and A at 1200 mg q3w.

[0197] Expansion Cohort B: V+C+A. Enroll patients into an expansion of the Cohort 4 regimen: 28-day V+C run-in (V 21 days at 960 mg BID and 7 days V at 720 mg BID + C at 60 mg QD 21 / 7), followed by: V at 720 mg BID + C at 60 mg QD 21 / 7 and A at 800 mg q2w, 28-day cycle.

[0198] Patients in any of the above cohorts can have dose adjustments based on protocol requirements for adverse events, vemurafenib dose reduction to 480 mg BID and / or cobimetinib reduction to 40 mg QD 21 / 7.

[0199] Results

[0200] Table 1 below provides the baseline characteristics of the patients. This Phase lb dose expansion study demonstrated promising antitumor activity and tolerability of vemurafenib + atezolizumab combination therapy in the treatment of BRAFV600 metastatic melanoma. Treatment resulted in SLD reduction in target lesions in 16 / 16 (100%) patients with assessable tumor response. For all patients, the median duration of response was 20.9 months and the median progression-free survival was 10.9 months.

[0201] Greater tolerability was observed with staggered vemurafenib + atezolizumab starting compared to parallel starting. In Cohort 1, the objective response rate (ORR) of parallel administration of vemurafenib and atezolizumab resulted in 1 / 3 complete responses and 0 / 3 partial responses, with an overall response rate of 33%. In Cohort 2, the ORR of a 56-day run-in of vemurafenib prior to administration of vemurafenib and atezolizumab resulted in 1 / 8 complete responses and 5 / 8 partial responses, with an overall response rate of 75%. In Cohort 3, the ORR of a 28-day run-in of vemurafenib prior to administration of vemurafenib and atezolizumab resulted in 1 / 6 complete responses and 5 / 6 partial responses, with an overall response rate of 100%. The higher ORR in Cohort 3 likely indicates that an ideal run-in schedule of 28 days (included in both expanded cohorts) would be better tolerated overall compared to parallel administration, as shown in Table 2 below.

[0202] Tissue samples were stained for CD8+ T cells and PD-L1 expression. Vemurafenib alone increased CD8+ T cell infiltration in immuno-tumor cells (data not shown). Vemurafenib alone or in combination with atezolizumab either increased PD-L1 expression on tumor-infiltrating immune cells and tumor cells (data not shown).

[0203]

[0204]

[0205] The initial early findings (as of September 2015) in Cohort 4, which followed the administration of vemurafenib and cobimetinib, followed by vemurafenib, cobimetinib and atezolizumab, showed that 8 patients had a total of 12 Grade 3 related AEs and 1 Grade 4 related AE, which were manageable and generally reversible. Six of the 12 Grade 3 related events were in the run-in period (prior to the addition of atezolizumab). Events included rash, photosensitivity, diarrhea, anemia, cellulitis and LFT elevations. Specifically, 3 patients had Grade 3 ALT or AST elevations: 2 patients in the run-in period (resolved with dose adjustments), while 1 patient in Cycle 2, resolved with dose adjustments including vemurafenib dose reduction to 480 mg BID and / or cobimetinib reduction to 40 mg QD 21 / 7. There was 1 patient who discontinued study drug treatment due to G3 AST / ALT / bilirubin and G4 ALT AEs attributed to atezolizumab and vemurafenib. Of the 9 assessable patients treated with vemurafenib and cobimetinib prior to the administration of vemurafenib, cobimetinib and atezolizumab in Cohort 4 as of September 2015, the best response was partial response in 7 / 9 patients, best response was stable disease in 1 / 9, and best response was progressive disease in 1 / 9. A 90% disease control rate was seen with vemurafenib, cobimetinib and atezolizumab in the early data. The study is ongoing and will enroll a total of 20 patients in an expanded cohort to be treated with the triple combination of vemurafenib, cobimetinib and atezolizumab and an additional 10 patients to be treated with vemurafenib and atezolizumab.

[0206] The present disclosure relates to the following embodiments.

[0207] 1. A method of treating cancer in an individual, the method comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor.

[0208] 2. A method of increasing the efficacy of a cancer treatment, the method comprising first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor.

[0209] 3. A method of treating cancer in an individual, wherein the cancer treatment comprises first administering to the individual an effective amount of a B-RAF inhibitor and second administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor, wherein the cancer treatment has increased efficacy compared to administering to the individual an effective amount of the B-RAF inhibitor and an effective amount of the immune checkpoint inhibitor alone.

[0210] 4. The method of any one of items 1-3, wherein the B-RAF inhibitor is propane-1- sulfonic acid {3-[5-(4-chlorophenyl)-1 H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4- difluoro-phenyl}-amide or a pharmaceutically acceptable salt thereof.

[0211] 5. The method of item 4, wherein the B-RAF inhibitor is Vemurafenib.

[0212] 6. The method of item 5, wherein the first administration and the second administration of the B-RAF inhibitor is at a dose of about 960 mg twice a day, about 720 mg twice a day, and / or about 480 mg twice a day.

[0213] 7. The method of item 6, wherein the first administration of the B-RAF inhibitor is at a greater dose than the second administration of the B-RAF inhibitor.

[0214] 8. The method of item 6, wherein the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor and the first dose is greater than the second dose.

[0215] 9. The method of any one of items 1-8, wherein the first administration of the B-RAF inhibitor is about 28 days or about 56 days.

[0216] 10. The method of item 9, wherein the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor, the first dose is greater than the second dose, and the first dose is administered for 21 days and the second dose is administered for 7 days.

[0217] 11. The method of any one of items 1-10, wherein the B-RAF inhibitor is administered orally.

[0218] 12. The method of any one of items 1-11, wherein the immune checkpoint inhibitor is a PD-1 axis binding antagonist.

[0219] 13. The method of item 12, wherein the PD-1 axis binding antagonist is an anti-PD-Ll antibody.

[0220] 14. The method of item 13, wherein the anti-PD-Ll antibody is Atezolizumab.

[0221] 15. The method of item 14, wherein Atezolizumab is administered at a dose of about 15 mg / kg q3w, about 20 mg / kg q3w, about 800 mg q2w, or about 1200 mg q3w.

[0222] 16. The method of any one of items 1-15, wherein the immune checkpoint inhibitor is administered intravenously.

[0223] 17. The method of any one of claims 1-16, wherein the first administration and the second administration further comprise an effective amount of a MEK inhibitor.

[0224] 18. The method of claim 17, wherein the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4- iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane or a pharmaceutically acceptable salt thereof.

[0225] 19. The method of claim 18, wherein the MEK inhibitor is (S)-[3,4-difluoro-2-(2-fluoro-4- iodophenylamino)phenyl][3-hydroxy-3-(piperidin-2-yl)azetidin-l-yl]methane, hemifumarate.

[0226] 20. The method of claim 18, wherein the MEK inhibitor is Cobimetinib.

[0227] 21. The method of claim 19, wherein the first administration and the second administration of the MEK inhibitor is at a dose of about 60 mg per day on a 21 days on / 7 days off schedule or about 40 mg per day on a 21 days on / 7 days off schedule.

[0228] 22. The method of any one of claims 17-20, wherein the first administration of the MEK inhibitor is about a 28 day schedule.

[0229] 23. The method of any one of claims 17-21, wherein the MEK inhibitor is administered orally.

[0230] 24. The method of any one of claims 1-23, wherein the cancer is melanoma.

[0231] 25. The method of claim 24, wherein the melanoma is unresectable or metastatic melanoma.

[0232] 26. The method of any one of claims 24-25, wherein the melanoma is a B-RAF V600 mutant melanoma.

[0233] 27. The method of claim 26, wherein the B-RAF V600E mutant melanoma or B-RAF V600K mutant melanoma.

[0234] 28. A method of treating melanoma in an individual, comprising first administering to the individual vemurafenib and cobimetinib on a 28-day schedule, wherein vemurafenib is administered at a dose of 960 mg twice a day for 21 days on the 28-day schedule, followed by 720 mg twice a day for 7 days on the 28-day schedule and cobimetinib is administered at a dose of 60 mg daily for 21 days off for 7 days on the 28-day schedule and second administering to the individual vemurafenib, cobimetinib, and atezolizumab, wherein vemurafenib is administered at a dose of 720 mg twice a day, cobimetinib is administered at a dose of 60 mg daily for 21 days off for 7 days, and atezolizumab is administered at a dose of 800 mg q2w.

[0235] 29. The method of item 28, wherein vemurafenib is administered orally as a tablet.

[0236] 30. The method of any one of items 28-29, wherein the cobimetinib is administered orally as a tablet.

[0237] 31. The method of any one of items 28-30, wherein the atezolizumab is administered intravenously.

[0238] 32. The method of any one of items 28-31, wherein the cancer is melanoma.

[0239] 33. The method of item 32, wherein the melanoma is unresectable or metastatic melanoma.

[0240] 34. The method of any one of items 32-33, wherein the melanoma is a B-RAF V600 mutant melanoma.

[0241] 35. The method of item 34, wherein the B-RAF V600 mutant melanoma is a B-RAF V600E mutant melanoma or a B-RAF V600K mutant melanoma.

Claims

1. A method for treating cancer in an individual, the method comprising first administering an effective amount of a B-RAF inhibitor to the individual and second administering an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor to the individual.

2. A method for improving the efficacy of cancer treatment, the method comprising first administering an effective amount of a B-RAF inhibitor to the individual and second administering an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor to the individual.

3. A method of treating cancer in an individual, wherein the cancer treatment comprises first administering an effective amount of a B-RAF inhibitor to the individual and second administering an effective amount of the B-RAF inhibitor and an effective amount of an immune checkpoint inhibitor to the individual, wherein the cancer treatment has improved efficacy compared to administering an effective amount of the B-RAF inhibitor and an effective amount of the immune checkpoint inhibitor to the individual alone.

4. The method of any one of claims 1-3, wherein the B-RAF inhibitor is propane-1-sulfonic acid {3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide or a pharmaceutically acceptable salt thereof.

5. The method of claim 4, wherein the B-RAF inhibitor is vemurafenib.

6. The method of claim 5, wherein the first and second administrations of the B-RAF inhibitor are at doses of about 960 mg twice daily, about 720 mg twice daily, and / or about 480 mg twice daily.

7. The method of claim 6, wherein the first administration of the B-RAF inhibitor is at a larger dose than the second administration of the B-RAF inhibitor.

8. The method of claim 6, wherein the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor, and the first dose is greater than the second dose.

9. The method of any one of claims 1-8, wherein the first administration of the B-RAF inhibitor is about 28 days or about 56 days.

10. The method of claim 9, wherein the first administration of the B-RAF inhibitor comprises a first dose and a second dose of the B-RAF inhibitor, the first dose being greater than the second dose, and the first dose being administered for 21 days and the second dose being administered for 7 days.

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