HK40007829A - Combination therapy with a mek inhibitor, a pd-1 axis inhibitor, and a vegf inhibitor - Google Patents

Description

Combination therapy of MEK inhibitors, PD-1 axis inhibitors, and VEGF inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial No. 62/374437 filed on 12.8.2016, hereby incorporated by reference in its entirety.

Technical Field

The field of the invention generally relates to cancer therapy with a combination of a MEK inhibitor, a PD-1 axis inhibitor, and a VEGF inhibitor.

Background

Gastrointestinal tumors are a common cause of cancer-related death worldwide. Colorectal cancer (CRC) is the third and second most frequently diagnosed cancer in men and women, respectively, and the leading cause of the fourth and third cancer deaths in men and women, respectively (Torre LA, Bray F, Siegel RL, et al, Global cancer statistics,2012, CACancer J clin.2015; 65: 87-108). Among patients diagnosed with CRC annually in the United states, about 40% are diagnosed with early disease, about 40% are diagnosed with regional disease, and about 20% are diagnosed with distant metastasis, with five-year survival rates of 90%, 70%, and 13%, respectively, with the majority of patients dying from Metastatic disease (Alberts SR and Wagman LD., chemotherapeutical for Metastatic cancers 2008, 13:1063-73, Kennecke H, YuJ, Gill S, et al, Effect of M1a and M1b Category in metabolic Cancer, oncologue 2014, 19:720-6, and American Cancer Society, Estimated Number of New and Cancer Cases, US,2013[ resources on Seal 20111; Internet 12 months).

Systemic cytotoxic chemotherapy is the therapeutic mainstay for the vast majority of metastatic crc (mcrc) patients and has a median overall survival of only around 30 months. anti-Vascular Endothelial Growth Factor (VEGF) therapies, such as Bevacizumab, ramucirumab, and ziv-aflibercept, and anti-epidermal growth factor receptor monoclonal antibodies (mAbs), such as cetuximab and panitumumab, may be used in combination with chemotherapy in First, second, and third line therapies, although the therapeutic combination varies by region (Petrelli F, Coinu A, Ghirardi M, et al, Efficacy of Oxaliplatin-basedhehermaph + Bevacizumab as First-line therapy for Advanced Coloralcarcell. am J Clin Oncol 2015; 38: 227-.

Despite recent advances, mCRC remains an incurable disease. The disease control period for patients with mCRC is continuously shortened in each next line of therapy until the disease becomes refractory and the patient yields to cancer. Bone marrow suppression, gastrointestinal toxicity, fatigue/debilitation, peripheral neurotoxicity, and skin toxicity (including hand and foot syndromes) are adverse events often observed in patients with mCRC undergoing treatment. These adverse events can significantly affect the quality of life of patients.

Thus, there is a need for improved therapies for CRC and metastatic CRC.

Summary of The Invention

The present disclosure provides a method of treating a subject having colorectal cancer. The method comprises administering to the subject a therapy comprising (i) a therapeutically effective amount of a MEK inhibitor, (ii) a therapeutically effective amount of a PD-1 axis inhibitor, and (iii) a therapeutically effective amount of a VEGF inhibitor.

The present disclosure further provides a kit for treating colorectal cancer in a human subject. The kit comprises a MEK inhibitor, a PD-1 axis inhibitor, a VEGF inhibitor, and a package insert comprising instructions for using a therapeutically effective amount of the MEK inhibitor, a therapeutically effective amount of the PD-1 axis inhibitor, and a therapeutically effective amount of the VEGF inhibitor to treat the subject.

The order of administration of the MEK inhibitor, the PD-1 axis inhibitor, and the VEGF inhibitor may vary. In some aspects, when the PD-1 axis inhibitor and the VEGF inhibitor are administered on the same day, the PD-1 axis inhibitor is administered at least 30 minutes prior to the VEGF inhibitor.

In some aspects of the invention, the MEK inhibitor is cobicisinib, or a pharmaceutically acceptable salt thereof; the PD-1 axis inhibitor is a PD-L1 inhibitor, and more particularly, astuzumab; and/or, the VEGF inhibitor is bevacizumab.

The disclosure further provides a cancer therapy pharmaceutical combination comprising (i) a MEK inhibitor in a dose of about 20mg to about 100mg, about 40mg to about 80mg, or about 80 mg; (ii) a PD-1 axis inhibitor in a dose of about 400mg to about 1200mg, about 600mg to about 1000mg, about 700mg to about 900mg, or about 840 mg; and (iii) a VEGF inhibitor in a dose from about 5mg/kg to about 15mg/kg, from about 5mg/kg to about 10mg/kg, about 5mg/kg, about 10mg/kg or about 15 mg/kg. In some particular aspects of the invention, the MEK inhibitor is cobicisinib, or a pharmaceutically acceptable salt thereof; the PD-1 axis inhibitor is a PD-L1 inhibitor, and more particularly, astuzumab; and/or, the VEGF inhibitor is bevacizumab.

Brief Description of Drawings

FIG. 1 shows a study protocol for run-in and extended cohort for a clinical trial.

Figure 2 shows a study protocol of a biopsy cohort for a clinical trial.

Detailed Description

The present invention relates to the treatment of cancer with a combination of a MEK inhibitor, a PD-1 axis inhibitor and a VEGF inhibitor, more particularly a combination of a MEK inhibitor, a PD-L1 inhibitor and a VEGF inhibitor, and still more particularly a combination of cobicistinib or a pharmaceutically acceptable salt thereof, atelizumab and bevacizumab. In some aspects, the cancer is CRC, and more particularly metastatic CRC (mcrc). It is believed that simultaneous inhibition of MEK, VEGF signaling, and the PD-1 axis, such as PD-L1, whereby targeting tumors in a multifactorial manner enhances the efficacy of immunotherapy components in patients with CRC. In some aspects, combination therapy is believed to enhance the efficacy of the immunotherapy component in patients with CRC who have received at least one prior line therapy containing fluoropyrimidine and oxaliplatin or irinotecan.

It is further believed that the addition of bevacizumab to cobicisinib and atuzumab provides a positive treatment with reduced toxicity to patients with CRC compared to chemotherapy-based regimens.

Since the mechanism of action of the combination therapy of the present invention is different from traditional chemotherapy regimens, it is further believed that the activity of further standard therapies is not significantly affected. This would allow patients with progressive disease to continue treatment.

Definition of

As used herein, "colorectal cancer" (CRC) refers to colon cancer, rectal cancer, and colorectal cancer (i.e., cancers of both the colon and rectal regions).

As used herein, the term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells.

As used herein, the terms "patient" and "subject" refer to an animal, such as a mammal, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In certain aspects, the patient or subject is a human.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated/treated individual or cell during the course of clinical pathology. Desirable effects of treatment/management include reducing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including but not limited to reducing the proliferation of cancerous cells (or destroying cancerous cells), reducing/alleviating symptoms resulting from the disease, improving the quality of life of those afflicted with the disease, reducing the dosage of other drugs required to treat the disease, and/or prolonging survival of the individual.

As used herein, the phrase "therapeutically effective amount" refers to an amount of one or more pharmaceutical compounds that (i) treats or prevents a particular disease, condition, or disorder described herein, (ii) alleviates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with cancer. To the extent that the drug can prevent the growth of cancer cells and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing Overall Response Rate (ORR). A therapeutically effective amount herein may vary with factors such as the disease state, age, sex, and weight of the patient, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount where the therapeutically beneficial effect exceeds the toxic or detrimental effect of the treatment. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during the onset of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing/alleviating one or more symptoms resulting from the disease, improving the quality of life of those suffering from the disease, reducing the dosage of another drug needed to treat the disease, enhancing the effect of another drug (such as via targeting), delaying disease progression, and/or prolonging survival. In the case of cancer or tumors, a therapeutically effective amount of the drug is effective in reducing the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent or expect to stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and expect to stop) tumor metastasis; inhibit tumor growth to some extent; and/or may have an effect in alleviating to some extent one or more symptoms associated with the condition. A therapeutically effective amount may be administered in one or more administrations. For the purposes of the present invention, a therapeutically effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in the clinical setting, a therapeutically effective amount of a drug, compound, or pharmaceutical composition may be achieved in combination with or without another drug, compound, or pharmaceutical composition. As such, a "therapeutically effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in a therapeutically effective amount if, in combination with one or more other agents, the desired result or results are achieved.

As used herein, "in combination with … …" refers to the administration of one therapeutic modality in addition to another. Thus, "in combination with … …" refers to the administration of one therapeutic modality before, during, or after the administration of another therapeutic modality to an individual.

As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form effective to allow for the biological activity of the active ingredient, and that is free of additional ingredients that are unacceptably toxic to a subject that will receive administration of the formulation. Such formulations are sterile. "pharmaceutically acceptable" excipients (vehicles, additives) are those which are reasonably administered to a subject mammal to provide an effective dose of the active ingredient employed.

As used herein, "immunohistochemistry" (IHC) refers to the process of detecting an antigen (e.g., a protein) in cells of a tissue section by utilizing the principle that an antibody specifically binds to an antigen in a biological tissue. Immunohistochemical staining can be used to diagnose abnormal cells, such as those found in cancerous tumors. Specific molecular markers are characteristic of specific cellular events such as proliferation or cell death (apoptosis). IHC can also be used to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Expression is detected using antibodies or antisera, such as polyclonal antisera and monoclonal antibodies, specific for each marker. The antibody may be detected by directly labeling the antibody itself, for example with a radiolabel, a fluorescent label, a hapten label such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. In one visualization method, the antibody is conjugated to an enzyme capable of catalyzing a chromogenic reaction, such as peroxidase (see immunoperoxidase staining). In another visualization method, the antibody may also be labeled with a fluorophore tag, such as fluorescein or rhodamine (see immunofluorescence). Alternatively, unlabeled primary antibodies are used in combination with labeled secondary antibodies, the latter comprising antisera, polyclonal antisera or monoclonal antibodies specific for the primary antibody. Immunohistochemical protocols and kits are well known in the art and are commercially available.

AS used herein, "anti-therapeutic antibody assessment" (ATA) refers to an immunogenicity assessment that uses a risk-based immunogenicity strategy to characterize ATA responses, such AS Rosenberg AS, Worobec AS., a risk-based adaptive immunity conjugates of therapeutic protein products, BioPharm int 2004; 17: 34-42; and Koren E, Smith HW, Shores E, et al, Recommendations on rise-basedsystems for detection and characterization of antibodies against microbiological analysis technology products, J Immuno Methods 2008; 333: 1-9). Each reference is incorporated herein by reference in its entirety.

As used herein, C_{Maximum of}Refers to the maximum plasma concentration.

As used herein, C_{Minimum size}Refers to the minimum plasma concentration.

As used herein, "area under the concentration curve" (AUC) refers to the area under the fitted plasma concentration versus time curve. AUC_0-∞Refers to the area under the baseline-infinity of the curve. AUC_0-TIs the total exposure.

As used herein, "response evaluation criteria in solid tumors" (RECIST) v1.1 refers to Eisenhauer, EA, et, New response evaluation criteria in solid tumors: Revised RECISTGUIDINE (version 1.1), Eur J Cancer 2009:45: 228-; topalian SL, et al, Safety, activity, and animal corrlates of anti-PD-L1anti in cancer, N EnglJ Med 2012:366: 2443-54; and Wolchok JD, et al, Guidelines for the evaluation of immunological activity in soluble tumors, animal-related stress criteria, ClinCan Res 2009; standard specifications for tumor response detailed in 15: 7412-20. Each reference is incorporated herein by reference in its entirety.

As used herein, "immunologically improved RECIST" (irRC) refers to a protein-related protein response obtained from RECIST v1.1 specification (Eisenhauer, EA, et al, (2009)) and Nishino M, et al, Optimizing the protein-related protein response, do reducing the number of versions of the protein response with the protein encoded with the protein complex with the antigen, J immunothera Can 2014; 2: 17; and Nishinom, Giobbie-Hurder A, Gargano M et al, development a common language for a tomorresponse to immunotherapy, animal-related stress criteria using unidimensional measures, Clin Can Res 2013; criteria derived from the immune response criteria detailed in 19: 3936-43. Each reference is incorporated herein by reference in its entirety. The RECIST v1.1 specification applies unless otherwise specified.

As used herein, "inhibit" refers to a decrease in the activity of the enzyme of interest as compared to the activity of the enzyme in the absence of the inhibitor. In some aspects, the term "inhibit" means a decrease in activity of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In other aspects, inhibition means a decrease in activity of about 5% to about 25%, about 25% to about 50%, about 50% to about 75%, or about 75% to 100%. In some aspects, inhibition means a decrease in activity of about 95% to 100%, e.g., a decrease in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such reduction can be measured using a variety of techniques as would be recognized by one skilled in the art.

As used herein, "progression free survival" (PFS) refers to the time from treatment of a disease to the first occurrence of disease progression or recurrence as determined by a investigator using recistv 1.1.

As used herein, "overall survival" (OS) refers to the time of randomization to death of any cause.

As used herein, "partial response" (PR) refers to a reduction in the sum of the diameters of the target lesions of at least 30%, referenced to the sum of the baseline diameters.

As used herein, "delaying disease progression" means delaying, hindering, slowing, delaying, stabilizing, and/or delaying the onset of a disease (such as cancer). This delay may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may essentially encompass prevention, as the individual does not develop disease. For example, the occurrence of late stage cancer, such as metastasis, may be delayed.

As used herein, "sustained response" refers to a sustained effect on reducing tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller than the size at the beginning of the administration phase. In some aspects, the sustained response has a duration at least the same as the duration of treatment, at least 1.5 times, 2 times, 2.5 times, or 3 times the length of the duration of treatment.

As used herein, "reducing or inhibiting cancer recurrence" means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and/or cancer progression includes, but is not limited to, cancer metastasis.

As used herein, "complete response" (CR) refers to the disappearance of all target lesions. The minor axis of any pathological lymph node (whether targeted or non-targeted) is shortened to less than 10 mm.

As used herein, "progressive disease" (PD) means an increase in the sum of the diameters of the target lesions of at least 20%, including baseline and absolute increase of at least 5mm, with the minimum sum (nadir) at the time of study as a reference. The appearance of one or more new lesions is also considered to be progression.

As used herein, "stable disease" (SD) refers to neither a contraction enough to comply with PR nor an enlargement enough to comply with PD, as a minimum and reference at the time of study.

As used herein, "overall response rate" (ORR) refers to the ratio of PR or CR that occurs after randomization and is confirmed after ≧ 28 days as determined by the investigator using RECIST v 1.1.

As used herein, "unconfirmed overall response rate" (ORR _ uc) refers to the ratio of PR or CR occurring after randomization, as determined by investigators using RECIST v1.1, where no confirmation is required.

As used herein, "duration of response" (DOR) refers to the time from the first occurrence of a recorded target response to the time of recurrence as determined by investigators using RECIST v1.1 or death of any cause during the study, whichever occurs first.

As used herein, "national cancer institute adverse event general term standard" (NCI CTCAE) refers to the adverse effect general term standard published by the U.S. department of health and human services, national institutes of health, 2009, on day 5-28, version 4.0 (v4.03:2010, on day 6-14) (incorporated by reference in its entirety).

As used herein, "general cancer Therapy Functional Assessment" (FACT-G) refers to a validated and reliable 27-item questionnaire consisting of four subscales that measure physical (7), social/home (7), emotional (6) and Functional health (7), and is deemed suitable for use with patients with any form of cancer (cell DF, Tuky DS, Gray G, Sarafian B, Linn E, Bonomi AE et al, The Functional Assessment of cancer Therapy scale: determination and evaluation of The cancer Therapy, Journal of clinical Oncology 1993; 11(3 supply.2): 570-9; and The master, K, Odom, L., Peterman, A., L., L.D. Therapy, Functional Assessment (Website of health, 2) income 1999, each of which refers to The complete version of The text 2: balance of health, 2: balance, 2, see The text, health of health, 2, and version of health, 2, see The text, 2: weight, 2, and version of health of The entire version of The query, 2, see The text for quality of health, 2: supplement, 2, The entire version of The text, 2, see The text of The text for quality of health of The text, 2: supplement, 2, The text of The text for references (see The text of Research). The patient evaluated the trueness to each statement on them in the previous 7 days on a 5-part scale (0, none at all; 1, little at all; 2, some; 3, quite many; 4, very many).

As used herein, the term "MEK inhibitor" refers to a molecule that inhibits MEK, such as the enzyme mitogen-activated protein kinase MEK1 (also known as MAP2K1), or MEK2 (also known as MAP2K 2). MEK inhibitors may be useful in affecting MAPK/ERK pathways that may be overactive in some cancers, such as CRC. MEK Inhibitors have been extensively reviewed (S.price, PutativeAllocatic MEK1and MEK 2Inhibitors, Expert Optin. Ther. patents,2008,18(6): 603; J.I.Trujillo, MEK Inhibitors: a patent review 2008-.

As used herein, the term "PD-1 axis inhibitor" or "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 of restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis inhibitors include PD-1 inhibitors, PD-L1 inhibitors, and PD-L2 inhibitors.

As used herein, the term "PD-1 inhibitor" or "binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1 and PD-L2). In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a particular aspect, the PD-1 inhibitor inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 inhibitors include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 inhibitor reduces negative co-stimulatory signaling (mediated signaling via PD-1) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

As used herein, the term "PD-L1 inhibitor" or "binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 inhibitor inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 inhibitors include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, a PD-L1 inhibitor reduces negative co-stimulatory signaling (mediated signaling via PD-L1) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody.

As used herein, the term "PD-L2 inhibitor" or "binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a particular aspect, the PD-L2 inhibitor inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 inhibitors include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with 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 inhibitor reduces negative co-stimulatory signaling (mediated signaling via PD-L2) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 inhibitor is an immunoadhesin.

As used herein, the term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. The term includes the common requirement that antigen recognition can occur, but that the subsequent immune response is exhausted and/or anergic, which is ineffective in controlling infection or tumor growth. As used herein, the term "dysfunctional" also includes an inability to sense or respond to antigen recognition, in particular, to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing, impaired ability.

As used herein, the term "anergy" refers to an incomplete or inadequate signal resulting from delivery via a T cell receptor (e.g., intracellular Ca in the absence of ras activation⁺²Elevation of) a non-responsive state to an antigenic stimulus. T cell anergy can also occur following stimulation with antigen in the absence of co-stimulation, causing the cells to become insensitive to subsequent antigen-induced activation even in the context of co-stimulation. The unresponsive state can often be overcome by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or gain effector function.

As used herein, the term "depletion" refers to the depletion of T cells as a state of T cell dysfunction resulting from sustained TCR signaling that occurs during many chronic infections and cancers. It differs from anergy in that it does not occur via incomplete or defective signaling, but rather as a result of sustained signaling. It is defined by poor effector function, sustained inhibitory receptor expression and a transcriptional state that is distinct from the transcriptional state of functional effector or memory T cells. Depletion prevents optimal control of infection and tumors. Depletion may result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and intracellular negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

By "enhancing T cell function" is meant inducing, causing or stimulating T cells to have sustained or amplified biological function, or renewing or reactivating exhausted or inactive T cells. Examples of enhanced T cell function include elevated levels of CD8 relative to such levels prior to intervention⁺Gamma-interferon secretion by T cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. One of ordinary skill in the art knows the manner in which this enhancement is measured.

A "T cell dysfunctional disorder" is a T cell disorder or condition characterized by decreased responsiveness to antigenic stimuli. In a particular embodiment, the T cell dysfunctional disorder is a disorder that is specifically associated with inappropriately elevated signaling via PD-1. In another embodiment, a T cell dysfunctional disorder is one in which the T cell is anergic or has reduced ability to secrete cytokines, proliferate, or perform cytolytic activity. In a particular aspect, the reduced responsiveness results in ineffective control of the pathogen or tumor expressing the immunogen. Examples of T cell dysfunctional disorders characterized by T cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.

As used herein, "VEGF" refers to a 165 amino acid human vascular endothelial growth factor and related 121, 189, and 206 amino acid human vascular endothelial growth factors, as described by Leung et al (1989) Science246:1306 and Houck et al (1991) mol. The term "VEGF" also refers to VEGF from non-human species such as mouse, rat, or primate. Sometimes, VEGF from a particular species is represented as follows, hVEGF for human VEGF, mVEGF for murine VEGF, and so on. The term "VEGF" is also used to refer to truncated forms of the polypeptide comprising 165 amino acids at amino acids 8 to 109 or 1 to 109 of human vascular endothelial growth factor. In the present application, it is possible to use, for example, "VEGF (8-109)", "VEGF (1-109)" or "VEGF₁₆₅"to identify any such form of VEGF. The amino acid positions of a "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid 17 (methionine) in truncated native VEGF is also amino acid 17 (methionine) in native VEGF. The truncated native VEGF has comparable binding affinity to the KDR and Flt-1 receptors as native VEGF.

As used herein, a "VEGF antagonist" refers to a molecule that is capable of neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with VEGF activity (including its binding to one or more VEGF receptors). VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof that specifically bind VEGF thereby sequestering it from binding to one or more receptorsReceptor molecules and derivatives, anti-VEGF receptor antibodies and VEGF receptor antagonists such as small molecule inhibitors of VEGFR tyrosine kinase, and fusion proteins (e.g., VEGF-trap (Regeneron)), VEGF₁₂₁-gelonin (Peregrine). VEGF antagonists also include antagonistic variants of VEGF, antisense molecules directed to VEGF, RNA aptamers specific for VEGF, and ribozymes directed to VEGF or VEGF receptors. Antagonists of VEGF act by interfering with VEGF binding to cell receptors, by disabling or killing cells that have been activated by VEGF, or by interfering with vascular endothelial cell activation following VEGF binding to cell receptors. All such intervention points for VEGF antagonists should be considered equivalent for the purposes of this invention. Preferred VEGF antagonists are anti-VEGF antagonist antibodies that are capable of inhibiting one or more biological activities of VEGF, such as its mitogenic, angiogenic or vascular permeability activity. anti-VEGF antagonistic antibodies include, but are not limited to, the antibodies a4.6.1, rhuMab VEGF (bevacizumab), Y0317 (ranibizumab), G6, B20,2C3, and e.g., WO98/45331, US2003/0190317, U.S. patent nos. 6,582,959 and 6,703,020; WO 98/45332; WO 96/30046; WO 94/10202; WO 2005/044853; EP 0666868B 1; and others described in Popkov et al, Journal of Immunological Methods,288:149-164 (2004). Each reference is incorporated herein by reference in its entirety. More preferably, the anti-VEGF antagonistic antibody of the invention is ranibizumab, a humanized, affinity matured anti-human VEGF antibody Fab fragment having the light and heavy chain variable domain sequences of Y0317 as described in WO98/45331 and Chen et al, J Mol Biol,293:865-881(1999), each of which is incorporated herein by reference in its entirety.

As used herein, the term "package insert" refers to instructions for use, typically included in commercial packaging for therapeutic products, that contain information regarding the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

The term "pharmaceutically acceptable salt" refers to salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition is chemically and/or toxicologically compatible with the other components comprising the formulation and/or the mammal being treated therewith. Acid addition salts are those formed with inorganic acids (such as hydrochloric, hydrobromic, sulfuric, nitric, carbonic, phosphoric) and organic acids (selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxyl, and sulfonic organic acids such as formic, acetic, propionic, glycolic/glycolic, gluconic, lactic, pyruvic, oxalic, malic, maleic/maleic, malonic, succinic, fumaric/fumaric, tartaric, citric, aspartic, ascorbic, glutamic, anthranilic, benzoic, cinnamic, mandelic, pamoic, phenylacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, and salicylic). Base addition salts are those formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, diatrizoate, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins).

The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment refer to substances that interfere with the research, diagnostic, or therapeutic uses of antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to homogeneity (1) by more than 95% antibody weight, and in some embodiments more than 99% weight, as determined by, for example, the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using, for example, a rotor cup sequencer, or (3) by SDS-PAGE under reducing or non-reducing conditions using, for example, coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies will typically be prepared by at least one purification step.

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end_H) Followed by a number of constant domains. Each light chain has a variable domain (V) at one end_L) And a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin (the variable domains that contain the antigen binding site). Constant domain heavy chain-containing C_H1,C_H2 and C_H3 domains (collectively CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". The variable domain of the light chain may be referred to as "V_L". These domains are generally the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen, however, the variability is not evenly distributed throughout the variable domains of the antibodies it focuses on three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains the more highly conserved portions of the variable domains are called Framework Regions (FRs) the variable domains of the native heavy and light chains each comprise four FR regions, mostly adopt an β -sheet conformation, are linked by three HVRs (which form loops connecting β -sheet structures and in some cases forming part of β -sheet structures), the HVRs in each chain are held together in close proximity by the FR regions and together with HVRs from the other chain contribute to the formation of the antigen binding site of the antibody (see kance, Sequences of Proteins of immunology international, fire Edition, nature of Health, the fact that they exhibit direct binding to the antigen, but are not involved in the antibody binding function, such as the antibody binding site.

Based on the amino acid sequences of their constant domains, the "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinctly different types, known as kappa ("κ") and lambda ("λ").

As used herein, the term IgG "isotype" or "subclass" means any immunoglobulin subclass defined by the chemical and antigenic characteristics of their constant regions.

Antibodies (immunoglobulins) can be assigned to different classes according to the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁,IgG₂,IgG₃,IgG₄,IgA₁And IgA₂The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described, for example, in Abbas et al, Cellular and mol.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, rather than an antibody fragment as defined below. The term particularly refers to antibodies whose heavy chains comprise an Fc region.

For purposes herein, "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In the single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a similar "dimer" structure as in the two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six HVRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

The Fab fragment comprises the heavy and light chain variable domains, and further comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry a free thiol group. F (ab')₂Antibody fragments were originally generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

For reviews on scFv see for example Pluckth ü n, in The Pharmacology of monoclonal antibodies, Vol.113, eds Rosenburg and Moore, Springer-Verlag, New York,1994, p.269-315.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) linked in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc.Natl.Acad.Sci.USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. As such, the modifier "monoclonal" indicates that the antibody is not characteristic of a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the target binding sequence selected may be further altered, for example to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are also advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, Monoclonal Antibodies to be used in accordance with the present invention may be generated by a variety of techniques, including, for example, the Hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al, Hybridoma 14(3):253-260 (1995); Harlow et al, Antibodies: ALaboratom Manual, (Cold Spring Harbor Laboratory Press,2nd ed.1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell hybrids 563-, methods 284(1-2):119-132(2004)), and techniques for producing human or human-like antibodies in animals having part or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; jakobovits et al, Proc.Natl.Acad.Sci.USA 90:2551 (1993); jakobovits et al, Nature 362:255-258 (1993); bruggemann et al, Yeast in Immuno.7:33 (1993); U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; and 5,661,016; marks et al, Bio/Technology 10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368: 812-; fishwild et al, Nature Biotechnol.14: 845-; neuberger, Nature Biotechnol.14:826 (1996); and Lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995)).

Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies includeAn antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing macaques with an antigen of interest.

A "humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin. In one embodiment, a humanized antibody refers to a human immunoglobulin (recipient antibody) in which residues from a recipient HVR are replaced with residues from a non-human species (donor antibody) HVR (such as mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and/or capacity. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. For additional details see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-; and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. Transactions23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.227:381 (1991); Marks et al, J.mol.biol.222:581 (1991)). Cole et al, Monoclonal Antibodies and cancer therapy, Alan R.Liss, p.77 (1985); the methods described in Boerner et al, J.Immunol.147(1):86-95(1991) can also be used for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, curr, opin, pharmacol.5:368-74 (2001). Human antibodies can be made by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomic), modified to produce such antibodies in response to an antigenic challenge, but whose endogenous genome has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XeNOMOUS^TMA technique). See also, e.g., Li et al, Proc. Natl.Acad.Sci.USA 103:3557-3562(2006), for human antibodies generated via human B-cell hybridoma technology.

"species dependent resistanceAn antibody "refers to an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it has for a homolog of the antigen from a second mammalian species. Normally, species-dependent antibodies "specifically bind" to human antigens (e.g., have no more than about 1x 10^-7M, preferably not more than about 1x 10^-8M, and preferably no more than about 1x 10^-9M, but has a binding affinity (Kd) for the antigen from the second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than its binding affinity for a human antigen. The species-dependent antibody may be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

As used herein, the term "hypervariable region", "HVR", or "HV" refers to the region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops. Typically, antibodies comprise six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 show the greatest diversity of six HVRs, and H3 is specifically thought to play a unique role in conferring precise specificity to antibodies. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, in: Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, NJ, 2003). Indeed, naturally occurring camelid (camelid) antibodies, consisting of only heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

Many HVR statements are used and encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the positioning of structural loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops, and results in the use of Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on analysis of the available complex crystal structure. Residues from each of these HVRs are recorded below.

HVRs may include "extended HVRs" from 24-36 or 24-34(L1),46-56 or 50-56(L2) and 89-97 or 89-96(L3) in the VL and 26-35(H1),50-65 or 49-65(H2) and 93-102,94-102, or 95-102(H3) in the VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues refer to those variable domain residues other than the HVR residues defined herein.

The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refers to Kabat et al, supra for the heavy chain variable domain or light chain variable domain numbering system for antibody editing. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or HVR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a,82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat residue numbering for a given antibody can be determined by aligning the sequence of the antibody at the region of homology with a "standard" Kabat numbered sequence.

The Kabat numbering system is generally used when referring to residues in the variable domain (about light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al, Sequences of Immunological interest.5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., Kabat et al, see EU index reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

The expression "linear antibody" refers to the antibody described in Zapata et al (1995) Protein Eng.8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandemly connected Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with a complementary light chain polypeptide. Linear antibodies can be bispecific or monospecific.

As used herein, the terms "bind," "specific binding," or "specific for … …" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds or specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

The term "detecting" includes any means of detection, including direct and indirect detection.

As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, e.g., predictive, diagnostic, and/or prognostic. Biomarkers can serve as indicators of particular subtypes of a disease or disorder (e.g., cancer) characterized by a particular, molecular, pathological, histological, and/or clinical characteristic. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.

Therapeutic agent

The present disclosure uses a combination of a MEK inhibitor, a PD-1 axis inhibitor, and a VEGF inhibitor to treat CRC in a subject. In some aspects, the MEK inhibitor is cobicisinib or a pharmaceutically acceptable salt thereof; the PD-1 axis inhibitor is a PD-L1 inhibitor, and more particularly the PD-L1 inhibitor is atelizumab; and/or, the VEGF inhibitor is bevacizumab. In some other aspects, cobicistinib isThe Abutilizumab isAnd/or bevacizumab is

The presently disclosed compounds may be administered in any suitable manner known in the art. In some aspects, the compound can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, in frame, by implantation, by inhalation, intrathecally, intraventricularly, intratumorally, or intranasally.

It is understood that the appropriate dosage of the active compound will depend upon a variety of factors within the knowledge of the ordinarily skilled practitioner. The dosage of the active compound will vary, for example, depending on the age, body weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination.

It will also be appreciated that the therapeutically effective dose of a compound of the disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, or derivative thereof, for treatment may be increased or decreased over the course of a particular treatment. Dose variation may result from the results of the diagnostic assay and become apparent.

MEK inhibitors

Examples of MEK inhibitors within the scope of the present disclosure include cobitinib, trametinib, binimetinib, selumetinib, pimasetinib, refametinib, PD-0325901, and BI-847325, or a pharmaceutically acceptable salt thereof.

In some particular aspects of the disclosure, the MEK inhibitor is cobicistinib or a pharmaceutically acceptable salt thereof (e.g., a pharmaceutically acceptable salt thereof)) Having the chemical name (S) [3, 4-difluoro-2- (2-fluoro-4-iodophenylamino) phenyl][ 3-hydroxy-3- (piperidin-2-yl) piperidine]Azetidin-1-yl) methanone, and having the structure:

is the fumarate salt of cobicistinib. Cobitinib is described in U.S. patent nos. 7,803,839 and 8,362,002, each of which is incorporated in its entirety by reference. Cobicisinib is a reversible, potent, and highly selective inhibitor of MEK1and MEK2 (central components of RAS/RAF/MEK/erk (mapk)) pathways and has single agent antitumor activity in multiple human cancer models.

Cobinib inhibits proliferation of various human tumor cell lines via inhibition of MEK1and MEK 2. In addition, cobicistinib inhibited ERK phosphorylation and stimulated apoptosis in xenograft tumor models. Cobinib accumulates in tumor xenografts and remains at high concentrations in tumors after plasma concentrations have declined. The activity of cobicistinib in inhibiting phosphorylation of ERK1 correlates more closely with its concentration in tumor tissue than in plasma; in general, there is a better correlation between reduced ERK1 phosphorylation and efficacy in tumor xenograft models. Tumor regression has been observed in several human tumor xenograft models. This regression was dose dependent with up to 100% regression at the highest dose tested. The models studied included CRC, malignant melanoma, breast cancer, and lung cancer.

The Pharmacokinetics (PK) of cobicistinib administered as a single agent has been characterized in phase Ia dose escalation studies MEK4592g after one and more doses in cancer patients following oral administration, which studies included evaluating cobicistinib doses at 60mg per day in patients containing BRAF, NRAS, or KRAS mutations. A total of 6 patients (all of them had melanoma; 6.2%) had confirmed Partial Response (PR),28 patients (28.9%) had Stable Disease (SD), and 40 patients (41.2%) had progressive disease. Of 14 colorectal cancer (CRC) patients, all patients experienced Progressive Disease (PD). In phase III of study MEK4592g, 18 patients were accumulated and the best overall response was assessed for 14 of the 18 patients. 4 patients (22.2%) had SD as their best overall response, and 2 patients (11.1%) had unconfirmed tumor response.

Cobicistinib has a moderate absorption rate (median time to maximum concentration [ t ] of 1 to 3 hours_{Maximum of}]) And a mean terminal half-life (t) of 48.8 hours (in the range of 23.1 to 80 hours)_1/2). Cobitinib binds plasma proteins (95%) in a concentration-independent manner. Cobicisinib exhibited linear pharmacokinetics in a dose range of 0.05mg/kg (approximately 3.5mg/kg for a 70kg adult) to 80mg and absolute bioavailability was determined to be 45.9% (90% CI: 39.74%, 53.06%) in healthy subjects in study MEK4952 g. Cobicistinib pharmacokinetics were unchanged in healthy subjects when administered in the fed state compared to administration in the fasted state. Since food does not alter cobicistinib pharmacokinetics, cobicistinib can be administered with or without food. The proton pump inhibitor rabeprazole (rabeprazole) appears to have minimal impact on cobinib pharmacokinetics, whether administered in the presence or absence of a high fat meal, compared to cobinib alone in the fasted state. Thus, an increase in gastric pH did not alter cobicistinib pharmacokinetics, indicating that it is not sensitive to changes in gastric pH.

Cobicistinib salts, crystal forms, and prodrugs are within the scope of the present disclosure. Cobitinib, a method of preparation, and therapeutic uses are disclosed in international publication nos. WO 2007/044515, WO 2007/044615, WO 2014/027056, and WO 2014/059422, each of which is incorporated herein by reference in its entirety. For example, in some aspects of the disclosure, the MEK inhibitor is crystalline hemifumarate cobitinib polymorph form a.

A dose of a MEK inhibitor (e.g., cobitinib) within the scope of the present disclosure is from about 20mg to about 100mg, from about 40mg to about 80mg, or about 60mg of the MEK inhibitor per day. In particular embodiments, the MEK inhibitor is cobicisinib and is administered at about 60mg, about 40mg or about 20 mg.

The MEK inhibitor is suitably administered once daily. In some aspects, the MEK inhibitor is administered once daily for 21 consecutive days of a 28 day treatment cycle. In some aspects, the MEK inhibitor is administered once daily on days 1 to 21 of a 28 day treatment cycle. In some aspects, the MEK inhibitor is administered once daily on days 3 to 23 of a 28 day treatment cycle.

PD-1 axis inhibitors

In accordance with the present disclosure, a PD-1 axis inhibitor may be more particularly referred to as a PD-1 inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 inhibitor is a molecule that inhibits the binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 inhibitor is a molecule that inhibits the binding of PD-L2 to its binding partner. In a particular aspect, the PD-L2 binding partner is PD-1. The inhibitor may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligosaccharide.

In some embodiments, theThe PD-1 inhibitor 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 nivolumab (nivolumab), pembrolizumab (pembrolizumab), lambrolizumab, and CT-011. In some embodiments, the PD-1 inhibitor 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., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 inhibitor is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, andis an anti-PD-1 antibody as described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab,and SCH-900475, an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT or hBAT-1, 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 WO2010/027827 and WO 2011/066342.

In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4). In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region amino acid sequence from SEQ ID NO:1 and/or a light chain variable region comprising a light chain variable region amino acid sequence from SEQ ID NO: 2. In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising heavy and/or light chain sequences, wherein:

(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 a heavy chain sequence of seq id no:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:1), or

(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 a light chain sequence that is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:2)。

in some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS registry number: 1374853-91-4). In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region amino acid sequence from SEQ ID NO. 3 and/or a light chain variable region comprising a light chain variable region amino acid sequence from SEQ ID NO. 4. In yet another embodiment, provided is an isolated anti-PD-1 antibody comprising heavy and/or light chain sequences, wherein:

(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 a heavy chain sequence of seq id no:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:3), or

(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 a light chain sequence that is:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4)。

in some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 inhibitor is selected from the group consisting of yw243.55.s70, MPDL3280A (atelizumab), MDX-1105, and MEDI 4736. MDX-1105, also known as BMS-936559, is an anti-PD-L1antibody described in WO 2007/005874. Antibody yw243.55.s70 (heavy and light chain variable region sequences shown in SEQ ID nos. 5 and 6, respectively) is an anti-PD-L1 described in WO2010/077634 a 1. MEDI4736 is an anti-PD-L1antibody described in WO2011/066389 and US 2013/034559.

Examples of anti-PD-L1 antibodies useful for the methods of this invention, and methods for producing the same, are described in PCT patent application WO2010/077634 a 1and U.S. patent No.8,217,149, which are incorporated herein by reference.

In some embodiments, the PD-1 axis inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1antibody is selected from the group consisting of Fab, Fab '-SH, Fv, scFv, and (Fab')₂Antibody fragments of the group consisting of fragments. In some embodiments, the anti-PD-L1antibody is a humanized antibody. In some embodimentsThe anti-PD-L1antibody is a human antibody.

anti-PD-L1 antibodies useful in this invention include compositions comprising such antibodies, such as those described in WO2010/077634 a 1. In some embodiments, the anti-PD-L1antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 or 8 (infra) and a light chain variable region comprising the amino acid sequence of SEQ ID NO:9 (infra).

In one embodiment, the anti-PD-L1antibody comprises a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

(a) the HVR-H1 sequence is GFTFSX₁SWIH(SEQ ID NO:10)；

(b) The HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG(SEQ ID NO:11)；

(c) The HVR-H3 sequence is RHWPGGFDY (SEQ ID NO: 12);

further wherein X₁Is D or G; x₂Is S or L; x₃Is T or S.

In a particular aspect, X₁Is D; x₂Is S and X₃Is T. In another aspect, the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between HVRs according to the formula (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VH subgroup III consensus framework. In yet another aspect, at least one of the framework sequences is the following:

HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:13)

HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:14)

HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:15)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO: 16).

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:

(a) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:17)；

(b) The HVR-L2 sequence is SASX₉LX₁₀S(SEQ ID NO:18)；

(c) The HVR-L3 sequence is QQQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:19)；

Further 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; x₁₀Is Y or A; x₁₁Is Y, G, F, or S; x₁₂Is L, Y, F or W; x₁₃Is Y, N, A, T, G, F or I; x₁₄Is H, V, P, T or I; x₁₅Is A, W, R, P or T.

In yet another aspect, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H; x₁₅Is A. In yet another aspect, the light chain further comprises variable region light chain framework sequences juxtaposed between HVRs according to the formula (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR 4). In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the framework sequence is a VL kappa I consensus framework. In yet another aspect, at least one of the framework sequences is the following:

LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:20)

LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO:21)

LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:22)

LC-FR4 is FGQGTKVEIKR (SEQ ID NO: 23).

In another embodiment, provided is an isolated anti-PD-L1antibody or antigen-binding fragment comprising heavy and light chain variable region sequences, wherein:

the heavy chain comprises HVR-H1, HVR-H2 and HVR-H3, wherein further:

(i) the HVR-H1 sequence is GFTFSX₁SWIH(SEQ ID NO:10)

(ii) The HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG(SEQ ID NO:11)

(iii) The HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:12), and

the light chain comprises HVR-L1, HVR-L2 and HVR-L3, wherein further:

(i) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A(SEQ ID NO:17)

(ii) The HVR-L2 sequence is SASX₉LX₁₀S (SEQ ID NO:18) and

(iii) the HVR-L3 sequence is QQQX₁₁X₁₂X₁₃X₁₄PX₁₅T(SEQ ID NO:19),

Further 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; x₁₀Is Y or A; x₁₁Is Y, G, F, or S; x₁₂Is L, Y, F or W; x₁₃Is Y, N, A, T, G, F or I; x₁₄Is H, V, P, T or I; x₁₅Is A, W, R, P or T.

In a particular aspect, X₁Is D; x₂Is S and X₃Is T. In another aspect, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H; x₁₅Is A. In yet another aspect, X₁Is D; x₂Is S and X₃Is T, X₄Is D; x₅Is V; x₆Is S; x₇Is A; x₈Is V; x₉Is F; x₁₀Is Y; x₁₁Is Y; x₁₂Is L; x₁₃Is Y; x₁₄Is H and X₁₅Is A.

In yet another aspect, the heavy chain variable region comprises one or more framework sequences (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4) juxtaposed between HVRs, and the light chain variable region comprises one or more framework sequences (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4) juxtaposed between HVRs. In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is the following:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:13)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:14)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:15)

HC-FR4 WGQGTLVTVSA(SEQID NO:16)。

in yet another aspect, the light chain framework sequence is derived from a Kabat kappa subgroup I, II, or IV sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:20)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:21)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:22)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:23)。

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 IgG1, IgG2, IgG2, IgG3, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from "effector-less Fc mutation" or aglycosylation. In yet another embodiment, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1antibody comprising heavy and light chain variable region sequences, wherein:

(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:24), AWISPYGGSTYYADSVKG (SEQ ID NO:25) and RHWPGGFDY (SEQ ID NO:12), respectively, or (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:26), SASFLYS (SEQ ID NO:27) and QQYLYHPAT (SEQ ID NO:28), respectively.

In a particular 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 (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4) juxtaposed between HVRs, and the light chain variable region comprises one or more framework sequences (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4) juxtaposed between HVRs. In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is the following:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:13)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:14)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:15)HC-FR4WGQGTLVTVSA(SEQ ID NO:16)。

in yet another aspect, the light chain framework sequence is derived from a Kabat kappa subgroup I, II, or IV sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:20)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:21)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:22)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:23)。

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 IgG1, IgG2, IgG2, IgG3, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from "effector minor Fc mutations" or aglycosylation. In yet another embodiment, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an isolated anti-PD-L1antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to a heavy chain sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:29), or

(b) The light chain sequence has at least 85% sequence identity with DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9).

In a particular 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 (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4) juxtaposed between HVRs, and the light chain variable region comprises one or more framework sequences (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4) juxtaposed between HVRs. In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is the following:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:13)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:14)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:15)HC-FR4WGQGTLVTVSA(SEQ ID NO:16)。

in yet another aspect, the light chain framework sequence is derived from a Kabat kappa subgroup I, II, or IV sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:20)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:21)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:22)LC-FR4FGQGTKVEIKR(SEQ ID NO:23)。

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 IgG1, IgG2, IgG2, IgG3, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. 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 "effector minor Fc mutations" or aglycosylation. In yet another embodiment, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In another further embodiment, provided is an isolated anti-PD-L1antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:7), or

(b) The light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:9)。

in yet another embodiment, provided is an isolated anti-PD-L1antibody comprising heavy and light chain variable region sequences, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:8), or

(b) The light chain sequence has at least 85% sequence identity to a light chain sequence of seq id no:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(SEQ ID NO:9)。

in a particular 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 (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4) juxtaposed between HVRs, and the light chain variable region comprises one or more framework sequences (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4) juxtaposed between HVRs. In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is the following:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:13)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:14)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:15)

HC-FR4 WGQGTLVTVSS(SEQ ID NO:30)。

in yet another aspect, the light chain framework sequence is derived from a Kabat kappa subgroup I, II, or IV sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:20)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:21)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:22)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:23)。

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 IgG1, IgG2, IgG2, IgG3, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. 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 "effector minor Fc mutations" or aglycosylation. In yet another embodiment, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another embodiment, the anti-PD-L1antibody is atelizumab, or MPDL3280A (CAS registry number: 1422185-06-5). In yet another embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain variable region from

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:7) or

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:8), the light chain variable region comprising

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 9). In yet another embodiment, provided is an isolated anti-PD-L1antibody comprising heavy and/or light chain sequences, wherein:

(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 a heavy chain sequence of seq id no:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:31), and/or

(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 a light chain sequence that is:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:32)。

in yet another embodiment, provided is an isolated nucleic acid encoding a light chain or heavy chain variable region sequence of an anti-PD-L1antibody, wherein:

(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:24), AWISPYGGSTYYADSVKG (SEQ ID NO:25) and RHWPGGFDY (SEQ ID NO:12), respectively, and (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:26), SASFLYS (SEQ ID NO:27) and QQYLYHPAT (SEQ ID NO:28), respectively.

In a particular aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In one aspect, the heavy chain variable region comprises one or more framework sequences (HC-FR1) - (HVR-H1) - (HC-FR2) - (HVR-H2) - (HC-FR3) - (HVR-H3) - (HC-FR4) juxtaposed between HVRs, and the light chain variable region comprises one or more framework sequences (LC-FR1) - (HVR-L1) - (LC-FR2) - (HVR-L2) - (LC-FR3) - (HVR-L3) - (LC-FR4) juxtaposed between HVRs. In yet another aspect, the framework sequence is derived from a human consensus framework sequence. In yet another aspect, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another aspect, one or more of the heavy chain framework sequences is the following:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:13)

HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:14)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:15)

HC-FR4 WGQGTLVTVSA(SEQ ID NO:16)。

in yet another aspect, the light chain framework sequence is derived from a Kabat kappa subgroup I, II, or IV sequence. In yet another aspect, the light chain framework sequence is a VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences is the following:

LC-FR1 DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO:20)

LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:21)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:22)

LC-FR4 FGQGTKVEIKR(SEQ ID NO:23)。

in yet another specific aspect, an antibody described herein (such as an anti-PD-1 antibody, an anti-PD-L1antibody, or an anti-PD-L2 antibody) further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG 4. In yet another specific aspect, the human constant region is IgG 1. In yet another aspect, the murine constant regions are selected from the group consisting of IgG1, IgG2A, IgG2B, IgG 3. In yet another aspect, the murine constant region is IgG 2A. 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 "effector minor Fc mutations" or aglycosylation. In yet another aspect, the effector minor Fc mutation is an N297A or D265A/N297A substitution in the constant region.

In yet another aspect, provided herein is a nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing a nucleic acid encoding any of the previously described anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies. In yet another specific aspect, the vector further comprises a host cell suitable for expression of the nucleic acid. In yet another particular aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary (CHO).

The antibody or antigen-binding fragment thereof can be generated using methods known in the art, for example, by a process comprising culturing a host cell containing a nucleic acid encoding any of the previously described anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies or antigen-binding fragments in a form suitable for expression under conditions suitable for the production of such antibodies or fragments, and recovering the antibody or fragment.

In some embodiments, the isolated anti-PD-L1antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the side chain of the carbohydrate moiety attached to the 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 module to the asparagine side chain. Thus, 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-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from the antibody is conveniently achieved by altering the amino acid sequence such that one of the tripeptide sequences (for N-linked glycosylation sites) described above is removed. Changes may be made by substituting an asparagine, serine or threonine residue within a glycosylation site with another amino acid residue (e.g., glycine, alanine or a conservative substitution).

It is noted in this regard that the pharmacokinetics of the atlizumab administered as a single agent have been characterized based on clinical data from the study PCD4989g and are consistent with the currently ongoing first line treatment of TNBC, phase III study WO 29522. The antitumor activity of astuzumab has been observed at doses ranging from 1 to 20 mg/kg. In summary, for doses ≧ 1mg/kg every three weeks (q3w), atlizumab exhibited its pharmacokinetics linear and consistent with that of the typical IgG1 antibody. Pharmacokinetic data (Bai S, Jorga K, Xin Y, et al, A guide to a systemic diagnosis of monoclonalantities, Clin Pharmacokinet 2012; 51:119-35, herein incorporated by reference in its entirety) do not suggest any clinically meaningful differences in exposure after a fixed dose or a dose adjusted for body weight. Attrituzumab dosing schedules q3w and q2w have been tested. A fixed dose of atezumab 800mg every two weeks (q2w) (equivalent to a body weight-based dose of 10mg/kg q2w) resulted in an exposure equivalent to 1200mg administered every three weeks (q3w) with the phase III dose. The q3w schedule is being used for multiple phase III studies of atzumab monotherapy in multiple tumor types while q2w is used primarily in combination with chemotherapy regimens. In study PCD4989g, the overall 24-week progression-free survival (PFS) rate estimated by Kaplan-Meier was 33% (95% CI: 12%, 53%).

The PD-1 axis inhibitor dose of the present disclosure is suitably from about 400mg to about 1200mg, from about 600mg to about 1000mg, from about 700mg to about 900mg, or about 840 mg. In some aspects, the PD-1 axis inhibitor is a PD-L1 inhibitor, and more particularly, altlizumab, administered at a dose of about 840 mg.

In particular embodiments, the PD-1 axis inhibitor, or more particularly the PD-L1 inhibitor, is administered intravenously every 14 days of a 28 day treatment cycle. In some aspects, the subject is treated with a PD-1 axis inhibitor, and more particularly a PD-L1 inhibitor, on days 1and 15 of a 28 day treatment cycle.

VEGF inhibitors

VEGF inhibitors within the scope of the disclosure include pazopanibbevacizumabsorafenibsunitinibaxitinibponatinibregorafenibcabozantinibvendetanibramucirumablenvatinibAnd ziv-aflibercept

In some aspects, the VEGF inhibitor drug is bevacizumab. Bevacizumab has been approved by the FDA for the treatment of metastatic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) in combination with a chemotherapy regimen. Bevacizumab is described in U.S. Pat. nos. 6,054,297 and 6,884,879, the contents of which are incorporated herein by reference, and is a recombinant humanized IgG1 monoclonal antibody that binds VEGF and neutralizes the biological activity of VEGF by preventing its interaction with its receptor.

In this or another aspect of the disclosure, the VEGF inhibitor is an antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO:33 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 34. In yet another aspect of the disclosure, there is provided an isolated VEGF-inhibiting antibody comprising a heavy chain and/or a light chain sequence, wherein:

(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 a heavy chain sequence that is:

EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTYAADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO: 33); or

(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 heavy chain sequence:

DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR(SEQ ID NO:34)。

in this or yet another aspect of the disclosure, the VEGF inhibitor is an antibody comprising a heavy chain and/or light chain variable region sequence, wherein:

(a) the heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3 sequences having at least 85% sequence identity to GYTFTNYGMN (SEQ ID NO:35), WINTYTGEPTYAADFKR (SEQ ID NO:36), and YPHYYGSSHWYFDV (SEQ ID NO:37), respectively; or (b) the light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences having at least 85% sequence identity to SASQDISNYLN (SEQ ID NO:38), FTSSLHS (SEQ ID NO:39), and QQYSTVPWT (SEQ ID NO:40), respectively.

In accordance with the present disclosure, the VEGF inhibitor dose is about 0.1 to about 15 mg/kg/week, about 0.5 to about 15 mg/kg/week, about 1 to about 15 mg/kg/week, about 5 to about 10 mg/kg/week, such as about 5 mg/kg/week, about 10 mg/kg/week or about 15 mg/kg/week. In some aspects, the VEGF inhibitor is bevacizumab. In some aspects, bevacizumab is administered weekly, and more particularly at a dose of about 5 mg/kg/week.

Colorectal cancer

In one aspect, provided herein is a method for treating colorectal cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a combination of a MEK inhibitor, a PD-1 axis inhibitor, and a VEGF inhibitor. mCRC is particularly suitable for combination therapy as described herein.

In some aspects of the disclosure, the treatment results in delaying the progression of CRC in the subject. In some other aspects, the treatment results in a complete response in the subject. In some other aspects, the response continues after treatment is discontinued. In still other aspects, the treatment extends the median progression-free survival time compared to CRC subjects receiving therapy comprising (i) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a MEK inhibitor and not administered a VEGF inhibitor, (ii) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a VEGF inhibitor and not administered a MEK inhibitor, and/or (iii) a therapeutically effective amount of a MEK inhibitor and a therapeutically effective amount of a VEGF inhibitor and not administered a PD-1 axis inhibitor.

Combination therapy

It is believed that the triple combination of MEK inhibitor, PD-1 axis inhibitor, and VEGF inhibitor (i) targets the hallmarks of cancer (i.e., proliferative signaling, immune evasion, and angiogenesis), (ii) elicits synergistic antitumor activity based on complex interactions and the activities exhibited by these agents, and/or (iii) offers the potential for substantial clinical benefit in patients with CRC.

It is still further believed that triple combination therapy of the present disclosure can extend the median progression-free survival time of subjects having CRC as compared to subjects having CRC receiving therapy comprising (i) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a MEK inhibitor and not administered a VEGF inhibitor, (ii) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a VEGF inhibitor and not administered a MEK inhibitor, and/or (iii) a therapeutically effective amount of a MEK inhibitor and a therapeutically effective amount of a VEGF inhibitor and not administered a PD-1 axis inhibitor.

Pharmaceutical combination

In some aspects of the disclosure, there is provided a cancer therapy pharmaceutical combination comprising (i) a MEK inhibitor in a dose of about 20mg to about 100mg, about 40mg to about 80mg, or about 80 mg; (ii) a PD-1 axis inhibitor in a dose of about 400mg to about 1200mg, about 600mg to about 1000mg, about 700mg to about 900mg, or about 840 mg; and (iii) a VEGF inhibitor in a dose from about 5mg/kg to about 15mg/kg, from about 5mg/kg to about 10mg/kg, about 5mg/kg, about 10mg/kg or about 15 mg/kg. In a particular aspect, the MEK inhibitor is cobitinib, the PD-L1 inhibitor is atelizumab, and the VEGF inhibitor is bevacizumab. In some aspects, the combination may be administered every two weeks. For example, the combination may be administered on days 1and 15 of a 28 day treatment cycle.

It is noted in this regard that any combination of the dosage ranges of the combination partners may be used without departing from the intended scope of the present disclosure. When the drug combinations (i.e., MEK inhibitor, PD-1 axis inhibitor, and VEGF inhibitor) are administered to a subject on the same day, the drugs may be administered in any order. For example, (i) the drugs may be administered separately in any order, or, (ii) the first drug and the second drug may be administered closely at the same time or time interval and the third drug may be administered before or after the administration of the first and second drugs. The administration of each drug of the drug combination may be separated by some period of time, such as 0.5 hours, 1 hour, 2 hours, 3 hours, or 4 hours. In some particular aspects, cobicistinib may be administered orally, atuzumab may be administered intravenously, and bevacizumab may be administered parenterally or intravenously at least 0.5 hours after atuzumab administration. In such aspects, cobicistinib may be administered before or after atezumab. In some aspects, the MEK inhibitor and the PD-1 axis inhibitor are each administered on days 1and 15 of a 28 day treatment cycle, while cobicisib is administered on days 1 to 21 of a 28 day treatment cycle.

Reagent kit

In some aspects of the disclosure, a kit for treating CRC in a human subject is provided. The kit comprises a MEK inhibitor, a PD-1 axis inhibitor, a VEGF inhibitor, and a package insert comprising instructions for using a therapeutically effective amount of the MEK inhibitor, a therapeutically effective amount of the PD-1 axis inhibitor, and a therapeutically effective amount of the VEGF inhibitor to treat the subject. In some aspects, the MEK inhibitor is cobicistinib, the PD-1 axis inhibitor is atelizumab, and the VEGF inhibitor is bevacizumab.

The kits of the present disclosure extend the median progression-free survival time compared to a CRC subject receiving therapy comprising (i) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a MEK inhibitor and no administration of a VEGF inhibitor, (ii) a therapeutically effective amount of a PD-1 axis inhibitor and a therapeutically effective amount of a VEGF inhibitor and no administration of a MEK inhibitor, and/or (iii) a therapeutically effective amount of a MEK inhibitor and a therapeutically effective amount of a VEGF inhibitor and no administration of a PD-1 axis inhibitor.

Examples

The examples relate to a two-stage, open label, multicenter, single arm, phase Ib study designed to assess safety, tolerability and pharmacokinetics of a combination of cobicistinib, atuzumab and bevacizumab in patients with mCRC who have received at least one prior line therapy with fluoropyrimidine and oxaliplatin or irinotecan and progressed for late stage disease.

Phase 1 would be a security insertion. Stage 2 would be a dose expansion and an expansion queue and a biopsy queue. The patient first enters a safety insertion period. Once the safety and tolerability of the treatment regimen is determined, the study will proceed to an expansion phase. If the results from the safety insertion phase require a dose reduction of cobicistinib, an additional phase 1 queue is opened. The patient may enroll in either a treatment or biopsy cohort during the augmentation phase, depending on the patient's suitability and willingness to undergo successive tumor biopsies. The study was concluded when all patients who had been enrolled had been tracked until death, consent was withdrawn, follow-up was lost, or the sponsor decided to end the trial, whichever occurred first.

The main objectives of the study include evaluation of the safety and tolerability of cobicistinib plus bevacizumab plus atelizumab and confirmation of suggested dosage regimens for further clinical development. Evaluation criteria and endpoints include (i) incidence, nature and severity of adverse events, graded according to the national cancer institute adverse event's commonly used terminology criteria (NCI CTCAE) v 4; (ii) laboratory data; and (iii) grade 3 adverse events, including adverse events of particular interest and adverse events that caused discontinuation of treatment.

Exploratory efficacy goals included assessing the efficacy of cobitinib plus bevacizumab plus atelizumab. Assessment criteria and endpoints include (i) confirmed overall response rates assessed by investigators, defined by response assessment criteria for solid tumors (RECIST) v 1.1; (ii) progression-free survival, defined by the time from day 1 of cycle 1 to the first occurrence of disease progression determined by investigators using RECIST v1.1 or death of any cause during the study, whichever occurs first; and (iii) duration of response, defined by the time from the first occurrence of a recorded objective response to disease progression as determined by investigators using RECIST v1.1 or the time to death of any cause during the study, whichever occurs first.

Study treatment will include cobitinib at a dose of 60mg on the 21/7 schedule, atbizumab on the 840mg every 2-week (q2w) schedule, and bevacizumab on the 5mg/kg q2w schedule. Patients in the safety insert and in the extended cohort will receive cobicistinib, altlizumab, and bevacizumab from day 1 onwards. Patients in the biopsy cohort in the expansion phase will start bevacizumab on day 1, followed by tumor biopsy on day 14(± 2 day window) and cobitinib on day 15, followed by tumor biopsy on day 28(± 2 day window) and attrituximab on day 29 (i.e., day 1 of cycle 2), followed by an optional tumor biopsy on day 56(± 2 day window). Biopsies will be performed before cobitinib and atuzumab, respectively, are initiated. From this point forward, patients in the biopsy queue will follow the same treatment protocols as those in the safety insert and treatment extension queue.

Pre-treatment biopsies will be required for all patients and will be formalin-fixed, paraffin-embedded tissues. Archival biopsies can be used for pre-treatment biopsies as long as they are collected no more than 3 months prior to screening. Fresh samples are preferred for patients in the biopsy cohort. As described above, only the remaining biopsies will be scheduled for the patients in the biopsy queue.

Safety and tolerability will be closely monitored for all patients during all cycles of therapy, at study treatment end visits, and during follow-up periods. NCI CTCAE v4.0 will be used to characterize the toxicity profile of study treatment for all patients.

Patients in both phases will continue to receive study therapy until disease progression according to recistv1.1, unacceptable toxicity, death, patient or physician decision to withdraw, or pregnancy, whichever occurs first. Any evaluable and measurable disease will be recorded at screening and re-evaluated at each subsequent tumor evaluation. Investigators will evaluate tumor response at 8 week intervals, regardless of any prolonged dosing.

Treatment will continue until the patient has disease progression according to RECIST v1.1, unacceptable toxicity, death, patient or physician decision to withdraw, or pregnancy, whichever occurs first. Elevated carcinoembryonic antigen levels alone are not considered disease progression. Patients are allowed to receive study treatment beyond disease progression if certain conditions are met.

Inclusion criteria for patients admitted to the study included the following. The age is at least 18 years. Eastern cooperative oncology panel performance status 0 or 1. Histologically confirmed unresectable metastatic colorectal adenocarcinoma. Progress was made on a prior line therapy containing fluoropyrimidine and oxaliplatin or irinotecan for unresectable metastatic colorectal adenocarcinoma. Adjuvant or neoadjuvant chemotherapy is allowed, provided it is completed at least 12 months prior to the start of study treatment. Measurable disease according to RECIST v 1.1. Note that the lesion intended for biopsy should not be the target lesion. Adequate hematologic and end organ function was defined by the following laboratory results obtained within 14 days prior to the first dose of study drug treatment (i) WBC ≧ 2.5 and ≤ 15.0 × 10⁹/L；(ii)ANC≥1.5×10⁹L; (iii) platelet count is not less than 100X 10⁹L; (iv) the hemoglobin is more than or equal to 9 g/dL; (v) albumin is more than or equal to 9 g/dL; serum bilirubin ≦ 1.5 × Upper Normal limit (ULN) (patients with known Gilbert's disease may have bilirubin ≦ 3.0 × ULN); (vi) INR and PTT are less than or equal to 1.5 multiplied by ULN; (vii) amylaseAnd lipase is less than or equal to 1.5 × ULN; (viii) AST, ALT, and alkaline phosphatase (ALP). ltoreq.3 × ULN with the exception of patients with documented liver metastasis (AST and/or ALT. ltoreq.5 × ULN) and patients with documented liver or bone metastasis (ALP. ltoreq.5 × ULN); (ix) creatine elimination is more than or equal to 30 mL/min.

Criteria for rejection in patients admitted to the study include the following. Major surgical procedures were required during surgical procedures (including open biopsy, surgical resection, wound revision, or any other major surgery) or major traumatic injury within 60 days prior to enrollment, or during the course of the intended study. Secondary surgical procedures (including placement of vascular access devices) within 15 days 1 of study cycle 1. Untreated CNS metastasis. Treatment of brain metastases by surgical or radiological techniques will be completed at least 4 weeks prior to initiation of study treatment. Treatment with any investigational agent or approved therapy within 28 days or two investigational agent half-lives (whichever is longer) prior to enrollment of the study (cycle 1, day 1). Malignancies other than colorectal cancer within 5 years prior to day 1 of cycle 1, with the exception of those treated with the expected outcome of cure with negligible risk of metastasis or death (e.g. expected 5 years overall survival > 90%) (such as properly treated orthotopic cervical cancer, basal or squamous cell skin cancer, to cure localized prostate cancer intended for surgical treatment, to cure orthotopic ductal carcinoma intended for surgical treatment.A prior radiotherapy and/or a persistent radiation related adverse effect within 30 days prior to day 1 of the study cycle 1.

Patient rejection criteria for study admission related to study medication include the following. Therapeutic oral or parenteral anticoagulants or thrombolytic agents are currently or recently (within 10 days of study enrollment) used for therapeutic purposes (> 325 mg/day), clopidogrel (> 75 mg/day) or currently or recently (within 10 days of the first dose of bevacizumab). History of severe allergic, anaphylactic, or other hypersensitivity reactions to the chimeric or humanized antibody or fusion protein. Known hypersensitivity or allergy to any ingredient of biopharmaceuticals or cobicistinib, atuzumab, or bevacizumab formulations produced in chinese hamster ovary cells. Prior treatment with CD137 agonists or immune checkpoint blockade therapy, anti-apoptosis-1, anti-programmed death ligand 1, MEK inhibitors.

Patient rejection criteria for study admission related to organ function and medical history include the following. History of clinically significant cardiac or pulmonary dysfunction. Severe non-healing wounds, active ulcers or untreated fractures. History of abdominal fistulae or gastrointestinal perforations within 6 months prior to day 1 of cycle 1. Hemoptysis (not less than)¹/₂Teaspoon of bright red blood per episode), or any other severe bleeding or bleeding risk (history of gastrointestinal bleeding, gastrointestinal ulcers, etc.). INR > 1.5 and aPTT > 1.5 × ULN 7 days before cycle 1 day 1. History or evidence of major coagulopathies or hereditary hemorrhagic diathesis with bleeding risk. Life expectancy <12 weeks. Any prior grade 3 venous thromboembolism. The proteinuria during screening is proved by that the proteinuria is more than or equal to 2+ or more than 1.0g in 24 hours by urine test paper. The left ventricular ejection fraction is below the normal lower limit of the system. Uncontrolled severe medical or psychiatric illness. Uncontrolled tumor pain; patients requiring narcotic pain medication during the screening period should be on a stable dosing schedule prior to cycle 1 day 1. Pregnancy or lactation, or intended to become pregnant during the study. Women that are not postmenopausal (amenorrhea ≧ 12 consecutive months, no cause other than menopause was identified) or surgically sterilized must have a negative serum pregnancy test within 14 days prior to day 1 of cycle 1. Idiopathic pulmonary fibrosis, organized pneumonia (e.g., obstructive bronchiolitis), drug-induced pneumonia, history of idiopathic pneumonia, or evidence of active pneumonia when screening chest CT scans. Is considered to be the history or evidence of retinopathy on ophthalmic examination of sensory neuroretinal detachment/central serous chorioretinopathy, retinal vein occlusion or risk factors for neovascular macular degeneration. Exclusion criteria based on infectious diseases include active infection requiring IV antibiotics at the time of screening; patients with active hepatitis b (chronic or acute); having a past hepatitis B Virus(HBV) infected or resolved HBV infected patients; patients with active hepatitis c; and known HIV infections. The rejection criteria based on an autoimmune condition include a history of autoimmune diseases 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.

Patient inclusion criteria included in the biopsy cohort include all inclusion criteria that meet study admission; and receiving no bevacizumab or the last bevacizumab treatment at least 12 months prior to day 1 of cycle 1.

Cobicistinib will be administered orally once daily at a dose of 60mg cobicistinib (three tablets of 20mg each) on days 1-21 of a 28-day cycle.

Alemtuzumab will be administered at a dose of 840mg by IV infusion on days 1and 15 of each 28 day cycle. The attrituzumab will be administered first, followed by bevacizumab for a minimum of 30 minutes between administrations.

Bevacizumab will be administered at a dose of 5mg/kg by IV infusion on days 1and 15 of each 28 day cycle.

Collection of archived and/or fresh tumor specimens, and biomarker assessment

The expanded RAS status involves treatment and prognosis in mCRC (karapatitis CS, Khamata-Ford S, Jonker DJ, et al, K-RAS Mutations and Benefit from Cetuximab in advanced dColorative Cancer, N Engl J Med 2008; 359: 1757-65; De Roock W, Claes B, Bernasonid, et al, Effects of KRAS, BRAF, NRAS, and PIK3CA Mutations of The expression of nuclear ployab syndrome in chemotherapy reactivity recovery expression of nucleic acid RAS: a reactive contract analysis, EGFR Lance 2010, 11: 753-62; Soichia MJ, sodium and epidermal growth expression, EGFR expression and expression of nucleic acidA metallic color of random controlled triangles, Ann Oncol 2015; 26: 13-21; and Allegra CJ, Rumble RB, Hamilton SR, et al, Extended RAS Gene Mutation Testing in metallic color selection to Predict Response to Anti-Epidermal Growth factor receptor Monoclonal Antibody Therapy, American Society of Clinical Oncology Clinical Opinion Update2015, J Clin Oncol 2016; 34:179-85. Each reference is incorporated herein by reference in its entirety). Several studies demonstrated that RAS mutations carry worse PFS and overall survival prognosis compared to the RAS wild-type cohort (Sorbye H, Dragomir a,m, et al, High BRAF mutationF request and market Survival Differences in Subgroups accessing to KRAS/BRAFMutation Status and Tumor Tissue Availability in a proactive position-Based metallic color Cancer code, PLoS One 2015; 10: e 0131046; sorich et al 2015; and Vincenzi B, Cremolini C, Sarore-Bianchi A, et al, scientific design of K-Ras mutation rate in quantitative color cancer patents 2015; 6:31604-12). Phase Ib study GP28363 (cobitinib administered with atuzumab in patients with locally advanced or metastatic tumors) assessed safety and efficacy in KRAS mutant mCRC and in biopsy cohorts including multiple solid tumors. In connection with the present disclosure, efficacy and safety of this regimen in all mCRC patients were evaluated regardless of the extended RAS status, as the mechanism of action would not predict differential effects. However, given the prognosis based on differences in RAS state, testing for extended RAS state will be performed on archived/baseline tumor tissue.

MSI status in CRC also implicates both treatment and prognosis in mCRC (Goldstein J, Tran B, EnsorJ, et al, Multicenter retrospecific analysis of metallic color candidate (CRC) with high-level microsomal availability (MSI-H), Annals of Oncology 2014; 25:1032-8, herein incorporated by reference in its entirety). It has a different ORR and duration of response (DOR) rate compared to checkpoint inhibitors such as PD-L1 and PD-1 antagonists (Li J, Qin S, Xu R, et al, Regorafenib plus post Supportive Care cover board plus post Supportive Care in asset Patents with a previous mental distinct mental color cancer (CONCUR): a random mixed, double-index, plate-controlled, phase 3 tertiary, Lancetocol 2015; 16: 619-29; and Oh DY, VenooP, food L., On the very version: Imotherapy for clinical color, J Natl Net net 970; 13: c-970). It is believed that the PD-L1antibody, atzumab, may be effective in patients with MSI high CRC, similar to other checkpoint inhibitors, but may have differential effects depending on MSI status. Thus, MSI status will be assessed from the archive/baseline organization to differentiate the efficacy of such a regimen in these different populations.

Tumor tissue samples will be collected at baseline for DNA and/or RNA extraction, enabling Next Generation Sequencing (NGS) to identify somatic mutations, increasing investigators' understanding of disease pathobiology. Gene-based CRC classification is increasingly suggested as a way to differentiate various subtypes of CRC and may have profound effects on both therapy and prognosis. These subtypes have been shown to have different immunomodulatory effects and may influence The efficacy of this protocol (Guinney J, Dienstmann R, Wangx, et al, The consensus molecular subsets of The synergistic cancerers, Nat Med 2015; 21: 1350-6; Kocarbonik JM, Shiovitz S, Phipps AI, molecular protocols of The synergistic cancerers and molecular clinical applications, Gastrontel Rep (Oxf) 2015; 3: 269-76; and Lal N, Beggs AD, Willcox BE, Middton GW, immunological targeting of The synergistic cancerers: immunological cancerers: expression for targeted imaging, therapeutic on 6052; 6052). Because these biomarkers may also have prognostic value, their potential association with disease progression is also explored. Archival/baseline tumor analysis and classification of different CRC subtypes will be performed to further assess this possible correlation.

One independent (stand-alone) cohort of tumor biopsies was scheduled before treatment, when treated with bevacizumab in combination with cobicistinib, and when triple-recombined with atuzumab. Comparison of biomarkers between biopsies at the time of staggered treatment will further elucidate the possible mechanism of action of this combination. Biomarker analysis will focus on assessment of CD8 positive T cell infiltrates, PD-L1 expression, and enhanced immune responses, MAPK inhibition, and other biomarkers involved in apoptosis or inflammation.

Stage 1 Security insert queue

Phase I study GP28363 has explored the amplified dose of cobicistinib administered with atuzumab. Cobicistinib administered with atezumab has been shown to be safe and tolerable. Bevacizumab was added to cobitinib and atuzumab in this study. Although bevacizumab had been previously administered with atuzumab, this study would begin with a safety insertion cohort because bevacizumab had not previously been administered to the patient along with cobitinib and atuzumab. Bevacizumab had some overlapping toxicity with cobitinib and atuzumab.

Patients who are safely enrolled in the cohort will receive cobitinib, altlizumab, and bevacizumab from day 1 onwards. The research scenario provided for security insertion and expansion of queues is depicted in fig. 1.

In the safety insertion cohort, a 28 day dosing cycle will be evaluated in which patients will be administered (i) the first 21 days of 60mg cobicistinib followed by a 7 day rest period without cobicistinib therapy; (ii) an infusion of 840mg of atuzumab on days 1and 15 of a 28 day cycle; and (iii) 5mg/kg bevacizumab infusion on days 1and 15 of a 28 day cycle.

After a patient in the safety insertion phase has completed at least one 28 day cycle of treatment, the clinical data will be reviewed to determine the safety and tolerability of the test dose. It is believed that this would take months to complete the registration of this group; thus, the safety review should contain data for patients who have accepted the regimen for several cycles at the time of the safety assessment. If it was determined that a 60mg cobicistinib dose was not tolerated during the safety insertion phase, the study team would enroll patients in an additional cohort with a reduced cobicistinib dose of 40mg QD (21/7). In such reduced cobicistinib dose assessments, after the additional cohort has completed one cycle of treatment, a clinical data review is conducted to determine whether the expansion phase can be initiated with such lower doses of cobicistinib.

Study treatment will be stopped immediately for the patient individual and thorough investigation and safety analysis will be performed if any of the following events occur and are assessed by the investigator as relevant (i) > grade 3 hypertension (> 180mmHg systolic or > 110mmHg diastolic); (ii) grade 3 or more bleeding (transfusion with symptoms and indications of 2 unit packed RBCs); (iii) grade 3 or more pneumonia (symptomatic, interfering with ADL; indicating oxygen); (iv) grade 3 or more left ventricular dysfunction (symptomatic CHF, responsive intervention); (v) grade 3 or more diarrhea, no response to antidiarrheal (7 more stool per day relative to baseline increase; incontinence; IV fluid > 24 hours; hospitalization); or (vi) ALT or AST >5x ULN in combination with total bilirubin >2x ULN. Further enrollment and study treatment will immediately cease until a thorough survey and safety analysis has been conducted if any of (i) any subject experienced death due to an AE (by investigator and/or sponsor) assessed as being relevant to study treatment, which would result in a temporary suspension of the pending study, reviewed by the study team; or (ii) more than 30% of patients meet the individual stopping rules defined above.

Stage 2 augmentation and biopsy queuing

Patients in the extended cohort will receive cobicistinib, altlizumab, and bevacizumab from day 1 onwards.

Patients in the biopsy cohort in the expansion phase will start bevacizumab on day 1, followed by tumor biopsy on day 14(± 2 day window) and cobitinib on day 15, followed by tumor biopsy on day 28(± 2 day window) and attrituximab on day 29 (i.e., day 1 of cycle 2), followed by an optional tumor biopsy on day 56(± 2 day window). Biopsies will be collected before cobitinib and atuzumab, respectively, are initiated. From this point forward, patients in the biopsy queue will follow the same treatment protocols as those in the safety insert and treatment extension queue.

The study protocol of the biopsy cohort is depicted in fig. 2.

Tumor response will be assessed according to RECIST v 1.1. Any evaluable and measurable disease will be recorded at screening and re-evaluated at each subsequent tumor evaluation. Baseline tumor assessments will be performed ≦ 28 days before cycle 1 day 1and assessed according to RECIST v1.1 as outlined below. The same protocol used to assess the disease site at baseline (e.g., the same contrast protocol used for CT scans or MRI scans) will be used throughout the study.

Tumor response assessments that followed RECIST v1.1 will be recorded every 8 weeks ± 1 week (regardless of where the patient was in the treatment cycle) until recorded, confirmed by investigators, progressive disease, loss of clinical benefit, withdrawal of consent, death, or termination of the study by the sponsor, whichever occurs first. The schedule of tumor assessment is independent of any changes in the study treatment administration schedule (e.g., dosing delay) and may occur in cycles depending on the length of the cycle. If tumor assessments have to be performed earlier or later, subsequent assessments should be performed according to the original schedule based on the date of the first study drug administration (cycle 1, day 1). Confirmation of response (PR or complete response [ CR ]) will be performed no earlier than 28 days since study entry. In the case of SD, the measurements must meet the SD criteria at least once at a minimum interval of not less than 6 weeks after study entry. Patients who discontinue study treatment for any reason other than disease progression will continue to undergo tumor response assessment (approximately every 8 weeks) until progressive disease. Tumor markers (e.g., CEA) that are elevated in the absence of radiological evidence of progression are not considered progressive disease.

Response assessment in solid tumors

At baseline, tumor lesions/lymph nodes would be classified as measurable or unmeasurable as follows. A tumor lesion will measure at least one dimension (the longest diameter of the measurement plane to be recorded) with the smallest dimension of (i) 10mm (CT/MRI scan slice thickness/spacing no greater than 5mm) by Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) scanning; (ii) measured by a clinical examination 10mm caliper (lesions that cannot be accurately measured with a caliper should be recorded as non-measurable); or (iii) 20mm by chest X-ray. Malignant lymph nodes would be considered pathologically enlarged and measurable lymph nodes with a short axis > 15mm when assessed by CT scan (CT scan slice thickness recommended no more than 5 mm). At baseline and during follow-up, only the short axis will be measured and tracked. Unmeasurable tumor lesions encompass small lesions (pathologic lymph nodes with a longest diameter <10mm or a minor axis ≧ 10 and ≦ 15 mm), as well as truly unmeasurable lesions. Lesions that are considered truly unmeasurable include pia mater disease identified by physical examination, unmeasurable by reproducible imaging techniques, ascites, pleural or pericardial effusion, inflammatory breast disease, lymphatic involvement of the skin or lungs, peritoneal spread, and abdominal mass/abdominal organ megaly.

In connection with bone lesions, bone scans, Positron Emission Tomography (PET) scans, or plain film are not considered sufficient imaging techniques to measure bone lesions. However, these techniques can be used to confirm the presence or absence of bone lesions. A lytic bone lesion or mixed lytic-osteogenic lesion with an identifiable soft tissue component that can be assessed by a transverse slice imaging technique such as CT or MRI can be considered a measurable lesion if the soft tissue component satisfies the measurable definition described above. Osteogenic bone lesions are not measurable. In connection with cystic lesions, lesions that meet the criteria for a radiologically defined simple cyst should not be considered malignant lesions (neither measurable nor unmeasurable) because they are by definition simple cysts. Cystic lesions that are representative of a cystic metastasis can be considered measurable lesions if they meet the measurable definition described above. However, these are preferred for selection as target lesions if non-cystic lesions are present in the same patient. Tumor lesions located in previously irradiated areas or areas subjected to other local regional therapies are generally not considered measurable unless there has been a recorded progression in the lesion. The study protocol should detail that such damage would be considered a measurable condition.

If clinically evaluated using calipers, lesion measurements are recorded in metric notation. All baseline assessments will be performed as close to the start of treatment as possible and never more than 4 weeks before starting treatment. The same assessment method and the same technique will be used to characterize each identified and reported lesion at baseline and during the study. Preferably based on an evaluation of the image.

Clinical lesions are considered measurable only if they are superficial and assessed for diameters ≧ 10mm (e.g., skin nodules) using calipers. For the case of skin lesions, the size of the lesion is preferably assessed by color photographic (including ruler) recordings.

Chest CT is preferred over chest X-ray, especially when progression is an important endpoint, as CT is more sensitive than X-ray, especially in identifying new lesions. However, lesions on chest X-rays may be considered measurable if they are well defined and surrounded by an inflated lung. CT is the best reproducible method currently available for measuring the lesions selected for response assessment. This guideline defines the scalability of lesions on CT scans based on the assumption that the CT slice thickness is 5mm or less. When the CT scan has a slice thickness greater than 5mm, the smallest dimension of the measurable lesion should be twice the slice thickness. MRI is also acceptable. The decision as to whether to use contrast-free CT or MRI (without IV contrast) at baseline and during the study to evaluate the patient should be guided by the anatomical location of the tumor type and disease under investigation if it is known that the patient could not undergo a CT scan with Intravenous (IV) contrast due to allergic or renal insufficiency prior to enrollment. For patients who have contraindications for contrast agents after performing baseline contrast CT, the decision as to whether to perform contrast-free CT or MRI (enhanced or non-enhanced) should also be based on the tumor type and anatomical location of the disease and should be optimized to allow comparison with prior studies if possible.

Assessment of tumor response involves assessment of the overall tumor burden at baseline and using this as a comparator for subsequent measurements. Measurable disease is defined by the presence of at least one measurable lesion as detailed above. When there is more than one measurable lesion at baseline, all lesions representing up to a total of up to five lesions (and up to two lesions per organ) for all involved organs should be identified as target lesions and will be recorded and measured at baseline. This means that a maximum of two lesions (one site) and four lesions (two sites) will be recorded in the case where the patient has only one or two organ sites involved. Other lesions (although measurable) in those organs will be recorded as unmeasurable lesions (even though of size >10mm by CT scan). The target lesions should be selected based on their size (lesions with the longest diameter) and represent all involved organs, but should otherwise be reproducibly repeated themselves. Lymph nodes with a short axis of <10mm are considered non-pathological and should not be recorded or tracked. The sum of the diameters of all target lesions (longest of the non-nodal lesions, short axis of the nodal lesion) is calculated and reported as the baseline diameter sum. If the lymph nodes are to be included in the sum, then only the minor axis is added to the sum, as described above. Any objective tumor regression using the baseline diameter and measurable dimensions as a reference to further characterize the disease.

A summary of the overall response status calculations at each time point for patients with measurable disease at baseline is provided in table 1 below. A summary of the overall response calculations is provided below in table 2, where confirmation of the response is required.

TABLE 1 time Point response- -patients with target lesions (with or without non-target lesions)

Target damage	Non-targetDamage or injury	New damage	Overall response
				CR	CR	Whether or not	CR
CR	non-CR/non-PD	Whether or not	PR
				CR	Not evaluated	Whether or not	PR
PR	non-PD or not all evaluations	Whether or not	PR
				SD	non-PD or not all evaluations	Whether or not	SD
Not all of them are evaluated	non-PD	Whether or not	Not evaluable
				PD	Any of	Yes or no	PD
Any of	PD	Yes or no	PD
				Any of	Any of	Is that	PD

TABLE 2 best overall response (when confirmation is required)

^aAny disease seen at a subsequent time point relative to baseline, even a disease that meets PR criteria, qualifies as PD at that time point if CR is actually met at the first time point (because the disease must reappear after CR). The best response will depend on whether the minimum duration of the SD is met. However, CR may sometimes be declared when a subsequent scan suggests that a small lesion is likely to still be present and that in fact the patient has PR at the first time point rather than CR. In these cases, the original CR should be changed to PR and the best response is PR.

Laboratory, biomarker, and other biological samples and assessments

Samples for the laboratory tests described below are sent to one or several central laboratories for analysis. For patients showing evidence of immune-mediated toxicity, additional samples can be collected and analyzed, including (i) antinuclear antibodies; (ii) anti-double-stranded DNA; (iii) circulating anti-neutrophil cytoplasmic antibodies; and (iv) perinuclear anti-neutrophil cytoplasmic antibodies. The pharmacokinetic assay would include (i) serum samples with validated immunoassay concentrations determined for attrituximab and bevacizumab following the schedule in table 3 below; and (ii) plasma samples of cobitinib concentration were measured using a validated liquid chromatography in combination with tandem mass spectrometry methods following the schedule in table 3 below. Blood samples will be collected from all eligible patients and analyzed for biomarkers, including but not limited to biomarkers related to CRC or tumor immunobiology, according to the schedule in table 3. The samples will be processed to obtain plasma for determination of the blood-based suggested biomarkers listed in table 4.

TABLE 3 Schedule of pharmacokinetic, immunogenicity, and biomarker samples

^aPatients who discontinue study medication will return to the clinic for a treatment discontinuation visit 30 (+ -7) days after the last dose of study medication. Visits showing progressive disease can be used as treatment discontinuation visits.

TABLE 4 suggested biomarkers for exploratory studies

Tumor tissue sample

Representative tumor specimens (preferred) or at least 20 serial sections in paraffin blocks with associated pathology reports will be submitted and unstained slides used to determine RAS status and MSI status.

Extended RAS mutations are defined as codons 12 and 13 of exon 2 of the KRAS and NRAS genes; codons 59 and 61 of exon 3; and mutations occurring in codons 117 and 146 of exon 4 (Allegra et al, 2016). Local RAS test results are accepted and the results and interpreted copies are part of the screening process, requiring central validation.

MSI states can be defined by the fraction of repeated MSI loci exhibiting different sizes by several methods, such as IHC detection of hMLH 1and hMSH2 gene products, NGS testing, or PCR testing (Lindor NM, Burgart LJ, Leontovich O, et al, immunological chemistry university microsalt microbiological specificity in photosynthetic color regulators, J Clin Oncol 2002; 20: 1043-8; SalipanteSJ, Scrogins SM, Hampel HL, et al, microscopic specificity detection by mutagenesis, Clin Chem 2014; 60: 1192-9. each reference is incorporated herein by reference in its entirety). Local MSI test results are accepted and a central validation is required with the results and interpreted copy as part of the screening process.

In addition, NGS can be evaluated for exploratory studies of non-genetic (or tumor-specific) biomarkers, including but not limited to cancer-associated genes and biomarkers associated with common molecular pathways, and exploratory biomarkers, including but not limited to markers associated with immunity, MAP kinase pathways, or CRC biology, such as T cell markers or tumor mutational status.

This specification uses examples to disclose the invention. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Sequence listing

<110> Gene Tak corporation (Genentech, Inc.)

<120> combination therapy of MEK inhibitors, PD-1 axis inhibitors, and VEGF inhibitors

<130>P33774 (33988-58)

<150>US 62/374437

<151>2016-08-12

<160>40

<170>PatentIn version 3.5

<210>1

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala LeuGly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>2

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>2

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>3

<211>447

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>3

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg PheAsp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210>4

<211>218

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>4

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>5

<211>117

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210>6

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>6

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

3540 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210>7

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>8

<211>122

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120

<210>9

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210>10

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> modified residue

<222>(6)..(6)

<223> Asp or Gly

<400>10

Gly Phe Thr Phe Ser Xaa Ser Trp Ile His

1 5 10

<210>11

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> modified residue

<222>(4)..(4)

<223> Ser or Leu

<220>

<221> modified residue

<222>(10)..(10)

<223> Thr or Ser

<400>11

Ala Trp Ile Xaa Pro Tyr Gly Gly Ser Xaa Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210>12

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>12

Arg His Trp Pro Gly Gly Phe Asp Tyr

1 5

<210>13

<211>25

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210>14

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>14

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

1 5 10

<210>15

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>15

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>16

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>16

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210>17

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> modified residue

<222>(5)..(5)

<223> Asp or Val

<220>

<221> modified residue

<222>(6)..(6)

<223> Val or Ile

<220>

<221> modified residue

<222>(7)..(7)

<223> Ser or Asn

<220>

<221> modified residue

<222>(9)..(9)

<223> Ala or Phe

<220>

<221> modified residue

<222>(10)..(10)

<223> Val or Leu

<400>17

Arg Ala Ser Gln Xaa Xaa XaaThr Xaa Xaa Ala

1 5 10

<210>18

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> modified residue

<222>(4)..(4)

<223> Phe or Thr

<220>

<221> modified residue

<222>(6)..(6)

<223> Tyr or Ala

<400>18

Ser Ala Ser Xaa Leu Xaa Ser

1 5

<210>19

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> modified residue

<222>(3)..(3)

<223> Tyr, Gly, Phe, or Ser

<220>

<221> modified residue

<222>(4)..(4)

<223> Leu, Tyr, Phe or Trp

<220>

<221> modified residue

<222>(5)..(5)

<223> Tyr, Asn, Ala, Thr, Gly, Phe, or Ile

<220>

<221> modified residue

<222>(6)..(6)

<223> His, Val, Pro, Thr, or Ile

<220>

<221> modified residue

<222>(8)..(8)

<223> Ala, Trp, Arg, Pro, or Thr

<400>19

Gln Gln Xaa Xaa Xaa Xaa Pro Xaa Thr

1 5

<210>20

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210>21

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>21

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210>22

<211>32

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>22

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 1015

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210>23

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>23

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

1 5 10

<210>24

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>24

Gly Phe Thr Phe Ser Asp Ser Trp Ile His

1 5 10

<210>25

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>25

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210>26

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>26

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210>27

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>27

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210>28

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>28

Gln Gln Tyr Leu Tyr His Pro Ala Thr

1 5

<210>29

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210>30

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>30

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>31

<211>447

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>31

Glu Val Gln Leu Val Glu SerGly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210>32

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>33

<211>123

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>34

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210>35

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>35

Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

1 5 10

<210>36

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>36

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys

1 5 10 15

Arg

<210>37

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>37

Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

1 5 10

<210>38

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>38

Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210>39

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>39

Phe Thr Ser Ser Leu His Ser

1 5

<210>40

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>40

Gln Gln Tyr Ser Thr Val Pro Trp Thr

1 5

Claims (30)

1. A method of treating a subject having colorectal cancer, the method comprising administering to the subject a therapy comprising (i) a therapeutically effective amount of a MEK inhibitor, (ii) a therapeutically effective amount of a PD-1 axis inhibitor, and (iii) a therapeutically effective amount of a VEGF inhibitor.

2. The method of claim 1, wherein the subject has metastatic colorectal cancer.

3. The method of claim 1 or claim 2, wherein the MEK inhibitor is cobicistinib (cobimetinib) or a pharmaceutically acceptable salt thereof.

4. The method of any one of claims 1 to 3, wherein the PD-1 axis inhibitor is a PD-L1 inhibitor.

5. The method of claim 4, wherein the PD-L1 inhibitor is an antibody comprising a heavy chain and a light chain, the heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:24), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:25), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:12) and the light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:26), the HVR-L2 sequence of SASFLYS (SEQ ID NO:27), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 28).

6. The method of claim 4, wherein the PD-L1 inhibitor is an antibody comprising a heavy chain variable region and a light chain variable region,

the heavy chain variable region comprises the amino acid sequence of EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAWISPYGGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSS (SEQ ID NO:7) and

the light chain variable region comprises

DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR (SEQ ID NO: 9).

7. The method of any one of claims 1 to 6, wherein the PD-L1 inhibitor is atelizumab (atezolizumab).

8. The method of any one of claims 1 to 7, wherein the VEGF inhibitor is an antibody comprising a heavy chain comprising the HVR-H1 sequence of GYTFTNYGMN (SEQ ID NO:35), the HVR-H2 sequence of WINTYTGEPTYAADFKR (SEQ ID NO:36), and the HVR-H3 sequence of YPHYYGSSHWYFDV (SEQ ID NO:37) and a light chain comprising the HVR-L1 sequence of SASQDISNYLN (SEQ ID NO:38), the HVR-L2 sequence of FTSSLHS (SEQ ID NO:39), and the HVR-L3 sequence of QQYSTVPWT (SEQ ID NO: 40).

9. The method of any one of claims 1 to 7, wherein the VEGF inhibitor is an antibody comprising a heavy chain variable region and a light chain variable region,

the heavy chain variable region comprises the amino acid sequence of EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVTVSS (SEQ ID NO:33) and

the light chain variable region comprises the amino acid sequence of DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYFTSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO: 34).

10. The method of any one of claims 1 to 9, wherein the VEGF inhibitor is bevacizumab (bevacizumab).

11. The method of any one of claims 1 to 10, wherein the subject is treated with about 20mg to about 100mg, about 40mg to about 80mg, or about 60mg of the MEK inhibitor per day.

12. The method of claim 11, wherein the MEK inhibitor is cobicisinib or a pharmaceutically acceptable salt thereof, and further wherein the subject is treated with about 60mg, about 40mg, or about 20 mg.

13. The method of any one of claims 1 to 12, wherein the MEK inhibitor is administered once daily for 21 consecutive days of a 28-day treatment cycle.

14. The method of claim 13, wherein the MEK inhibitor is administered on days 3 to 23 of the 28 day treatment cycle.

15. The method of any one of claims 1 to 14, wherein the subject is treated intravenously with about 400mg to about 1200mg, about 600mg to about 1000mg, about 700mg to about 900mg, or about 840mg of the PD-1 axis inhibitor every 14 days of a 28-day treatment cycle.

16. The method of claim 15, wherein the PD-1 axis inhibitor is atelizumab, and further wherein the subject is treated with about 840 mg.

17. The method of claim 15 or claim 16, wherein the subject is treated with the PD-1 axis inhibitor on days 1and 15 of the 28-day treatment cycle.

18. The method of any one of claims 1 to 17, wherein the subject is treated with about 3mg per kg body weight to about 7mg per kg body weight, about 4mg per kg body weight to about 6mg per kg body weight, or about 5mg per kg body weight of the VEGF inhibitor every 14 days of a 28-day treatment cycle.

19. The method of claim 18, wherein the VEGF inhibitor is bevacizumab, and further wherein the subject is treated with about 5mg per kg body weight.

20. The method of claim 18 or claim 19, wherein the subject is treated with the VEGF inhibitor on days 1and 15 of the 28-day treatment cycle.

21. The method of any one of claims 1 to 20, wherein the MEK inhibitor, the PD-1 axis inhibitor, and the VEGF inhibitor are each administered on days 1and 15 of a 28 day treatment cycle.

22. The method of any one of claims 1 to 21, wherein the colorectal cancer is microsatellite-stabilized colorectal cancer.

23. The method of any one of claims 1 to 22, wherein each of the PD-1 axis inhibitor and the VEGF inhibitor is administered on days 1and 15 of a 28 day treatment cycle and wherein the PD-1 axis inhibitor is administered to the subject prior to administration of the VEGF inhibitor to the subject.

24. The method of any one of claims 1 to 23, wherein the MEK inhibitor is administered on days 1 to 21 of the 28 day treatment cycle.

25. A method of treating a subject having colorectal cancer, the method comprising administering to the subject a therapy comprising:

(i) a therapeutically effective amount of cobicistinib, or a pharmaceutically acceptable salt thereof;

(ii) a therapeutically effective amount of a PD-L1 inhibitor which is an antibody comprising:

(a) a heavy chain comprising the HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:24), the HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:25), and the HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 12); and a light chain comprising the HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:26), the HVR-L2 sequence of SASFLYS (SEQ ID NO:27), and the HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:28), or

(b) A heavy chain variable region comprising the amino acid sequence of EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYYADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSS (SEQ ID NO:7) and a light chain variable region comprising the amino acid sequence of DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKPGKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR (SEQ ID NO: 9); and

(iii) a therapeutically effective amount of a VEGF inhibitor, which is an antibody comprising:

(a) a heavy chain comprising the HVR-H1 sequence of GYTFTNYGMN (SEQ ID NO:35), the HVR-H2 sequence of WINTYTGEPTYAADFKR (SEQ ID NO:36), and the HVR-H3 sequence of YPHYYGSSHWYFDV (SEQ ID NO: 37); and a light chain comprising the HVR-L1 sequence of SASQDISNYLN (SEQ ID NO:38), the HVR-L2 sequence of FTSSLHS (SEQ ID NO:39), and the HVR-L3 sequence of QQYSTVPWT (SEQ ID NO:40), or

(b) A heavy chain variable region comprising the amino acid sequence of EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTYAADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:33) and a light chain variable region comprising the amino acid sequence of DIQMTQSPSS LSASVGDRVT ITCSASQDISNYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQGTKVEIKR (SEQ ID NO: 34).

26. The method of claim 25, wherein the total amount of cobicistinib or a pharmaceutically acceptable salt thereof is administered in an amount of about 60 mg; about 840mg of the PD-L1 inhibitor; and about 5mg per kg body weight of the VEGF inhibitor to treat the subject.

27. A kit for treating colorectal cancer in a human subject, the kit comprising a MEK inhibitor, a PD-1 axis inhibitor, a VEGF inhibitor, and a package insert comprising instructions for using a therapeutically effective amount of the MEK inhibitor, a therapeutically effective amount of the PD-1 axis inhibitor, and a therapeutically effective amount of the VEGF inhibitor to treat the subject.

28. The kit of claim 27, wherein the MEK inhibitor is cobitinib or a pharmaceutically acceptable salt thereof, the PD-1 axis inhibitor is the PD-L1 inhibitor atelizumab, and the VEGF inhibitor is bevacizumab.

29. A colorectal cancer therapy drug combination comprising:

(i) a MEK inhibitor in a dose of about 20mg to about 100mg, about 40mg to about 80mg, or about 80 mg;

(ii) a PD-1 axis inhibitor in a dose of about 400mg to about 1200mg, about 600mg to about 1000mg, about 700mg to about 900mg, or about 840 mg; and

(iii) a dose of about 5mg/kg to about 15mg/kg, about 5mg/kg to about 10mg/kg, about 5mg/kg, about 10mg/kg or about 15mg/kg of a VEGF inhibitor.

30. The colorectal cancer therapy drug combination of claim 29, wherein the MEK inhibitor is cobicistinib or a pharmaceutically acceptable salt thereof at a dose of about 60mg, the PD-1 axis inhibitor is the PD-LI inhibitor atezumab at a dose of about 840mg, and the VEGF inhibitor is bevacizumab at a dose of about 5mg per kg body weight.