Treatment of LAG-3 positive tumors

Abstract

SOLUTION: The present invention provides a method for treating tumors in a human patient, the method including: (i) identifying a patient as having a LAG-3 positive tumor; and (ii) administering, to the patient, a PD-1 pathway inhibitor, a combination of a PD1 pathway inhibitor and an immune checkpoint inhibitor, a combination of a LAG-3 inhibitor and a PD-1 pathway inhibitor, or an anti-CTLA4 antibody. In some embodiment, the method further includes identifying the patient as having the LAG-3 positive PD-L1 positive tumors. In some embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody.EFFECT: The method of the present invention can improve a response to a treatment with the PD-1 pathway inhibitor, the combination of the PD1 pathway inhibitor and the immune checkpoint inhibitor, or the combination of the LAG-3 inhibitor and the PD-1 pathway inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 512,648, filed May 30, 2017; U.S. Provisional Application No. 62 / 513,813, filed June 1, 2017; U.S. Provisional Application No. 62 / 555,176, filed September 7, 2017; and U.S. Provisional Application No. 62 / 582,178, filed November 6, 2017, which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION The invention disclosed herein relates to methods of treating LAG-3 positive malignancies in human patients with a PD-1 pathway inhibitor, a combination of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, a combination of a LAG-3 inhibitor and a PD-1 pathway inhibitor, or an anti-CTLA-4 antibody. [Background technology]

[0003] Background of the Invention Lymphocyte-activation gene-3 (LAG-3; CD223) is a type I transmembrane protein expressed on the cell surface of activated CD4+ and CD8+ T cells and subsets of NK and dendritic cells (Triebel F, et al., J. Exp. Med. 1990; 171:1393-1405; Workman CJ, et al., J. Immunol. 2009; 182(4):1885-91). LAG-3 is closely related to CD4, the coreceptor for T helper cell activation. Both molecules possess four extracellular Ig-like domains and require binding to their ligand, major histocompatibility complex (MHC) class II, for functional activity. In contrast to CD4, LAG-3 is expressed only on the cell surface of activated T cells, and its cleavage from the cell surface terminates LAG-3 signaling. LAG-3 has also been found as a soluble protein, but it does not bind to MHC class II and the function of soluble LAG-3 is unknown.

[0004] LAG-3 has been reported to play an important role in promoting regulatory T cell (Treg) activity and negatively regulating T cell activation and proliferation (Workman CJ, et al., J. Immunol. 2005;174:688-695). Both natural and induced Tregs express increased levels of LAG-3, which is required for their maximal suppressive function (Camisaschi C, et al., J. Immunol. 2010;184:6545-6551 and Huang CT, et al., Immunity. 2004;21:503-513). Furthermore, ectopic expression of LAG-3 in CD4+ effector T cells reduces their proliferation potential and confers regulatory capabilities on third-party T cells (Huang CT, et al., Immunity. 2004;21:503-513). Recent studies have shown that high LAG-3 expression on exhausted lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells contributes to their unresponsive state and limits CD8+ T cell antitumor responses (Blackburn SD, et al., Nat. Immunol. 2009;10:29-37 and Grosso JF, et al., J. Clin. Invest. 2007;117:3383-3392). In fact, LAG-3 maintains tolerance to self and tumor antigens through direct effects on CD8+ T cells in two mouse models (Grosso JF, et al., J. Clin. Invest. 2007;117:3383-3392).

[0005] However, the immune tolerance observed in the setting of tumor initiation and tumor recurrence appears to be mediated not solely by LAG-3 but by the co-expression of various T cell negative regulatory receptors. Chronic viral infection models (Blackburn SD, et al., Nat. Immunol. 2009; 10:29-37, Grosso JF, et al., J. Clin. Invest. 2007; 117:3383-3392 and Lyford-Pike S, et al., Cancer Res. 2013; 73(6):1733-41), knockout mice (Woo SR, et al., Cancer Res. 2012; 72:917-927; Okazaki T, et al., J. Exp Med. 2011; 208:395-407, and Bettini M. et al., J. Immunol. 2011; 187:3493-3498), tumor recurrence models (Goding SR, et al., J. Immunol. 2013; 190(9):4899-4909) and, to a lesser extent, from human cancer patients (Goding SR, et al., J. Immunol. 2013; 190(9):4899-4909, Matsuzaki J, et al., Proc. Natl. Acad. Sci., USA. 2010; 107:7875-7880, and Gandhi MK, et al., Blood. 2006; 108:2280-2289) support a model in which T cells continuously exposed to antigen gradually become inactivated through a process termed "exhaustion." Exhausted T cells are characterized by the expression of T cell negative regulatory receptors, primarily cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death 1 (PD-1), and LAG-3, whose effects limit the cells' ability to proliferate, produce cytokines, kill target cells, and / or increase Treg activity. However, the timing and sequence of expression of these molecules during tumor development and recurrence are not fully understood.

[0006] PD-1 is a cell surface signaling receptor that plays an important role in regulating T cell activation and tolerance (Keir ME, et al., Annu Rev Immunol 2008; 26:677-704). It is a type I transmembrane protein and, together with BTLA, CTLA-4, ICOS, and CD28, constitutes the CD28 family of T cell costimulatory receptors. PD-1 is primarily expressed on activated T cells, B cells, and myeloid cells (Dong H, et al., Nat Med. 1999; 5:1365-1369). It is also expressed on natural killer (NK) cells (Terme M, et al., Cancer Res 2011; 71:5393-5399). Binding of PD-1 to its ligands, PD-L1 and PD-L2, results in phosphorylation of tyrosine residues in the proximal intracellular immunoreceptor tyrosine inhibitory domain, followed by recruitment of the phosphatase SHP-2, ultimately leading to downregulation of T cell activation. One important role of PD-1 is to restrict T cell activity in peripheral tissues during the inflammatory response to infection, thereby limiting the development of autoimmunity (Pardoll D M., Nat Rev Cancer 2012; 12:252-264). Evidence for this negative regulatory role comes from the discovery that PD-1-deficient mice develop lupus-like autoimmune disease, including arthritis and nephritis along with cardiomyopathy (Nishimura H, et al., Immunity, 1999; 11:141-151; and Nishimura H, et al., Science, 2001; 291:319-322). In the tumor context, this results in the development of immune tolerance within the microenvironment. PD-1 is highly expressed on tumor-infiltrating lymphocytes, and its ligand is upregulated on the cell surface of various tumors (Dong H, et al., Nat Med 2002; 8:793-800). Multiple mouse cancer models have shown that ligand binding to PD-1 leads to immune evasion. Furthermore, blocking this interaction results in antitumor activity (Topalian SL, et al. NEJM 2012; 366(26):2443-2454; Hamid O, et al., NEJM 2013; 369:134-144).Furthermore, inhibition of PD-1 / PD-L1 interaction has been shown to mediate potent antitumor activity in preclinical models (U.S. Patents 8,008,449 and 7,943,743).

[0007] Recently, several immune checkpoint pathway inhibitors have begun to provide novel immunotherapeutic approaches for cancer treatment, including the development of ipilimumab (Yervoy®), an antibody (Ab) that binds to and inhibits cytotoxic T-lymphocyte antigen-4 (CTLA-4) for the treatment of patients with advanced melanoma; antibodies such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement, (2013), pembrolizumab: Statement on a nonproprietary name adopted by the USAN Council (ZZ-165), November 27, 2013), which specifically bind to the programmed death-1 (PD-1) receptor and block the inhibitory PD-1 / PD-1 ligand pathway; and BMS-986016 (described in U.S. Patent 9,505,839), an antibody that specifically binds to LAG-3 and can stimulate an immune response. Summary of the Invention [Problem to be solved by the invention]

[0008] The emerging field of personalized medicine promises that advances in pharmacogenomics will increasingly be used to tailor therapeutic agents to defined subpopulations and ultimately individual patients to enhance efficacy and minimize adverse effects. Recent successes include, for example, imatinib mesylate (Gleevec®), a protein tyrosine kinase inhibitor that inhibits the bcr-abl tyrosine kinase for Philadelphia chromosome-positive chronic myeloid leukemia (CML); crizotinib (Xalkori®) for treating the 5% of patients with late-stage non-small cell lung cancer who express the mutant anaplastic lymphoma kinase (ALK) gene; and vemurafenib (Zelboraf®), an inhibitor of the mutant B-RAF protein (V600E-BRAF) expressed in approximately half of melanoma tumors. However, unlike the clinical development of small molecule agents targeting individual activating mutations found in select cancer populations, a particular obstacle in cancer immunotherapy is the identification of predictive biomarkers that enable patient selection and guide treatment management. It is therefore an object of the present invention to provide improved methods of treating tumors. [Means for solving the problem]

[0009] Summary of the Invention Certain aspects of the presently disclosed invention relate to methods for selecting malignant tumors in a human patient for treatment with a PD-1 pathway inhibitor, a LAG-3 inhibitor, a combination of a PD1 pathway inhibitor and an immune checkpoint inhibitor, or a combination of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the method comprises detecting LAG-3 expression in the tumor. In certain embodiments, the method comprises detecting LAG-3 expression and PD-L1 expression in the tumor. Also disclosed herein are methods for treating LAG-3-positive tumors in a human patient, comprising administering a LAG-3 inhibitor and a PD-1 pathway inhibitor.

[0010] One embodiment of the presently disclosed invention is a method for selecting malignant tumors in a human patient for immunotherapy, comprising determining the expression level of LAG-3 in a tumor sample and, if the tumor is a LAG-3-positive tumor, selecting the tumor for immunotherapy. Another embodiment of the presently disclosed invention is a method for identifying malignant tumors in a human patient as being eligible for immunotherapy, comprising determining the expression level of LAG-3 in a tumor sample and, if the tumor is a LAG-3-positive tumor, identifying the tumor as being eligible for immunotherapy. Another embodiment of the presently disclosed invention is a method for identifying malignant tumors in a human patient that may be responsive to immunotherapy, comprising determining the expression level of LAG-3 in a tumor sample and, if the tumor is a LAG-3-positive tumor, identifying the tumor as being likely to be responsive to treatment. Another aspect of the presently disclosed invention relates to a method for classifying a malignant tumor in a human patient as likely to be responsive to immunotherapy, the method comprising determining the LAG-3 expression level in a tumor sample, and if the tumor is a LAG-3-positive tumor, classifying the tumor as likely to be responsive to immunotherapy. In certain embodiments, the presently disclosed method further comprises determining the PD-L1 expression level in the tumor sample. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the presently disclosed method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the presently disclosed method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In some embodiments, the methods disclosed herein comprise contacting a tumor with a therapeutically effective amount of a PD-1 pathway inhibitor.In certain embodiments, the methods disclosed herein comprise contacting a tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the methods disclosed herein comprise administering to a patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the methods disclosed herein comprise administering to a patient a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the methods disclosed herein comprise administering to a patient a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the methods disclosed herein comprise administering to a patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

[0011] Another embodiment of the presently disclosed invention relates to a method for identifying a patient having a malignant tumor that is likely to respond to immunotherapy, the method comprising determining the LAG-3 expression level in a tumor sample, and if the tumor is a LAG-3-positive tumor, identifying the patient as likely to respond to treatment. Another embodiment of the presently disclosed invention relates to a method for selecting a patient having a malignant tumor for immunotherapy, the method comprising determining the LAG-3 expression level in a tumor sample, and if the tumor is a LAG-3-positive tumor, selecting the patient for immunotherapy. In certain embodiments, the methods disclosed herein further comprise determining the PD-L1 expression level in the tumor sample. In certain embodiments, the methods disclosed herein comprise administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the methods disclosed herein comprise administering to the patient a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the methods disclosed herein comprise administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the methods disclosed herein comprise administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor.

[0012] Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is predicted to respond to treatment with the LAG-3 inhibitor and the PD-1 pathway inhibitor based on LAG-3 expression in a tumor sample from the patient. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor, wherein the patient is predicted to respond to treatment with the LAG-3 inhibitor based on LAG-3 expression in a tumor sample from the patient. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient is predicted to respond to treatment with the PD-1 pathway inhibitor based on LAG-3 expression in a tumor sample from the patient. Another aspect of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is predicted to respond to treatment with the PD-1 pathway inhibitor and the immune checkpoint inhibitor based on LAG-3 expression in a tumor sample from the patient. In one embodiment, the patient is predicted to respond to treatment based on LAG-3 and PD-L1 expression in a tumor sample from the patient.

[0013] Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising determining the expression level of LAG-3 in a tumor sample, and if the tumor is a LAG-3-positive tumor, administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising determining the expression level of LAG-3 in a tumor sample, and if the tumor is a LAG-3-positive tumor, administering to the patient a therapeutically effective amount of a LAG-3 inhibitor. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising determining the expression level of LAG-3 in a tumor sample, and if the tumor is a LAG-3-positive tumor, administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. Another aspect of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need thereof, comprising determining the expression level of LAG-3 in a tumor sample, and, if the tumor is a LAG-3-positive tumor, administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the presently disclosed method further comprises determining the expression level of PD-L1 in the tumor sample.

[0014] Another embodiment of the presently disclosed invention relates to a method of treating a malignancy in a human patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has been identified as having a LAG-3-positive malignancy prior to administration.Another embodiment of the presently disclosed invention relates to a method of treating a malignancy in a human patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor, wherein the patient has been identified as having a LAG-3-positive malignancy prior to administration.Another embodiment of the presently disclosed invention relates to a method of treating a malignancy in a human patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient has been identified as having a LAG-3-positive malignancy prior to administration. Another aspect of the presently disclosed invention is a method of treating a malignancy in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient has been identified as having a LAG-3-positive malignancy prior to administration. In certain embodiments, the patient has been identified as having a LAG-3-positive, PD-L1-positive malignancy prior to administration. In certain embodiments, the patient has been identified as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

[0015] Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising identifying the patient as having a LAG-3-positive malignant tumor and administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising identifying the patient as having a LAG-3-positive malignant tumor and administering to the patient a therapeutically effective amount of a LAG-3 inhibitor. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising identifying the patient as having a LAG-3-positive malignant tumor and administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. Another embodiment of the presently disclosed invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising identifying the patient as having a LAG-3-positive malignant tumor and administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy.

[0016] Another embodiment of the presently disclosed invention relates to a method of extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has been identified prior to administration as having a LAG-3-positive malignant tumor and the patient exhibits a progression-free survival of greater than 12 months. Another embodiment of the presently disclosed invention relates to a method of extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient a LAG-3 inhibitor, wherein the patient has been identified prior to administration as having a LAG-3-positive malignant tumor and the patient exhibits a progression-free survival of greater than 12 months. Another embodiment of the presently disclosed invention relates to a method of extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient a PD-1 pathway inhibitor, wherein the patient has been identified prior to administration as having a LAG-3-positive malignant tumor and the patient exhibits a progression-free survival of greater than 12 months. Another embodiment of the invention disclosed herein is a method for extending progression-free survival to more than 12 months in a human patient with a malignant tumor, comprising administering to the patient a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the patient exhibits progression-free survival of more than 12 months. In certain embodiments, the patient is identified as having a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the patient is identified as having a LAG-3-positive, PD-L1-negative malignant tumor prior to administration. In certain embodiments, the patient's progression-free survival is extended by more than about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration. In certain embodiments, the patient's progression-free survival is extended by more than 14 months.

[0017] Another embodiment of the presently disclosed invention relates to a method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. Another embodiment of the invention disclosed herein relates to a method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. Another embodiment of the presently disclosed invention relates to a method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. Another aspect of the presently disclosed invention is a method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. In one embodiment, the patient is identified as having a LAG-3-positive, PD-L1-positive malignant tumor prior to administration.In certain embodiments, the patient is identified as having a LAG-3-positive, PD-L1-negative malignancy prior to administration. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive malignancy prior to administration. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy prior to administration. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy prior to administration. In certain embodiments, the patient experiences (i) an extended progression-free survival of more than 12 months, (ii) at least about a 10%, about 20%, about 30%, about 40%, or about 50% reduction in tumor size compared to tumor size prior to administration, or (iii) both.

[0018] Another embodiment of the invention disclosed herein relates to a method of increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%.Another embodiment of the invention disclosed herein relates to a method of increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. Another embodiment of the invention disclosed herein relates to a method for increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients a therapeutically effective amount of a PD-1 pathway inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. Another embodiment of the invention disclosed herein relates to a method for increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. In one embodiment, each patient is identified prior to administration as having a LAG-3-positive, PD-L1-positive malignant tumor. In certain embodiments, each patient is identified as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

[0019] Another embodiment of the invention disclosed herein relates to a method for increasing the disease control rate to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. Another embodiment of the invention disclosed herein relates to a method for increasing the disease control rate to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. Another embodiment of the invention disclosed herein relates to a method for increasing the disease control rate to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients a therapeutically effective amount of a PD-1 pathway inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. Another embodiment of the invention disclosed herein relates to a method for increasing the disease control rate to greater than 50% in a population of human patients each having a malignant tumor, the method comprising administering to the patients therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein each patient is identified prior to administration as having a LAG-3-positive malignant tumor, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. In one embodiment, each patient is identified prior to administration as having a LAG-3-positive, PD-L1-positive malignant tumor. In certain embodiments, each patient is identified as having a LAG-3-positive, PD-L1-negative malignant tumor prior to administration. In certain embodiments, the median duration of response is ≧3 months, ≧6 months, ≧12 months, or ≧18 months.

[0020] In certain embodiments, the methods disclosed herein further comprise identifying each patient in the patient population as having a LAG-3-positive malignancy prior to administration. In certain embodiments, the methods disclosed herein further comprise identifying each patient in the patient population as having a LAG-3-positive, PD-L1-positive malignancy prior to administration. In certain embodiments, the methods disclosed herein further comprise identifying each patient in the patient population as having a LAG-3-positive, PD-L1-negative malignancy prior to administration. In certain embodiments, each patient in the patient population is further characterized by (i) prolonged progression-free survival of greater than 12 months, (ii) at least about a 10%, about 20%, about 30%, about 40%, or about 50% reduction in tumor size compared to tumor size prior to administration, or (iii) both. In certain embodiments, the patient population comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 patients with a LAG-3-positive malignancy.

[0021] Another embodiment of the presently disclosed invention relates to a method for selecting a human patient suitable for combination therapy, the method comprising identifying the patient as having a LAG-3-positive malignancy; and instructing a healthcare professional to administer to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. Another embodiment of the presently disclosed invention relates to a method for selecting a human patient suitable for combination therapy, the method comprising identifying the patient as having a LAG-3-positive malignancy; and instructing a healthcare professional to administer to the patient a therapeutically effective amount of a LAG-3 inhibitor. Another embodiment of the presently disclosed invention relates to a method for selecting a human patient suitable for combination therapy, the method comprising identifying the patient as having a LAG-3-positive malignancy; and instructing a healthcare professional to administer to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. Another aspect of the presently disclosed invention relates to a method for selecting a human patient suitable for combination therapy, the method comprising: identifying the patient as having a LAG-3-positive malignancy; and instructing a healthcare professional to administer therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor to the patient. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy. In certain embodiments, the administering treats the malignancy.

[0022] In certain embodiments, identifying a patient as having a LAG-3-positive malignancy comprises determining LAG-3 expression in the malignancy. In certain embodiments, identifying a patient as having a LAG-3-positive, PD-L1-positive malignancy comprises determining PD-L1 expression in the malignancy. In certain embodiments, identifying a patient as having a LAG-3-positive, PD-L1-negative malignancy comprises determining PD-L1 expression in the malignancy. In certain embodiments, LAG-3 expression is determined by examining the results of an assay capable of determining LAG-3 expression. In certain embodiments, LAG-3 expression is determined by examining the results of an immunohistochemistry assay capable of detecting LAG-3 expression. In certain embodiments, PD-L1 expression is determined by examining the results of an assay capable of determining PD-L1 expression. In certain embodiments, PD-L1 expression is determined by examining the results of an immunohistochemistry assay capable of detecting PD-L1 expression.

[0023] In some embodiments, LAG-3-positive tumors contain at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% LAG-3-expressing cells. In some embodiments, LAG-3-positive tumors contain at least about 1% LAG-3-expressing cells. In some embodiments, LAG-3-positive tumors contain at least about 5% LAG-3-expressing cells. In some embodiments, LAG-3-expressing cells include tumor-infiltrating lymphocytes. In some embodiments, LAG-3-expressing cells are the total number of cells. In other embodiments, cells express LAG-3 on the cell surface.

[0024] In some embodiments, the malignant tumor is liver cancer, bone cancer, pancreatic cancer, skin cancer, oral cancer, head and neck cancer, breast cancer, lung cancer including small cell and non-small cell lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's tumour and any combination thereof.

[0025] In certain embodiments, the malignant tumor is selected from melanoma, non-small cell lung cancer (NSCLC), human papillomavirus (HPV)-associated tumors, and gastric adenocarcinoma.

[0026] In certain embodiments, the malignant tumor is NSCLC, a virus-associated cancer-associated tumor, or gastric adenocarcinoma.

[0027] In certain embodiments, the malignant tumor is melanoma, gastric cancer, gastroesophageal junction cancer, non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, or renal cell carcinoma.

[0028] In some embodiments, the malignant tumor is lung cancer, melanoma, head and neck squamous cell carcinoma, renal cancer, gastric cancer, or hepatocellular carcinoma.

[0029] In certain embodiments, the LAG-3 positive malignant tumor is a melanoma tumor that contains about 1% or more LAG-3 expressing cells.

[0030] In certain embodiments, the LAG-3 positive malignant tumor is a gastric cancer tumor that contains about 1% or more LAG-3 expressing cells.

[0031] In certain embodiments, the malignant tumor is refractory to treatment with an immune checkpoint inhibitor. In certain embodiments, the malignant tumor is refractory to treatment with an anti-PD-1 antibody. In certain embodiments, the malignant tumor is refractory to treatment with an anti-PD-L1 antibody.

[0032] Another aspect of the presently disclosed invention relates to a method of treating melanoma in a human patient, the method comprising: identifying the patient as having a LAG-3-positive melanoma; and administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, identifying the patient as having a LAG-3-positive melanoma comprises determining LAG-3 expression in the melanoma tumor. In certain embodiments, LAG-3 expression is determined by examining the results of an assay capable of determining LAG-3 expression. In certain embodiments, LAG-3 expression is determined by an immunohistochemistry assay capable of detecting LAG-3 expression. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy. In certain embodiments, the methods disclosed herein further comprise identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy.

[0033] Another embodiment of the invention disclosed herein relates to a method of treating melanoma in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive melanoma prior to administration. Another embodiment of the invention disclosed herein relates to a method of extending progression-free survival to more than 12 months in a human patient with melanoma, comprising administering to the patient a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive melanoma prior to administration, and wherein the patient exhibits progression-free survival of more than 12 months. In certain embodiments, the patient is identified as having a LAG-3-positive, PD-L1-positive melanoma prior to administration. In certain embodiments, the patient is identified as having a LAG-3-positive, PD-L1-negative melanoma prior to administration.

[0034] Another embodiment of the presently disclosed invention relates to a method for increasing the objective response rate to cancer treatment to greater than 15% in a population of human patients each having melanoma, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignancy prior to administration, and the objective response rate is greater than 15%. Another embodiment of the presently disclosed invention relates to a method for increasing the disease control rate to cancer treatment to greater than 70% in a population of human patients each having melanoma, the method comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive melanoma prior to administration, and the objective response rate is greater than 70%. In certain embodiments, the methods disclosed herein further comprise identifying each patient in the patient population as having a LAG-3-positive melanoma prior to administration. In certain embodiments, the median duration of response is greater than 3 months, greater than 6 months, greater than 12 months, or greater than 18 months. In certain embodiments, each patient is identified as having LAG-3-positive, PD-L1-positive melanoma prior to administration. In certain embodiments, each patient is identified as having LAG-3-positive, PD-L1-negative melanoma prior to administration.

[0035] In certain embodiments, the melanoma is refractory to treatment with an immune checkpoint inhibitor. In certain embodiments, the melanoma is refractory to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0036] In some embodiments, determining the expression level of LAG-3 and / or PD-L1 comprises providing a test tissue sample from a patient, wherein the test tissue sample comprises tumor cells and / or tumor-infiltrating immune cells. In some embodiments, the test tissue sample is a tumor biopsy sample. In some embodiments, the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample.

[0037] In some embodiments, the determining comprises detecting LAG-3 and / or PD-L1 protein or RNA expression in a test tissue sample.

[0038] In one embodiment, LAG-3 and / or PD-L1 expression is detected by an assay capable of detecting LAG-3 and / or PD-L1 protein levels, respectively, in a test tissue sample.

[0039] In some embodiments, LAG-3 and / or PD-L1 expression is detected by immunohistochemistry. In some embodiments, the immunohistochemistry assay is a monoplex assay (an assay designed to detect / measure the presence of a single analyte, e.g., an antigen / antibody pair). In some embodiments, the immunohistochemistry assay is a multiplex assay (an assay designed to detect / measure the presence of multiple analytes, simultaneously or sequentially). In some embodiments, the immunohistochemistry assay comprises contacting the tumor sample with 17B4, SP346, 11E3, 874501, or EPR4392 (2) anti-human LAG-3 monoclonal antibody. In some embodiments, the immunohistochemistry assay comprises contacting the tumor sample with an anti-LAG-3 antibody comprising heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 3 and 5, respectively.

[0040] In some embodiments, the immunohistochemistry assay uses a black or brown chromogen. In some embodiments, the immunohistochemistry assay uses a red chromogen. In some embodiments, the immunohistochemistry assay uses a blue chromogen. In some embodiments, the immunohistochemistry assay uses a green chromogen. In some embodiments, the immunohistochemistry assay uses a purple chromogen. In some embodiments, the immunohistochemistry assay uses a yellow chromogen.

[0041] In some embodiments, immunohistochemistry assays are scored at low magnification (e.g., 4x or 10x). In some embodiments, low magnification is about 20x.

[0042] In some embodiments, immunohistochemistry assays are scored at high magnification, in some embodiments, high magnification is about 40x or greater (60x, 100x).

[0043] In some embodiments, the immunohistochemistry assay is scored by image analysis software. In some embodiments, the immunohistochemistry assay is scored manually by a pathologist.

[0044] In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of cells expressing LAG-3 in the test tissue sample and / or assessing the proportion of cells expressing PD-L1 in the test tissue sample. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of tumor cells expressing LAG-3 in the test tissue sample and / or assessing the proportion of tumor cells expressing PD-L1 in the test tissue sample. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of immune cells expressing LAG-3 in the test tissue sample and / or assessing the proportion of immune cells expressing PD-L1 in the test tissue sample. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of T cells expressing LAG-3 in the test tissue sample and / or assessing the proportion of T cells expressing PD-L1 in the test tissue sample. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of CD8+ T cells expressing LAG-3 in the test tissue sample and / or assessing the proportion of CD8+ T cells expressing PD-L1 in the test tissue sample. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of CD4+ T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of CD4+ T cells in the test tissue sample that express PD-L1. In certain embodiments, scoring the immunohistochemistry assay comprises assessing the proportion of FOXP3+ T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of FOXP3+ T cells in the test tissue sample that express PD-L1.

[0045] In some embodiments, cells with partial membrane / cytoplasmic LAG-3 localization are scored as LAG-3 expressing cells. In some embodiments, cells with dot-like LAG-3 localization are scored as LAG-3 expressing cells. In some embodiments, cells with complete membrane / cytoplasmic LAG-3 localization are scored as LAG-3 expressing cells. In some embodiments, cells with any LAG-3 localization pattern are scored as LAG-3 expressing cells.

[0046] In some embodiments, the immunohistochemistry assay is a multiplex assay that further comprises detecting MHC class II expression by tumor cells.In some embodiments, the scoring of the immunohistochemistry assay comprises evaluating the proportion of cells that express MHC class II in the test tissue sample.In some embodiments, the scoring of the immunohistochemistry assay comprises evaluating the proportion of non-immune cells that express MHC class II in the test tissue sample.

[0047] In some embodiments, LAG-3 and / or PD-L1 protein expression is detected by flow cytometry. In some embodiments, the test tissue sample obtained from the patient contains tumor-infiltrating immune cells. In some embodiments, the malignant tumor is a hematopoietic tumor, and the tissue sample contains circulating lymphocytes. In some embodiments, the flow cytometry is a multiplex assay. In some embodiments, the flow cytometry includes detecting the expression of markers including LAG-3, PD-L1, CD4, CD8, FOXP3, MHC class II, and any combination thereof.

[0048] In some embodiments, flow cytometry scoring comprises assessing the proportion of T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises assessing the proportion of CD8+ T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises assessing the proportion of CD4+ T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises assessing the proportion of FOXP3+ T cells expressing LAG-3 in the test tissue sample.

[0049] In certain embodiments, LAG-3 and / or PD-L1 expression is detected by an assay capable of detecting the level of LAG-3 and / or PD-L1 RNA, respectively, in a tumor sample. In certain embodiments, LAG-3 and / or PD-L1 expression is detected by an RT-PCR-based assay. In certain embodiments, scoring of the RT-PCR-based assay involves assessing the level of LAG-3 and / or PD-L1 RNA expression in the test tissue sample relative to a predetermined level.

[0050] In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-LAG-3 antibody is a bispecific antibody.

[0051] In one embodiment, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 7; (b) a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 8; (c) a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 9; (d) a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 10; (e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 11; and (f) a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 12.

[0052] In certain embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 3 and 5, respectively.

[0053] In certain embodiments, the anti-LAG-3 antibody is MK-4280 (28G-10), REGN3767, GSK2837781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-525), IMP321, FS-118, Sym022, TSR-033, MGD013, FS118, or GSK2831781.

[0054] In some embodiments, the PD-1 pathway inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:23; (b) a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:24; (c) a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:25; (d) a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:26; (e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:27; and (f) a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:28.

[0055] In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 19 and 21, respectively.

[0056] In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the sequences set forth in SEQ ID NOs: 17 and 18, respectively.

[0057] In certain embodiments, the anti-PD-1 antibody is pembrolizumab (Keytruda; MK-3475), pidilizumab (CT-011), or nivolumab (Opdivo; BMS-936558).

[0058] In some embodiments, the PD-1 pathway inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody is atezolizumab (Tecentriq or RG7446), durvalumab (Imfinzi or MEDI4736), avelumab (Bavencio), or BMS-936559.

[0059] In certain embodiments, the PD-1 pathway inhibitor is an anti-PD-L2 antibody or antigen-binding fragment thereof.

[0060] In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist, CD80 antagonist, CD86 antagonist, Tim-3 antagonist, TIGIT antagonist, CD20 antagonist, CD96 antagonist, IDO1 antagonist, STING antagonist, GARP antagonist, CD40 antagonist, A2aR antagonist, CEACAM1 (CD66a) antagonist, CEA antagonist, CD47 antagonist, PVRIG antagonist, TDO antagonist, VISTA antagonist, or KIR antagonist.

[0061] In certain embodiments, the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-LAG-3 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg, or 240 mg.

[0062] In certain embodiments, the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-PD-1 antibody is administered four times at a dose of 80 mg or 240 mg.

[0063] In certain embodiments, the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-PD-L1 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg, or 240 mg.

[0064] In certain embodiments, the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-LAG-3 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg, or 240 mg, and the anti-PD-1 antibody is administered four times at a dose of 80 mg or 240 mg.

[0065] In certain embodiments, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered in the following doses: (a) 3 mg of anti-LAG-3 antibody and 80 mg of anti-PD-1 antibody; (b) 3 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (c) 20 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (d) 80 mg of anti-LAG-3 antibody and 160 mg of anti-PD-1 antibody; (e) 80 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (f) 160 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody, or (g) 240 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0066] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at doses of 80 mg of anti-LAG-3 antibody and 160 mg of anti-PD-1 antibody.

[0067] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at doses of 80 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0068] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 160 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0069] In certain embodiments, the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated for intravenous administration.

[0070] In certain embodiments, the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated together. In certain embodiments, the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated separately.

[0071] In some embodiments, the treatment consists of up to 12 cycles.

[0072] In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered on days 1, 15, 29, and 43 of each cycle.

[0073] In certain embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered on days 1, 15, 29, and 43 of each cycle.

[0074] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered prior to administration of the anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is administered within 30 minutes prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered after administration of the anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered prior to administration of the anti-LAG-3 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is administered simultaneously with the anti-LAG-3 antibody or antigen-binding fragment thereof.

[0075] In certain embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof and the PD-1 pathway inhibitor are administered as a first line treatment. In certain embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof and the PD-1 pathway inhibitor are administered as a second line treatment.

[0076] In some embodiments, the methods disclosed herein further comprise administering at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the at least one additional therapeutic agent is an immune checkpoint inhibitor.

[0077] In certain embodiments, the method results in at least one therapeutic effect selected from a reduction in tumor size, a reduction in the number of metastatic lesions over time, a complete response, a partial response, and stable disease.

[0078] In certain embodiments, administration of an anti-LAG-3 antibody, or antigen-binding fragment thereof, and a PD-1 pathway inhibitor activates T cells in the patient. In certain embodiments, administration of an anti-LAG-3 antibody, or antigen-binding fragment thereof, and a PD-1 pathway inhibitor induces expression of activation markers by T cells in the patient.

[0079] In some embodiments, administration of an anti-LAG-3 antibody or its antigen-binding fragment results in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% occupancy of the LAG-3 receptor on the patient's T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are tumor-infiltrating T cells.

[0080] In certain embodiments, the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

[0081] Another embodiment of the presently disclosed invention relates to a kit for treating a patient with a malignant tumor, the kit comprising: a dose of an anti-LAG-3 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; a dose of an anti-PD-1 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; and instructions for using the anti-LAG-3 antibody and the anti-PD-1 antibody or antigen-binding fragment thereof in any of the methods disclosed herein.

[0082] Another embodiment of the presently disclosed invention relates to a kit for treating a patient with a malignant tumor, the kit comprising: a dose of an anti-PD-1 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; a dose of an immune checkpoint inhibitor; and instructions for using the anti-PD-1 antibody or antigen-binding fragment thereof and the immune checkpoint inhibitor in any of the methods disclosed herein.

[0083] Another embodiment of the presently disclosed invention relates to a kit for treating a patient with a malignant tumor, the kit comprising a dose of an anti-LAG-3 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; and instructions for using the anti-LAG-3 antibody or antigen-binding fragment thereof in any of the methods disclosed herein.

[0084] Another embodiment of the presently disclosed invention relates to a kit for treating a patient with a malignant tumor, the kit comprising a dose of an anti-PD-1 antibody or antigen-binding fragment in the range of 0.1 to 10 mg / kg body weight; and instructions for using the anti-PD-1 antibody or antigen-binding fragment thereof in any of the methods disclosed herein.

[0085] Certain aspects of the invention relate to methods for identifying patients refractory to treatment with a PD-1 antagonist, the method comprising determining LAG-3 expression levels, wherein an increase in LAG-3 expression levels after treatment with a PD-1 antagonist relative to the LAG-3 expression levels before treatment with the PD-1 antagonist indicates that the patient is refractory to PD-1 antagonist therapy. Other aspects of the invention relate to methods for identifying patients at risk of becoming refractory to treatment with a PD-1 antagonist, the method comprising determining LAG-3 expression levels, wherein an increase in LAG-3 expression levels after treatment with a PD-1 antagonist relative to the LAG-3 expression levels before treatment with the PD-1 antagonist indicates that the patient is at risk of becoming refractory to treatment with a PD-1 antagonist. Certain aspects of the present invention relate to methods for identifying patients who are likely to respond to LAG-3 therapy, comprising determining the expression level of LAG-3 in the patient, wherein an increase in the expression level of LAG-3 after treatment with a PD-1 antagonist relative to the expression level before treatment with the PD-1 antagonist indicates that the patient is likely to respond to LAG-3 therapy. Certain aspects of the present invention relate to methods for selecting patients for treatment with LAG-3 therapy, comprising determining the expression level of LAG-3 in the patient, wherein an increase in the expression level of LAG-3 after treatment with a PD-1 antagonist relative to the expression level before treatment with the PD-1 antagonist indicates that the patient is likely to respond to LAG-3 therapy. In certain embodiments, the PD-1 antagonist is a PD-1 inhibitor. In certain embodiments, the PD-1 antagonist is a PD-1 antibody. In certain embodiments, the LAG-3 therapy is a LAG-3 inhibitor. In certain embodiments, the LAG-3 therapeutic agent is an anti-LAG-3 antibody. In certain embodiments, the LAG-3 therapy is a combination therapy. In certain embodiments, the LAG-3 combination therapy is a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody. [Brief explanation of the drawings]

[0086] [Figure 1]Figure 1 shows the staining pattern observed in monoplex LAG-3 immunohistochemistry (IHC) samples.

[0087] [Figure 2] Figure 2 shows the frequency distribution of LAG-3+ cells as a proportion of total tumor cells in samples analyzed by monoplex LAG-3 IHC.

[0088] [Figure 3] Figures 3A-B show (Figure 3A) the study design and endpoints. (Figure 3B) Key eligibility criteria for patients in melanoma before the IO expansion cohort.

[0089] [Figure 4] Figure 4 shows the baseline study population and disease overview.

[0090] [Figure 5] Figure 5 shows the prior treatment.

[0091] [Figure 6] Figure 6 shows the LAG-3 expression status of the first 40 IO-experienced melanomas.

[0092] [Figure 7] Figure 7. Investigator-assessed response in patients with melanoma that progressed on prior anti-PD1 / PD-L1 therapy.

[0093] [Figure 8] Figure 8 LAG-3 expression and response enrichment.

[0094] [Figure 9] FIG. 9 shows the depth and duration of response due to LAG-3 expression.

[0095] [Figure 10] Figure 10 shows progression-free survival.

[0096] [Figure 11]Figure 11 shows response (investigator-assessed) by baseline characteristics.

[0097] [Figure 12] Figure 12 shows the LAG-3 expression status of gastric tumor samples. 48% (10 / 21) of the samples were scored as LAG-3 positive using a 1% cutoff in the monoplex IHC assay.

[0098] [Figure 13] Figure 13 shows the change in target lesion size in gastric cancer patients in response to treatment with a combination of anti-LAG-3 and anti-PD-1 antibodies. LAG-3-positive tumors were enriched in patients who responded to treatment. Tumor response was determined according to RECIST. Patients in this trial had not previously been exposed to anti-PD-1 / PD-L1 treatment.

[0099] [Figure 14] FIG. 14 shows the LAG-3 expression status of squamous cell carcinoma of the head and neck (SCCHN), renal carcinoma, hepatocellular carcinoma (HCC) and NSCLC tumor samples determined by monoplex IHC assay.

[0100] [Figure 15A] Figure 15A is a pigmented melanoma section. Nuclei were counterstained with hematoxylin with or without bleaching. Figure 15B is a pigmented melanoma LAG-3 IHC with or without prior bleaching. Nuclei were counterstained with hematoxylin. [Figure 15B] Figure 15A is a pigmented melanoma section. Nuclei were counterstained with hematoxylin with or without bleaching. Figure 15B is a pigmented melanoma LAG-3 IHC with or without prior bleaching. Nuclei were counterstained with hematoxylin.

[0101] [Figure 16] Figure 16 shows the updated study design and endpoints.

[0102] [Figure 17]Figure 17 shows the updated baseline study population and disease overview.

[0103] [Figure 18] Figure 18 shows the updated prior treatment.

[0104] [Figure 19] Figure 19 shows the updated antitumor activity of the combination treatment of BMS-986016 and nivolumab.

[0105] [Figure 20] Figure 20 is an updated baseline summary and response by LAG-3 expression.

[0106] [Figure 21] Figure 21 shows the best change in target lesion size by updated LAG-3 and PD-L1 expression.

[0107] [Figure 22] Figure 22: Depth and duration of response by updated LAG-3 and PD-L1 expression.

[0108] [Figure 23] Figure 23 shows the updated ongoing clinical follow-up.

[0109] [Figure 24] Figure 24. Role of LAG-3 and PD-1 in T cell exhaustion and proposed clinical benefit of combination with nivolumab.

[0110] [Figure 25] FIG. 25 shows the LAG-3 pattern of expression by IHC staining of total nucleated cells in melanoma tumor specimens.

[0111] [Figure 26]Figures 26A-F show correlations between LAG-3 and immune and inflammatory biomarkers: (A) LAG-3 vs. CD8, (B) LAG-3 vs. FOXP3, (C) LAG-3 vs. CD163, (D) LAG-3 vs. CD68, (E) LAG-3 vs. PD-L1, (F) LAG-3 vs. MHC II.

[0112] [Figure 27] FIG. 27 shows the percentage of LAG-3 positive tumor infiltrating lymphocytes (TILs) in tumors containing <1% or ≦1% MHC II positive tumor cells.

[0113] [Figure 28-1] Figures 28-C show correlations between inflammation clusters and biomarker expression in (A) urothelial carcinoma, (B) NSCLC, and (C) all tumor types. [Figure 28-2] Figures 28-C show correlations between inflammation clusters and biomarker expression in (A) urothelial carcinoma, (B) NSCLC, and (C) all tumor types.

[0114] [Figure 29] Figures 29A-C show heterogeneous MHC II tumor cell expression and LAG-3+ TILs. (A) Number of LAG-3+ TILs in MHC II-high and MHC II-low tumor cell regions in urothelial carcinoma. (B-C) Ratio of LAG-3+ TIL cells in MHC II-high and MHC II-low tumor cell regions in urothelial and gastric cancer samples.

[0115] [Figure 30A] Figures 30A and B show LAG-3 mRNA levels at screening and weeks 2 to 4 after nivolumab monotherapy. [Figure 30B] Figures 30A and B show LAG-3 mRNA levels at screening and weeks 2 to 4 after nivolumab monotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0116] Detailed Description of the Invention In certain embodiments, the present invention relates to improved methods for treating malignant tumors in human patients. In particular, the present invention demonstrates that administration of a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody achieves surprisingly improved treatment outcomes in a patient population with LAG-3-positive malignant tumors compared to a population including patients with LAG-3-positive and LAG-3-negative tumors. Accordingly, in certain embodiments, the invention described herein relates to methods for identifying patients with LAG-3-positive tumors, e.g., melanoma. In other embodiments, the invention described herein relates to methods for treating LAG-3-positive malignant tumors by administering a combination of a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody).

[0117] In other aspects, the invention described herein relates to methods of treating LAG-3 positive malignancies by administering a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody) or a combination of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

[0118] In another embodiment, the invention described herein relates to methods of treating LAG-3 positive malignancies by administering anti-CTLA4 antibodies.

[0119] 1. Definition In order that the present invention may be more readily understood, certain terms are first defined. As used herein, unless otherwise expressly provided herein, each of the following terms has the meaning indicated below. Additional definitions are set forth throughout this specification.

[0120] An "antibody" (Ab) specifically binds to an antigen and includes, but is not limited to, a glycoprotein immunoglobulin comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region contains three constant domains: C H1 , C H2 and C H3 Each light chain contains a light chain variable region (herein VL The light chain constant region contains one constant domain, C L Includes V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable regions are numbered using the Kabat numbering system, and amino acids in the constant regions are numbered using the EU system.

[0121] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and indicated otherwise by context, the term "antibody" includes monospecific, bispecific, or multispecific antibodies and single-chain antibodies. In certain embodiments, an antibody is a bispecific antibody. In other embodiments, an antibody is a monospecific antibody.

[0122] As used herein, an "IgG antibody" has the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-ICOS IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), where the two heavy and light chains are linked by the same number and positions of disulfide bridges as in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

[0123] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds PD-1 is substantially free of antibodies that specifically bind antigens other than PD-1), however, an isolated antibody that specifically binds PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0124] The antibody may be an antibody that has been modified (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, the antibody may contain one or more variant amino acids (compared to a naturally occurring antibody) that alter the properties (e.g., functional properties) of the antibody. Many such changes are known in the art that affect, for example, half-life in a patient, effector functions and / or the immune response to the antibody. The term antibody also includes artificial polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0125] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, which have essentially identical primary sequence and which display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0126] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0127] A "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In some embodiments of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within the CDR domains remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0128] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0129] An "anti-antigen" antibody refers to an antibody that specifically binds to that antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-LAG-3 antibody specifically binds to LAG-3.

[0130] An "antigen-binding portion" (also referred to as an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability of the whole antibody to specifically bind to the antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments or by portions of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody, for example, the anti-LAG-3 antibody described herein, are: (1) Fab fragment (a fragment obtained by papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (2) F(ab')2 fragment (pepsin cleavage fragment) or similar bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment consisting of the VH and CH1 domains; (4) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (5) single-domain antibody (dAb) fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-46); (6) bi-single domain antibodies (dual affinity retargeting antibodies (DARTs)), consisting of two VH domains linked by a hinge; (7) dual variable domain immunoglobulins; (8) an isolated complementarity-determining region (CDR); and (9) Combinations of two or more isolated CDRs, optionally connected by synthetic linkers. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0131] The term "LAG-3," "LAG3," or "lymphocyte-activation gene-3" refers to lymphocyte-activation gene-3. As used herein, the term LAG-3 includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, and analogs sharing at least one epitope with hLAG-3. As used herein, the term LAG-3 includes variants, isoforms, homologs, orthologs, and paralogs. For example, an antibody specific for human LAG-3 protein may, in some cases, cross-react with LAG-3 protein from species other than human. In other embodiments, an antibody specific for human LAG-3 protein may be completely specific for human LAG-3 protein and may not exhibit species or other types of cross-reactivity, or may cross-react with LAG-3 from some but not all other species (e.g., cross-react with monkey LAG-3 but not mouse LAG-3). The term "human LAG-3" refers to human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 having GenBank Accession No. NP_002277 (SEQ ID NO: 13). The term "mouse LAG-3" refers to mouse sequence LAG-3, such as the complete amino acid sequence of mouse LAG-3 having GenBank Accession No. NP_032505. LAG-3 is also known in the art, for example, as CD223. A human LAG-3 sequence may differ from human LAG-3 of GenBank Accession No. NP_002277, for example, by having conserved mutations or mutations in non-conserved regions, and the LAG-3 has substantially the same biological function as human LAG-3 of GenBank Accession No. NP_002277. For example, the biological function of human LAG-3 is that it has an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody of the present invention, or the biological function of human LAG-3 is binding to an MHC class II molecule.

[0132] A particular human LAG-3 sequence generally has at least 90% amino acid sequence identity with the human LAG-3 of Genbank Accession No. NP_002277 and contains amino acid residues that identify the amino acid sequence as human when compared with the LAG-3 amino acid sequence of another species (e.g., mouse). In some cases, the human LAG-3 may have at least 95%, or at least 96%, 97%, 98%, or 99% amino acid sequence identity with the LAG-3 of Genbank Accession No. NP_002277. In some embodiments, the human LAG-3 sequence exhibits no more than 10 amino acid differences with the LAG-3 sequence of Genbank Accession No. NP_002277. In some embodiments, the human LAG-3 may exhibit no more than 5, or no more than 4, 3, 2, or 1 amino acid differences with the LAG-3 sequence of Genbank Accession No. NP_002277. Percent identity may be determined as described herein.

[0133] As used herein, the terms "programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PD1," "PDCD1," "hPD-1," and "hPD-I" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one epitope with PD-1. The complete PD-1 sequence is available at GenBank Accession No. U64863 (SEQ ID NO: 29).

[0134] The protein programmed death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8). The earliest members of this family, CD28 and ICOS, were discovered by their functional effect of enhancing T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. Nature (1999); 397:263-266; Hansen et al. Immunogenics (1980); 10:247-260). PD-1 was discovered through screening for differential expression in apoptotic cells (Ishida et al. EMBO J (1992); 11:3887-95). Other members of this family, CTLA-4 and BTLA, were discovered through screening for differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all possess unpaired cysteine ​​residues that allow homodimerization. In contrast, PD-1 lacks the unpaired cysteine ​​residue characteristic of other CD28 family members, suggesting that it exists as a monomer.

[0135] The PD-1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). PD-1 is structurally similar to CTLA-4 but lacks the MYPPPY motif (SEQ ID NO: 32), which is important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and shown to downregulate T cell activation by binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not other CD28 family members. PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction of PD-1 and PD-L1 results in a reduction of tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100).Immune suppression can be reversed by inhibiting the local interaction of PD-1 and PD-L1, and the effect is additive when the interaction of PD-1 and PD-L2 is similarly blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0136] Consistent with PD-1 being an inhibitory member of the CD28 family, PD-1-deficient animals develop various autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndromes with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510). In mouse B-cell tumor lines, the ITSM of PD-1 inhibits BCR-mediated Ca 2+ It has been shown to be essential for blocking the flow and tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0137] "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as five analogs that share at least one shared epitope with hPD-L1. The complete hPD-L1 sequence can be obtained from GenBank Accession No. Q9NZQ7.

[0138] As used herein, the terms "programmed death-ligand-2" and "PD-L2" include human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs that share at least one epitope with hPD-L2. The complete hPD-L2 sequence can be obtained from GenBank Accession No. Q9BQ51.

[0139] As used herein, a "patient" includes any patient with cancer (e.g., melanoma). The terms "subject" and "patient" are used interchangeably herein.

[0140] "Administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered by a non-parenteral route, in some embodiments, orally. Other non-parenteral routes include topical, epithelial, or mucosal administration routes, for example, intranasal, vaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times and / or over one or more extended periods.

[0141] "Treatment" or "treatment" of a subject refers to any type of intervention or procedure or administration of an active agent performed on a subject with the purpose of ameliorating, alleviating, ameliorating, arresting, slowing or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition or the biochemical manifestations associated with a disease.

[0142] As used herein, "effective treatment" refers to a treatment that produces a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over measurements or observations made before treatment with the method is initiated. A beneficial effect can also take the form of halting, slowing, delaying, or stabilizing the adverse progression of markers of a solid tumor. Effective treatment can refer to the alleviation of at least one symptom of a solid tumor. Such effective treatment can, for example, reduce a patient's pain, reduce the size and / or number of lesions, reduce or prevent tumor metastasis, and / or slow tumor growth.

[0143] The term "effective amount" refers to an amount of an agent that provides a desired biological, therapeutic, and / or preventative result. The result can be reduction, amelioration, alleviation, reduction, delay, and / or alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., tumor growth inhibition) or prevent or slow other unwanted cell proliferation. In certain embodiments, an effective amount is an amount sufficient to delay tumor progression. In certain embodiments, an effective amount may be administered in one or more doses, which is an amount sufficient to prevent or slow tumor recurrence. An effective amount of a drug or composition can (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) prevent, delay, slow, or stop to some extent cancer cell invasion into peripheral organs; (iv) prevent (i.e., slow down to some extent or stop) tumor metastasis; (v) prevent tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. In one example, an "effective amount" is an amount of a combined anti-LAG-3 antibody and an anti-PD-1 antibody that has been clinically proven to affect a significant reduction in cancer or delay in the progression of cancer, such as advanced solid tumors. As used herein, the terms "fixed dose," "flat dose," and "flat-fixed dose" are used interchangeably and refer to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). A fixed or flat dose is therefore provided as an absolute dose of agent (e.g., anti-LAG-3 antibody and / or anti-PD-1 antibody) rather than a mg / kg dose.

[0144] As used herein, the term "progression-free survival," which may be abbreviated as PFS, refers to the period during and after treatment of a solid tumor (i.e., melanoma) during which a patient lives with disease but does not get worse.

[0145] As used herein, "dosing interval" refers to the time that elapses between multiple administrations of the formulations disclosed herein to a subject. The dosing interval can therefore be expressed as a time span.

[0146] As used herein, the term "dosing frequency" refers to the frequency of administration of the formulations disclosed herein over a given period of time. Dosing frequency can be expressed as a number of times over a given period of time, for example, once a week or once every two weeks.

[0147] The use of the term "fixed dose" with respect to compositions of the invention means that two or more different antibodies in a single composition are present in the composition in a specific (fixed) ratio to each other. In certain embodiments, the fixed dose is based on the weight (e.g., mg) of the antibody. In certain embodiments, the fixed dose is based on the concentration (e.g., mg / ml) of the antibody. In certain embodiments, the ratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 1:210, about 1:220, about 1:230, about 1:240, about 1:250, about 1:260, about 1:270, about 1:280, about 1:290, about 1:300, about 1:310, about 1:320, about 1:330, about 1:340, about 1:350, about 1:360, about 1:370, about 1:380, about 1:390, about 1:400, about 1:410, about 1:420, about 1:430, about 1:440, about 1:450, about 1:460, about 1:470, about 1:480, about 1:490, about 1 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg of first antibody to mg of second antibody. For example, a 3:1 ratio of first antibody to second antibody could mean that a vial could contain about 240 mg of first antibody and 80 mg of second antibody, or about 3 mg / ml of first antibody and 1 mg / ml of second antibody.

[0148] The use of the term "flat dose" with respect to compositions of the invention refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). A flat dose is therefore provided as an absolute dose of agent (e.g., anti-LAG-3 antibody and / or anti-PD-1 antibody) rather than a mg / kg dose. For example, a 60 kg person and a 100 kg person would receive the same dose of a composition (e.g., 240 mg of anti-PD-1 antibody and 80 mg of anti-LAG-3 antibody in a single fixed dose formulation vial containing both 240 mg of anti-PD-1 antibody and 80 mg of anti-LAG-3 antibody (or two fixed dose formulation vials containing 120 mg of anti-PD-1 antibody and 40 mg of anti-LAG-3 antibody, etc.)).

[0149] The term "weight-based dose" as used herein means that the dose administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires a combination of 3 mg / kg of an anti-LAG-3 antibody and 3 mg / kg of an anti-PD-1 antibody, the appropriate amount of anti-LAG-3 antibody (i.e., 180 mg) and anti-PD-1 antibody (i.e., 180 mg) can be readily derived from a 1:1 ratio fixed-dose formulation of the anti-LAG-3 antibody and the anti-PD-1 antibody.

[0150] As used herein, the terms "about once a week," "about once a week," "about once every two weeks," or any other similar administration period approximation refer to approximately every seven days ± 2 days, i.e., every five to nine days. Thus, an administration frequency of "once a week" can be every five days, six days, seven days, eight days, or nine days. "About once every two weeks" can include every 14 days ± 3 days, i.e., every 11 to 17 days. Similar approximations can apply, for example, to about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, and about once every 12 weeks. In certain embodiments, an administration interval of about once every six weeks or about once every 12 weeks means that the first dose can be administered on any day of the first week, followed by the next dose on any day of the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every 6 weeks or about once every 12 weeks means that the first dose is administered on a particular day of the week (e.g., Monday) in the first week, and the next dose is administered on the same day of the week (i.e., Monday) in the sixth or twelfth week, respectively.

[0151] "Cancer" refers to a diverse group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that can invade nearby tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0152] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0153] The term "LAG-3 positive" or "LAG-3 expression positive" in relation to LAG-3 expression is determined by the ratio of cells in the test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, and the tissue sample is scored as LAG-3 expression.In some embodiments, for LAG-3 expression measured by immunohistochemistry (IHC), LAG-3 positive tumor or LAG-3 expression positive tumor means that at least about 0.01%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100% of the total cell number expresses LAG-3. In other embodiments, for LAG-3 expression assayed by immunohistochemistry (IHC) or flow cytometry, a LAG-3-positive tumor or a LAG-3 expression-positive tumor means that at least about 0.01%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the total number of tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3. A LAG-3-positive tumor or a LAG-3 expression-positive tumor may also be referred to herein as a cell expressing LAG-3. In certain embodiments, this means that at least about 0.1% to at least about 20% of the total number of cells in a LAG-3 positive tumor or tumor positive for LAG-3 expression express LAG-3.In certain embodiments, a LAG-3-positive tumor or a LAG-3 expression-positive tumor means that at least about 0.1% to at least about 20% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3. In certain embodiments, a LAG-3-positive tumor or a LAG-3 expression-positive tumor means that at least about 0.1% to at least about 10% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3. In certain embodiments, a LAG-3-positive tumor or a LAG-3 expression-positive tumor means that at least about 0.1% to at least about 10% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3. In certain embodiments, a LAG-3-positive tumor or a LAG-3 expression-positive tumor means that at least about 1% of the total cells express LAG-3 on the cell surface. In certain embodiments, a LAG-3-positive or LAG-3-expression-positive tumor means that at least about 1% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3 on their cell surface. In other embodiments, a LAG-3-positive or LAG-3-expression-positive tumor means that at least about 5% of the total cell population express LAG-3 on their cell surface. In other embodiments, a LAG-3-positive or LAG-3-expression-positive tumor means that at least about 5% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3 on their cell surface. In certain embodiments, a LAG-3-positive or LAG-3-expression-positive tumor means that at least about 1% or in the range of 1-5% of the total cell population express LAG-3 on their cell surface. In certain embodiments, a LAG-3 positive or LAG-3 expression positive tumor means that at least about 1% or in the range of 1-5% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express LAG-3 on the cell surface.

[0154] "LAG-3 negative" or "LAG-3 expression negative" refers to the absence of detectable LAG-3 expression. In certain embodiments, for LAG-3 expression assayed by IHC, a LAG-3 negative tumor or a LAG-3 expression negative tumor means that less than 0.01% of the total cell number expresses detectable levels of LAG-3. In certain embodiments, for LAG-3 expression assayed by IHC or flow cytometry, a LAG-3 negative tumor or a LAG-3 expression negative tumor means that less than 0.01% of the total tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express detectable levels of LAG-3. In certain embodiments, for LAG-3 expression assayed by IHC, a LAG-3 negative tumor or a LAG-3 expression negative tumor means that less than 1% of the total cell number expresses detectable levels of LAG-3. In some embodiments, for LAG-3 expression assayed by IHC or flow cytometry, LAG-3 negative tumor or LAG-3 expression negative tumor means that less than 1% of the total number of tumor-infiltrating inflammatory cells (e.g., T cells, CD8+ T cells, CD4+ T cells, FOXP3+ cells) express detectable levels of LAG-3. In some embodiments, LAG-3 negative tumor or LAG-3 expression negative tumor means that zero (0) cells express detectable levels of LAG-3. In some embodiments, LAG-3 negative or LAG-3 expression negative tumor is any tumor other than LAG-3 positive or LAG-3 expression positive tumor.

[0155] The term "PD-L1 positive" or "PD-L1 expression positive" with respect to cell surface PD-L1 expression refers to the proportion of cells in a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, that exceeds the percentage that would score the tissue sample as having cell surface PD-L1 expression. For example, for a cell surface expression assay assayed by immunohistochemistry (IHC) using mAb 28-8, a PD-L1-positive tumor or a PD-L1 expression-positive tumor means that at least about 0.01%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% of the total cell population expresses PD-L1. A PD-L1-positive tumor or a PD-L1 expression-positive tumor may also be referred to herein as a tumor expressing PD-L1. In other embodiments, a PD-L1-positive tumor or a PD-L1-expressing tumor means that at least about 0.1% to at least about 20% of the total cell population express PD-L1. In certain embodiments, a PD-L1-positive tumor or a PD-L1-expressing tumor means that at least about 0.1% to at least about 10% of the total cell population express PD-L1. In certain embodiments, a PD-L1-positive tumor or a PD-L1-expressing tumor means that at least about 1% of the total cell population express PD-L1 on the cell surface. In other embodiments, a PD-L1-positive tumor or a PD-L1-expressing tumor means that at least about 5% of the total cell population express PD-L1 on the cell surface. In certain embodiments, a PD-L1-positive tumor or a PD-L1-expressing tumor means that at least about 1% or in the range of 1-5% of the total cell population express PD-L1 on the cell surface.

[0156] The terms "PD-L1 negative" or "PD-L1 expression negative" with respect to cell surface PD-L1 expression is determined by the proportion of cells in the test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, that are not PD-L1 positive or PD-L1 expression positive.

[0157] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or in the liver that result in the selective targeting, binding, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0158] "Tumor-infiltrating inflammatory cells" generally refer to any type of cell that participates in the inflammatory response in a subject and infiltrates tumor tissue. Such cells include tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, eosinophils, histiocytes, and dendritic cells.

[0159] The term "and / or," as used herein, is to be construed as a specific disclosure of two each specified property or component, with or without the other. Thus, herein, the term "and / or," as used in terms such as "A and / or B," includes "A and B," "A or B," "A" (single) and "B" (single). Similarly, the term "and / or" as used in terms such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0160] When embodiments are described herein using the term "comprising," it is understood that equivalent embodiments other than those described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0161] The terms "about" or "essentially consisting of" refer to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which depends, in part, on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within one or more standard deviations according to practice in the art. Alternatively, "about" or "essentially consisting of" can mean within a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any value between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in the specification and claims, unless otherwise indicated, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable error range for that particular value or composition.

[0162] Any concentration range, percentage range, ratio range, or integer range used herein is understood to include every integer value within the recited range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0163] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used herein.

[0164] Units, prefixes, and symbols are written in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein do not limit various aspects of the disclosure, which may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0165] Various aspects of the invention are described in further detail in the following subsections.

[0166] 2. The method of the present invention In some embodiments, the present invention relates to a method for treating a LAG-3-positive malignancy (e.g., melanoma) in a subject in need of such treatment. Combination therapy with a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody) results in better treatment outcomes (e.g., objective response rate and disease control rate) in a patient population with a LAG-3-positive malignancy (e.g., melanoma) than in the general patient population with a mixture of LAG-3-negative and LAG-3-positive malignancies. To improve malignancy treatment, in some embodiments, the present invention identifies a patient as having a LAG-3-positive tumor and provides immunotherapy with a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody).

[0167] In other embodiments, the invention relates to identifying a patient as having a LAG-3-positive tumor and treating the LAG-3-positive tumor by administering a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody) or a combination of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the invention includes a method of identifying a patient as having a LAG-3-positive tumor and treating the LAG-3-positive tumor by administering an anti-PD-1 antibody. In certain embodiments, the invention includes a method of identifying a patient as having a LAG-3-positive tumor and treating the LAG-3-positive tumor by administering an anti-PD-L1 antibody.

[0168] In other embodiments, the invention relates to identifying a patient as having an LAG-3 positive tumor and treating the LAG-3 positive tumor by administering an anti-CTLA-4 antibody.

[0169] In some embodiments, the present invention includes a method for selecting a malignant tumor in a human patient for immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) selecting the tumor for immunotherapy if the tumor is a LAG-3-positive tumor. In some embodiments, the present invention includes a method for identifying a malignant tumor in a human patient as being eligible for immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) identifying the tumor as being eligible for immunotherapy if the tumor is a LAG-3-positive tumor. In some embodiments, the present invention includes a method for identifying a malignant tumor in a human patient that may respond to immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) identifying the tumor as being likely to respond to treatment if the tumor is a LAG-3-positive tumor. In certain embodiments, the present invention includes a method for identifying a malignant tumor in a human patient that is likely to respond to immunotherapy, the method comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor, identifying the tumor as likely to respond to treatment. In certain embodiments, the present invention includes a method for classifying a malignant tumor in a human patient as likely to respond to immunotherapy, the method comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor, classifying the tumor as likely to respond to immunotherapy. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-L1 antibody. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody.In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-PD-L1 antibody. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody. In certain embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In certain embodiments, any of the present methods further comprises determining PD-L1 expression in the tumor sample.

[0170] In certain embodiments, the present invention includes a method for identifying a patient with a malignant tumor that is likely to respond to immunotherapy, the method comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor, identifying the patient as likely to respond to treatment. In certain embodiments, the present invention includes a method for selecting a patient with a malignant tumor for immunotherapy, the method comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor, selecting the patient for immunotherapy. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-PD-L1 antibody. In some embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-PD-1 antibody. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-PD-L1 antibody. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the method comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor.In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the method comprises administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody. In certain embodiments, the method comprises administering to the patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In certain embodiments, any of the present methods further comprises determining PD-L1 expression in the tumor sample.

[0171] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient, comprising administering to the patient an immunotherapy disclosed herein, wherein the patient is predicted to respond to treatment with a LAG-3 inhibitor and a PD-1 pathway inhibitor based on LAG-3 expression or LAG-3 and PD-L1 expression in a tumor sample from the patient. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody.

[0172] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) determining the LAG-3 expression level or LAG-3 and PD-L1 expression levels in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor or a LAG-3-positive, PD-L1-positive tumor, administering a therapeutically effective amount of a LAG-3 inhibitor to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor; and (b) administering a therapeutically effective amount of a LAG-3 inhibitor to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising administering a therapeutically effective amount of a LAG-3 inhibitor to the patient, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In some embodiments, the LAG-3 positive tumor is a LAG-3 positive PD-L1 negative tumor. In some embodiments, the LAG-3 positive malignant tumor is a LAG-3 positive PD-L1 negative malignant tumor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody.

[0173] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) determining the LAG-3 expression level or LAG-3 and PD-L1 expression levels in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor or a LAG-3-positive, PD-L1-positive tumor, administering a therapeutically effective amount of a PD-1 pathway inhibitor to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor; and (b) administering a therapeutically effective amount of a PD-1 pathway inhibitor to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising administering a therapeutically effective amount of a PD-1 pathway inhibitor to the patient, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some embodiments, the LAG-3 positive tumor is a LAG-3 positive, PD-L1 negative tumor. In some embodiments, the LAG-3 positive malignant tumor is a LAG-3 positive, PD-L1 negative malignant tumor.

[0174] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) determining the LAG-3 expression level or LAG-3 and PD-L1 expression levels in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor or a LAG-3-positive, PD-L1-positive tumor, administering a therapeutically effective amount of an anti-CTLA-4 antibody to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor; and (b) administering a therapeutically effective amount of an anti-CTLA-4 antibody to the patient. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising administering a therapeutically effective amount of an anti-CTLA-4 antibody to the patient, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the LAG-3 positive tumor is a LAG-3 positive, PD-L1 negative tumor. In certain embodiments, the LAG-3 positive malignant tumor is a LAG-3 positive, PD-L1 negative malignant tumor.

[0175] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) determining the LAG-3 expression level or LAG-3 and PD-L1 expression levels in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor or a LAG-3-positive, PD-L1-positive tumor, administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In some embodiments, the LAG-3 positive tumor is a LAG-3 positive PD-L1 negative tumor. In some embodiments, the LAG-3 positive malignant tumor is a LAG-3 positive PD-L1 negative malignant tumor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody.

[0176] In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) determining the LAG-3 expression level or LAG-3 and PD-L1 expression levels in a tumor sample; and (b) if the tumor is a LAG-3-positive tumor or a LAG-3-positive, PD-L1-positive tumor, administering to the patient a therapeutically effective amount of a PD1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising: (a) identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of a PD1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the present invention relates to a method of treating a malignant tumor in a human patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a PD1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In some embodiments, the LAG-3 positive tumor is a LAG-3 positive PD-L1 negative tumor. In some embodiments, the LAG-3 positive malignant tumor is a LAG-3 positive PD-L1 negative malignant tumor. In some embodiments, the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some embodiments, the PD-1 pathway inhibitor is an anti-PD-1 antibody.

[0177] In other embodiments, the present invention includes a method of treating a malignant tumor in a human patient in need of treatment, comprising administering to the patient an immunotherapy disclosed herein, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the LAG-3-positive malignant tumor is a LAG-3-positive, PD-L1-negative malignant tumor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody.

[0178] In certain embodiments, the present invention includes a method of extending progression-free survival to more than 12 months in a human patient with a malignant tumor, comprising administering to the patient an immunotherapy disclosed herein, wherein the patient has been identified as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration, and the patient exhibits progression-free survival of more than 12 months. In certain embodiments, the LAG-3-positive malignant tumor is a LAG-3-positive, PD-L1-negative malignant tumor. In certain embodiments, the patient's progression-free survival may be extended to more than about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0179] In yet another embodiment, the present invention includes a method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient an immunotherapy disclosed herein, wherein the patient has been identified as having a LAG-3-positive malignant tumor (e.g., melanoma) or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration. In certain embodiments, the method comprises identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the LAG-3-positive malignant tumor is a LAG-3-positive, PD-L1-negative malignant tumor. In certain embodiments, the immunotherapy comprises administering therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-L1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administration of therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0180] The present invention also includes a method of preventing recurrence and / or inducing remission in a patient, comprising administering to the patient an immunotherapy disclosed herein, wherein the patient has been identified as having a LAG-3-positive malignancy (e.g., melanoma) or a LAG-3-positive, PD-L1-positive malignancy prior to administration. In certain embodiments, the methods of the present invention comprise: (i) identifying the patient as having a LAG-3-positive malignancy or a LAG-3-positive, PD-L1-positive malignancy; and (ii) administering to the patient an immunotherapy disclosed herein. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the LAG-3-positive malignancy is a LAG-3-positive, PD-L1-negative malignancy. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-L1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0181] In certain embodiments, the present invention includes a method of increasing the objective response rate in cancer treatment to greater than 55% in a patient population, each patient in the patient population having a malignant tumor, comprising administering to the patient an immunotherapy disclosed herein, wherein each patient is identified as having a LAG-3-positive malignant tumor (e.g., melanoma) or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration, and the objective response rate is greater than 55%, 60%, 65%, 70%, or 75%. In certain embodiments, the method comprises identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the LAG-3-positive malignant tumor is a LAG-3-positive, PD-L1-negative malignant tumor. In certain embodiments, the immunotherapy comprises administering therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administering therapeutically effective amounts of a LAG-3 inhibitor and an anti-PD-1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor and an anti-PD-L1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0182] In certain embodiments, the present invention includes a method of increasing the disease control rate in cancer treatment to greater than 55% in a patient population, each patient of the patient population having a malignant tumor, comprising administering to the patient an immunotherapy disclosed herein, wherein each patient is identified as having a LAG-3-positive malignant tumor (e.g., melanoma) or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration, and the disease control rate is greater than 55%, 60%, 65%, 70%, or 75%. In certain embodiments, the method comprises identifying the patient as having a LAG-3-positive malignant tumor or a LAG-3-positive, PD-L1-positive malignant tumor prior to administration. In certain embodiments, the LAG-3-positive malignant tumor is a LAG-3-positive, PD-L1-negative malignant tumor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-PD-L1 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administration of a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0183] In other embodiments, each patient in the method experiences (i) an extended progression-free survival of more than 12 months, (ii) at least about a 10%, about 20%, about 30%, about 40%, or about 50% reduction in tumor size compared to the tumor size before administration, or (iii) both. In certain embodiments, the patient population can be at least 100 patients with a LAG-3-positive malignancy (e.g., melanoma) or a LAG-3-positive, PD-L1-positive malignancy. In certain embodiments, the patient population can be at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 patients with a LAG-3-positive malignancy or a LAG-3-positive, PD-L1-positive malignancy. In certain embodiments, the LAG-3-positive malignancy is a LAG-3-positive, PD-L1-negative malignancy.

[0184] In further embodiments, the present invention provides a method for selecting a human patient suitable for combination therapy, comprising: (a) identifying the patient as having a LAG-3-positive malignancy or a LAG-3-positive, PD-L1-positive malignancy; and (b) instructing a healthcare professional to administer the immunotherapy disclosed herein to the patient. In certain embodiments, the LAG-3-positive malignancy is a LAG-3-positive, PD-L1-negative malignancy. The method may further comprise administering the immunotherapy disclosed herein. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of an anti-PD-1 antibody to the patient. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of an anti-PD-L1 antibody to the patient. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody. In some embodiments, the administration treats malignant tumors.

[0185] The methods of the present invention can treat malignant tumors, reduce tumor size, inhibit tumor growth, eliminate tumors from patients, prevent tumor recurrence, induce remission in patients, or any combination thereof, as a result of administering the immunotherapies disclosed herein. In certain embodiments, administration of the immunotherapies disclosed herein induces a complete response. In other embodiments, administration of the immunotherapies disclosed herein induces a partial response. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a LAG-3 inhibitor. In certain embodiments, the immunotherapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of an anti-PD-L1 antibody. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody. In some embodiments, the immunotherapy comprises administering a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody, and the PD-1 pathway inhibitor is an anti-PD-L1 antibody.

[0186] In some embodiments, LAG-3-positive tumors contain at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% LAG-3-expressing cells. In some embodiments, LAG-3-expressing cells comprise tumor-infiltrating lymphocytes.

[0187] In certain embodiments, the identifying comprises determining LAG-3 expression in a malignant tumor.

[0188] In certain embodiments, LAG-3 expression is determined by examination of the results of an assay that allows for the determination of LAG-3 expression.

[0189] In certain embodiments, any of the present methods further comprises determining PD-L1 expression in the tumor sample.

[0190] In certain embodiments, any of the methods further comprises identifying the patient as having a PD-L1-positive malignancy prior to administering. In certain embodiments, any of the methods further comprises identifying the patient as having a PD-L1-negative malignancy prior to administering.

[0191] In certain embodiments, any of the present methods further comprises determining PD-L1 expression in the malignant tumor.

[0192] In certain embodiments of any of the methods, the patient is identified as having a PD-L1-positive malignancy prior to administering. In certain embodiments of any of the methods, the patient is identified as having a PD-L1-negative malignancy prior to administering.

[0193] Methods for determining PD-L1 expression in tumor samples, methods for identifying patients as having PD-L1-positive malignancies, and methods for determining PD-L1 expression in malignancies are disclosed in PCT / US2016 / 029878.

[0194] In certain embodiments, the methods of the present invention include methods of treating a human patient with unresectable or metastatic melanoma in need of treatment with a combination of a PD-1 pathway inhibitor and a LAG-3 inhibitor, wherein the patient has previously been treated with an anti-PD-1 inhibitor and / or an anti-PD-L1 inhibitor. In certain embodiments, the PD-1 pathway inhibitor is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody. In certain embodiments, the LAG-3 antibody is BMS-986016. In certain embodiments, the melanoma is a LAG-3-expressing tumor. In certain embodiments, the melanoma is a LAG-3-expressing tumor where LAG-3 expression is ≧1%.

[0195] Measurement of LAG-3 expression In certain embodiments, identifying patients suitable for LAG-3 inhibitor / PD-1 pathway inhibitor combination therapy, PD-1 pathway inhibitor (e.g., anti-PD-1 antibody) therapy, or anti-CTLA-4 antibody therapy for the methods of the present invention involves measuring or assessing LAG-3 expression in a sample, e.g., a malignant tumor test tissue sample containing tumor cells and tumor-infiltrating inflammatory cells. The terms "LAG-3-expressing tumor," "LAG-3-expressing tumor," "LAG-3-positive tumor," and "LAG-3-expressing positive tumor" are used interchangeably herein and include tumors containing LAG-3-expressing tumor-infiltrating lymphocytes. The meanings of these terms are provided elsewhere herein. Measuring or assessing LAG-3 expression can be accomplished by any applicable method.

[0196] In some embodiments, test tissue samples are obtained from patients who need treatment to evaluate LAG-3 expression.In some embodiments, test tissue samples include but are not limited to tumor biopsy, core biopsy tissue samples, fine needle aspiration or any clinically relevant tissue sample, such as blood, plasma, serum, lymph, ascites fluid, cyst fluid or urine.In some embodiments, test tissue samples are from primary tumor.In some embodiments, test tissue samples are from metastasis.In some embodiments, test tissue samples are taken from subject at multiple time points, for example, before treatment, during treatment and / or after treatment.In some embodiments, test tissue samples are taken from different locations in subject, for example, from primary tumor sample and from distant metastasis sample.

[0197] In some embodiments, the test tissue sample is a paraffin-embedded, fixed tissue sample. In some embodiments, the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample. In some embodiments, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a frozen tissue sample. In some embodiments, the test tissue sample is a fresh-frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is cells isolated from a bodily fluid. In some embodiments, the test tissue sample contains circulating tumor cells (CTCs). In some embodiments, the test tissue sample contains tumor-infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample contains tumor cells and tumor-infiltrating lymphocytes (TILs). In some embodiments, the test tissue sample contains circulating lymphocytes. In some embodiments, the test tissue sample is an archival tissue sample. In some embodiments, the test tissue sample is an archival tissue sample with a known diagnosis, treatment, and / or outcome history. In some embodiments, the sample is a tissue mass. In some embodiments, the test tissue sample is dispersed cells. In one embodiment, the sample size is from about 1 cell to about 1 x 10 6In one embodiment, the sample size is from about 1 cell to about 1 x 10 5 In some embodiments, the sample size is about 1 cell to about 10,000 cells. In some embodiments, the sample size is about 1 cell to about 1,000 cells. In some embodiments, the sample size is about 1 cell to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0198] In other embodiments, assessing LAG-3 expression can be performed without obtaining a test tissue sample. In one embodiment, selecting a suitable patient includes (i) providing a test tissue sample, optionally obtained from a patient with cancer of the tissue, containing tumor cells and / or tumor-infiltrating inflammatory cells; and (ii) assessing the proportion of cells in the test tissue sample that express LAG-3 on their cell surface based on assessing that the proportion of cells in the test tissue sample that express LAG-3 on their cell surface is higher than a predetermined threshold level.

[0199] However, it should be understood that in any of the methods involving measuring LAG-3 expression in a test tissue sample, the step involving providing a test tissue sample obtained from a patient is an optional step. That is, in certain embodiments, the method includes this step, while in other embodiments, this step is not included in the method. It should also be understood that in certain embodiments, the "measuring" or "assessing" step to identify or determine the number or proportion of cells expressing LAG-3 in the test tissue sample is performed by performing a novel method to assay LAG-3 expression, such as a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In certain other embodiments, the novel method step is not included, and LAG-3 expression is assessed, for example, by reviewing laboratory test results. In certain embodiments, LAG-3 expression is assessed by reviewing laboratory immunohistochemistry assay results. In certain embodiments, the method steps up to and including assessing LAG-3 expression provide intermediate results that can be provided to a physician or other healthcare professional for use in selecting appropriate candidates for combination therapy with a LAG-3 inhibitor and a PD-1 pathway inhibitor. In certain embodiments, the method steps up to and including assessing LAG-3 expression provide intermediate results that can be provided to a physician or other healthcare professional for use in selecting appropriate candidates for PD-1 pathway inhibitor (e.g., anti-PD-1 antibody) therapy. In certain embodiments, the method steps up to and including assessing LAG-3 expression provide intermediate results that can be provided to a physician or other healthcare professional for use in selecting appropriate candidates for anti-CTLA-4 antibody therapy. In certain embodiments, the step of providing intermediate results is performed by a physician or a person working under the supervision of a physician. In other embodiments, these steps are performed in an independent laboratory or by an independent person, such as a laboratory technician.

[0200] In certain embodiments of any of the methods, the proportion of cells expressing LAG-3 is assessed by performing an assay to detect the presence of LAG-3 RNA. In further embodiments, the presence of LAG-3 RNA is detected by RT-PCR, in situ hybridization, or RNase protection. In certain embodiments, the presence of LAG-3 RNA is detected by an RT-PCR-based assay. In certain embodiments, scoring the RT-PCR-based assay involves assessing the level of LAG-3 RNA expression in the test tissue sample relative to a predetermined level.

[0201] In other embodiments, the proportion of cells expressing LAG-3 is assessed by performing an assay to detect the presence of LAG-3 polypeptide. In further embodiments, the presence of LAG-3 polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, LAG-3 expression is assayed by IHC. In other embodiments of all of these methods, cell surface expression of LAG-3 is assayed using, for example, IHC or in vivo imaging.

[0202] In some embodiments, the biomarkers measured are LAG-3, CD4, CD8, FOXP3, CD163 CD68, and any combination thereof. In some embodiments, biomarkers are measured using any detection method disclosed herein. In other embodiments, the proportion of cells expressing LAG-3 in a test tissue sample is evaluated by flow cytometry. In some embodiments, the test tissue sample assayed by flow cytometry contains tumor-infiltrating immune cells. In some embodiments, the malignant tumor is a hematopoietic tumor, and the tissue sample assayed by flow cytometry contains peripheral blood cells. In some embodiments, the flow cytometry is a multiplex assay. In some embodiments, flow cytometry scoring includes detecting the expression of markers including LAG-3, CD4, CD8, FOXP3, and any combination thereof. In some embodiments, LAG-3, CD4, CD8, and FOXP3 are detected as a single marker. In some embodiments, flow cytometry scoring includes evaluating the proportion of T cells expressing LAG-3 in a test tissue sample. In some embodiments, flow cytometry scoring comprises evaluating the proportion of CD8+ T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises evaluating the proportion of CD4+ T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises evaluating the proportion of FOXP3+ T cells expressing LAG-3 in the test tissue sample. In some embodiments, flow cytometry scoring comprises detecting the expression of markers including CD163 and / or CD68. In some embodiments, flow cytometry scoring comprises evaluating the proportion of cells expressing CD163 and / or CD68 in the test tissue sample.

[0203] In some embodiments of any of the methods, the proportion of cells expressing LAG-3 in the test tissue sample is assessed by performing an assay to detect the presence of LAG-3 polypeptide.In some embodiments, the presence of LAG-3 polypeptide is detected by immunohistochemistry assay.In some embodiments, the test tissue sample is a tumor biopsy sample.In some embodiments, the test tissue sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0204] In some embodiments, the immunohistochemical assay is a monoplex assay.In some embodiments, the immunohistochemical assay is a multiplex assay.In some embodiments, the multiplex immunohistochemical assay can detect the presence of CD4, CD8, FOXP3, CD163, CD68, or any combination thereof.

[0205] In some embodiments, the immunohistochemistry assay involves contacting the tumor sample with 17B4 mouse anti-human LAG-3 IgG1 monoclonal antibody. In some embodiments, the immunohistochemistry assay involves contacting the tumor sample with an anti-LAG-3 antibody comprising heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 3 and 5, respectively. In some embodiments, the immunohistochemistry assay involves contacting the tumor sample with SP346 rabbit anti-human LAG-3 IgG monoclonal antibody. In some embodiments, the immunohistochemistry assay involves contacting the tumor sample with 11E3 (Novusbio), 874501 (Novusbio), or EPR4392(2) (Abcam) anti-human LAG-3 monoclonal antibody.

[0206] For example, melanin in melanoma tumor samples can interfere with histological analysis by obscuring histological features and interfering with and / or masking staining during immunohistochemistry (IHC). Melanin can be removed by bleaching the sample. See, e.g., Shen & Wu, Appl Immunohistochem Mol Morphol, 23(4): 303-307 (2015); Orchard & Calonje, Am J Dermatopathol, 20(4): 357-61 (1998). In some embodiments, immunohistochemistry assays involve melanin bleaching prior to contacting the sample with an anti-LAG-3 antibody. See, e.g., FIG. 15. In some embodiments, melanin bleaching involves contacting the sample with dilute hydrogen peroxide (0.1-30% v / v), trichloroisocyanuric acid (TCCA), potassium permanganate / oxalic acid, or other traditional oxidative methods for decolorizing (i.e., removing melanin from) tissue samples.

[0207] In some embodiments, the immunohistochemistry assay uses a black or brown chromogen. In some embodiments, the immunohistochemistry assay uses a red chromogen. In some embodiments, the immunohistochemistry assay uses a blue chromogen. In some embodiments, the immunohistochemistry assay uses a green chromogen. In some embodiments, the immunohistochemistry assay uses a purple chromogen. In some embodiments, the immunohistochemistry assay uses a yellow chromogen.

[0208] In some embodiments, the immunohistochemistry assay is scored at low magnification. In some embodiments, the low magnification is about 20x. In some embodiments, the immunohistochemistry assay is scored at high magnification. In some embodiments, the high magnification is about 40x.

[0209] In some embodiments, the immunohistochemistry assay is scored using image analysis software. In some embodiments, the immunohistochemistry assay is scored by a pathologist's visual immunoscore. In some embodiments, the immunohistochemistry assay is scored manually.

[0210] In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of cells expressing LAG-3 in a test tissue sample. In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of immune cells expressing LAG-3 in a test tissue sample. In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of T cells expressing LAG-3 in a test tissue sample. In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of CD8+ T cells expressing LAG-3 in a test tissue sample. In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of CD4+ T cells expressing LAG-3 in a test tissue sample. In some embodiments, scoring an immunohistochemistry assay involves assessing the proportion of FOXP3+ T cells expressing LAG-3 in a test tissue sample.

[0211] LAG-3 polypeptide localization includes partial membrane / cytoplasmic localization, dot-like localization, perinuclear and completely membrane / cytoplasmic localization.In some embodiments, cells with partial membrane / cytoplasmic LAG-3 localization are scored.In some embodiments, cells with dot-like LAG-3 localization are scored.In some embodiments, cells with completely membrane / cytoplasmic LAG-3 localization are scored.In some embodiments, cells with perinuclear LAG-3 localization are scored.In some embodiments, cells with any LAG-3 localization pattern are scored.

[0212] In some embodiments, the immunohistochemistry assay is a multiplex assay that further comprises detecting MHC class II expression by tumor cells.In some embodiments, the scoring of the immunohistochemistry assay comprises evaluating the proportion of cells that express MHC class II in the test tissue sample.In some embodiments, the scoring of the immunohistochemistry assay comprises evaluating the proportion of non-immune cells that express MHC class II in the test tissue sample.In some embodiments, the distribution of MHC II-expressing cells is heterogeneous in tumor samples.In some embodiments, the scoring of the immunohistochemistry assay comprises evaluating the proportion of cells that express MHC class II in the area of ​​the tumor sample that contains a high density of MHC class II-expressing cells.

[0213] In some embodiments, the immunohistochemistry assay is a multiplex assay that further includes detecting the expression of CD163 and / or CD68 by tumor-infiltrating lymphocytes (TILs). In some embodiments, the scoring of the immunohistochemistry assay includes evaluating the proportion of TILs that express CD163 and / or CD68 in the test tissue sample.

[0214] Imaging techniques have provided important tools in cancer research and treatment. Recent developments in molecular imaging systems, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence molecular tomography (FMT), bioluminescence imaging (BLI), laser scanning confocal microscopy (LSCM), and multiphoton microscopy (MPM), are likely to herald even greater use of these techniques in cancer research. Some of these molecular imaging systems not only allow physicians to see where tumors are located in the body, but also allow visualization of the expression and activity of specific molecules, cells, and biological processes that affect tumor behavior and / or responsiveness to therapeutic agents (Condeelis and Weissleder, Cold Spring Harb. Perspect. Biol. 2(12):a003848 (2010)). Antibody specificity combined with the sensitivity and resolution of PET makes immunoPET imaging particularly attractive for monitoring and assaying antigen expression in tissue samples (McCabe and Wu, Cancer Biother. Radiopharm. 25(3):253-61 (2010); Olafsen et al., Protein Eng. Des. Sel. 23(4):243-9 (2010)). In certain embodiments of any of the present methods, LAG-3 expression is assayed by immunoPET imaging. In certain embodiments, immunoPET is performed using a zirconium-89 radiolabeled anti-LAG-3 antibody. In certain embodiments of any of the present methods, the proportion of cells expressing LAG-3 in a test tissue sample is assessed by performing an assay that determines the presence of LAG-3 polypeptide on the cell surface of the test tissue sample. In certain embodiments, the test tissue sample is an FFPE tissue sample. In other embodiments, the presence of LAG-3 polypeptide is determined by an IHC assay. In further embodiments, the IHC assay is performed using automated methods. In certain embodiments, the IHC assay is performed using an anti-LAG-3 mAb that binds to a LAG-3 polypeptide.

[0215] LAG-3 expression assay by automated IHC In one embodiment of the method of the present invention, an automated IHC method is used to assay LAG-3 expression in FFPE tissue specimens. The present invention provides a method for detecting the presence of human LAG-3 antigen in a test tissue sample or quantifying the level of human LAG-3 antigen or the proportion of cells expressing the antigen in a sample, which method comprises contacting a test sample and a negative control sample with a mAb that specifically binds to human LAG-3 under conditions that allow the antibody or a portion thereof to form a complex with human LAG-3. In one embodiment, the test and control tissue samples are FFPE samples. The formation of the complex is then detected, and the difference in complex formation between the test sample and the negative control sample is an indication of the presence of human LAG-3 antigen in the sample. Various methods can be used to quantify LAG-3 expression.

[0216] In certain embodiments, the automated IHC method comprises: (a) deparaffinizing and rehydrating mounted tissue sections in an autostainer; (b) retrieving antigens in the autostainer; (c) setting reagents in the autostainer; and (d) running the autostainer to include the following steps: neutralizing endogenous peroxidase in the tissue specimen; blocking nonspecific protein binding sites on the slide; incubating the slide with primary Ab; incubating with a post-primary blocking agent; incubating with a post-primary antibody detection agent, such as another antibody that may or may not be conjugated with a detection enzyme; incubating with a polymer-enzyme detection agent; adding a chromogen substrate and developing; and counterstaining with hematoxylin. In some embodiments, antigen retrieval comprises the use of any heat-based antigen retrieval device.

[0217] In one embodiment, to evaluate LAG-3 expression in tumor tissue samples, pathologists examine the number of LAG-3+ tumor cells in each field under a microscope, calculate the percentage of positive cells, and then average them to arrive at a final percentage.Various staining intensities are defined as 0 / negative, 1+ / weak, 2+ / moderate, and 3+ / strong.Generally, percentage values ​​are first assigned to 0 and 3+ buckets, and then the intermediate 1+ and 2+ intensities are considered.For highly heterogeneous tissues, the specimen is divided into zones, and each zone is scored separately, and then combined into a single set of percentage values.The percentage of positive cells for negative and various staining intensities is determined from each region, and the median value is assigned to each zone.Final percentage values ​​are assigned for each staining intensity category: negative, 1+, 2+, and 3+ tissue.The sum of all staining intensities must equal 100%.

[0218] In some embodiments, staining is also evaluated for tumor-infiltrating inflammatory cells such as macrophages and lymphocytes. Macrophages and lymphocytes are evaluated for LAG-3 staining, and all samples are recorded as positive or negative for each cell category. Staining is also characterized by the designation of external / internal tumor immune cells. "Internal" means that immune cells are located within tumor tissue without physical interposition between tumor cells and / or on the border of the tumor area. "External" means that there is no physical relationship with the tumor, and immune cells are found in the periphery associated with connective tissue or any related adjacent tissue.

[0219] In certain embodiments of these scoring methods, samples are scored by two or more pathologists working independently, and the scores are then consolidated. In certain other embodiments, the identification of positive and negative cells is scored using appropriate software.

[0220] The histoscore (H-score) is used as a more quantitative index of the IHC data. The histoscore is calculated as follows: Histoscore = [(% tumor × 1 (low intensity)) + (% tumor × 2 (moderate intensity)) + (% tumor × 3 (high intensity)]

[0221] To determine the Histoscore, pathologists estimate the percentage of staining cells in each intensity category within a specimen. Because expression of most biomarkers is heterogeneous, the Histoscore is a true representation of overall expression. Final Histoscore ranges from 0 (lowest score, no expression) to 300 (highest score, strong, global expression).

[0222] 3. LAG-3 inhibitors In some embodiments, the present invention relates to methods of using LAG-3 inhibitors in the treatment of malignant tumors. As used herein, LAG-3 inhibitors include, but are not limited to, LAG-3 binding agents and soluble LAG-3 polypeptides. LAG-3 binding agents include antibodies that specifically bind to LAG-3.

[0223] In some embodiments, the LAG-3 inhibitor is a LAG-3 binding agent, e.g., an anti-LAG-3 antibody. In some embodiments, the LAG-3 inhibitor is a soluble LAG-3 polypeptide, e.g., a LAG-3-Fc fusion polypeptide capable of binding to MHC class II.

[0224] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. In some embodiments, the LAG-3 inhibitor comprises an anti-LAG-3 bispecific antibody. In some embodiments, the anti-LAG-3 antibody binds to LAG-3 and PD-1.

[0225] In certain embodiments, the anti-LAG-3 antibody is BMS-986016 or an antigen-binding fragment or variant thereof, comprising a heavy chain and a light chain comprising the sequences set forth in SEQ ID NOs: 1 and 2, respectively, of PCT / US13 / 48999.

[0226] In other embodiments, the antibody has the heavy and light chain CDRs or variable regions of BMS-986016. Thus, in certain embodiments, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of BMS-986016 having the sequence set forth in SEQ ID NO:3 and the CDR1, CDR2, and CDR3 domains of the VL region of BMS-986016 having the sequence set forth in SEQ ID NO:5. In other embodiments, the antibody comprises CDR1, CDR2, and CDR3 domains comprising the sequences set forth in SEQ ID NOs:7, 8, and 9, respectively, and CDR1, CDR2, and CDR3 domains comprising the sequences set forth in SEQ ID NOs:10, 11, and 12, respectively. In other embodiments, the antibody comprises VH and / or VL regions comprising the amino acid sequences set forth in SEQ ID NO:3 and / or SEQ ID NO:5, respectively. In other embodiments, the antibody comprises heavy chain variable (VH) and / or light chain variable (VL) regions encoded by the nucleic acid sequences set forth in SEQ ID NO:4 and / or SEQ ID NO:6, respectively. In other embodiments, the antibody competes for binding with the above-mentioned antibody and / or binds to the same epitope of LAG-3. In other embodiments, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 14). In other embodiments, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 15) or PAAPSSWG (SEQ ID NO: 16).

[0227] In other embodiments, the antibody has at least about 90% variable region amino acid sequence identity to the above antibodies (eg, at least about 90%, 95%, or 99% variable region identity to SEQ ID NO:3 or SEQ ID NO:5).

[0228] In some embodiments, art-recognized anti-LAG-3 antibodies can be used in the therapeutic methods of the present invention. For example, the anti-human LAG-3 antibody described in US2011 / 0150892A1 and designated monoclonal antibody 25F7 (also known as "25F7" and "LAG-3.1") can be used. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 (H5L7BW) described in US2011 / 007023, MK-4280 (28G-10) described in WO2016028672, REGN3767 described in Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supp. Supplement 1 Abstract Number: P195, BAP050 described in WO2017 / 019894, IMP-701 (LAG-525), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781. These and other anti-LAG-3 antibodies useful in the present invention are described in, for example: WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO20 US2017 / 0260271, WO2017 / 086367, WO2017 / 086419, WO2018 / 034227 and WO2014 / 140180. In some embodiments, the LAG-3 inhibitor is IMP321 (Eftilagimod Alpha). The contents of each of these cited documents are incorporated herein by reference in their entirety.

[0229] Antibodies that compete with any of the art-recognized control antibodies listed above for binding to LAG-3 can also be used.

[0230] In one embodiment, an anti-LAG-3 antibody is used to determine LAG-3 expression. In one embodiment, the anti-LAG-3 antibody is selected for its ability to bind to LAG-3 in formalin-fixed, paraffin-embedded (FFPE) tissue specimens. In another embodiment, the anti-LAG-3 antibody can bind to LAG-3 in frozen tissue. In a further embodiment, the anti-LAG-3 antibody can distinguish between membrane-bound, cytoplasmic, and / or soluble forms of LAG-3.

[0231] In one embodiment, an anti-LAG-3 antibody useful for assaying, detecting, and / or quantifying LAG-3 expression using the methods described herein is the 17B4 mouse IgG1 anti-human LAG-3 monoclonal antibody or an antigen-binding fragment thereof. See, e.g., J. Matsuzaki, et al.; PNAS 107, 7875 (2010).

[0232] 4. PD-1 Pathway Inhibitors In one embodiment, the present invention relates to methods of using PD-1 inhibitors in the treatment of malignancies. As used herein, "PD-1 pathway inhibitors" includes, but is not limited to, PD-1-binding agents, PD-L1-binding agents, and PD-L2-binding agents. PD-1-binding agents include antibodies that specifically bind to PD-1. PD-L1 and PD-L2-binding agents include antibodies that specifically bind to PD-L1 and / or PD-L2, and soluble PD-1 polypeptides that bind to PD-L1 and / or PD-L2.

[0233] In certain embodiments, the PD-1 pathway inhibitor is a PD-1 binding agent, e.g., an anti-PD-1 antibody. In certain embodiments, the PD-1 pathway inhibitor is a PD-L1 binding agent, e.g., an anti-PD-L1 antibody. In certain embodiments, the PD-1 pathway inhibitor is a PD-L2 binding agent, e.g., an anti-PD-L2 antibody. In further embodiments, the PD-L1 binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L1. In further embodiments, the PD-L2 binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L2.

[0234] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-PD-1 antibodies can be used. For example, monoclonal antibodies 5C4 (referred to herein as nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO2006 / 121168, can be used. Other known PD-1 antibodies include lambrolizumab (MK-3475), described in WO2008 / 156712, and AMP-514, described in WO2012 / 145493. Further known PD-1 antibodies and other PD-1 inhibitors include, for example, those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, and WO2011 / 161699, which are incorporated herein by reference. In one embodiment, the anti-PD-1 antibody is REGN2810. In one embodiment, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011).

[0235] In one embodiment, the anti-PD-1 antibody is nivolumab. (登録商標)"; formerly named 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor antibody that selectively inhibits interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent 8,008,449; Wang et al., Cancer Immunol Res. 2(9):846-56 (2014)). In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0236] In certain embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain comprising the sequences set forth in SEQ ID NOs: 17 and 18, respectively, or antigen-binding fragments and variants thereof.

[0237] In other embodiments, the antibody has the heavy and light chain CDRs or variable regions of nivolumab. Thus, in some embodiments, the antibody comprises the VH CDR1, CDR2, and CDR3 domains of nivolumab having the sequence set forth in SEQ ID NO: 19 and the VL CDR1, CDR2, and CDR3 domains of nivolumab having the sequence set forth in SEQ ID NO: 21. In other embodiments, the antibody comprises CDR1, CDR2, and CDR3 domains comprising the sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and the CDR1, CDR2, and CDR3 domains set forth in SEQ ID NOs: 26, 27, and 28, respectively. In other embodiments, the antibody comprises VH and / or VL regions comprising the amino acid sequences set forth in SEQ ID NO: 19 and / or SEQ ID NO: 21, respectively. In other embodiments, the antibody comprises heavy chain variable (VH) and / or light chain variable (VL) regions encoded by the nucleic acid sequence set forth in SEQ ID NO: 20 and / or SEQ ID NO: 22, respectively. In other embodiments, the antibody competes for binding to PD-1 with the above-mentioned antibodies and / or binds to the same epitope. In other embodiments, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibodies (eg, at least about 90%, 95%, or 99% variable region identity to SEQ ID NO: 19 or SEQ ID NO: 21).

[0238] Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity are disclosed in U.S. Patents 8,008,449 and 8,779,105. Other anti-PD-1 mAbs are described, for example, in U.S. Patents 6,808,710, 7,488,802, 8,168,757, and 8,354,509 and PCT Publication WO 2012 / 145493, which are incorporated herein by reference. In certain embodiments, the anti-PD-1 antibodies are shown to exhibit one or more of the following characteristics: (a) a cytoplasmic affinity of 1×10 or greater, as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(b) binds to human PD-1 in a mixed lymphocyte reaction (MLR) assay; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an antibody response; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies useful in the present invention include mAbs that specifically bind human PD-1 and exhibit at least one, at least two, at least three, at least four, or at least five of the above characteristics. Anti-PD-1 antibodies exhibiting one or more of these characteristics are disclosed in U.S. Patents 8,008,449, 8,779,105, 6,808,710, 7,488,802, 8,168,757, and 8,354,509 and PCT Publication WO 2012 / 145493, which are incorporated herein by reference. In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4(S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patents 8,354,509 and 8,900,587, which are incorporated herein by reference.

[0239] In certain embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In certain embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab ("Keytruda") (登録商標)", also known as lambrolizumab and MK-3475), is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patents 8,354,509 and 8,900,587; see also http: / / www.cancer.gov / drugdictionary?cdrid=695789 (last accessed: December 14, 2014). Pembrolizumab is approved by the FDA for the treatment of recurrent or refractory melanoma.

[0240] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In yet other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as MEDI0608. In certain embodiments, the anti-PD-1 antibody has the same CDRs as MEDI0608. In other embodiments, the anti-PD-1 antibody is the monoclonal antibody MEDI0608 (formerly AMP-514). MEDI0608 is described, for example, in U.S. Patent 8,609,089 B2 or http: / / www.cancer.gov / drugdictionary?cdrid=756047 (last accessed: December 14, 2014).

[0241] In some embodiments, the first antibody is an anti-PD-1 antagonist. One example of an anti-PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is described in U.S. Publication 2013 / 0017199 or http: / / www.cancer.gov / publications / dictionaries / cancer-drug?cdrid=700595 (last accessed July 8, 2015).

[0242] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In certain embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. In certain embodiments, the anti-PD-1 antibody is the humanized monoclonal antibody BGB-A317. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0243] In one embodiment, the antibody is pidilizumab (CT-011), an antibody previously reported to bind to PD-1 but believed to bind to a different target. Pidilizumab is described in U.S. Patent No. 8,686,119 B2 or WO 2013 / 014668 A1.

[0244] In some embodiments, the antibody that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of human PD-1 is a mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, humanized, or human antibodies. Such chimeric, humanized, or human mAbs can be produced and isolated by methods well known in the art.

[0245] Other anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patents 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO2012 / 145493, WO2008 / 156712, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, and WO2017 / 133540, WO2017 / 024465, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2014 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, WO2017 / 025016, WO2017 / 106061, WO2017 / 19846, WO2017 / 024465, WO2017 / 025016, WO2017 / 132825 and WO2017 / 133540, each of which is incorporated herein by reference in its entirety.

[0246] In certain embodiments, the anti-PD-1 antibody is nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008 / 156712), PDR001 (Novartis; see WO2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), and IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540).

[0247] Anti-PD-1 antibodies useful in the compositions of the invention also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments that fall within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; and (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody.

[0248] Anti-PD-1 antibodies useful in the disclosed methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein, e.g., nivolumab, for binding to human PD-1 (see, e.g., U.S. Patents 8,008,449 and 8,779,105; WO 2013 / 173223). In certain embodiments, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies described herein, e.g., nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the control antibody, e.g., nivolumab, due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0249] Anti-PD-1 antibodies suitable for use in the methods disclosed herein are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of the whole antibody in terms of ligand binding inhibition and immune system upregulation. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is a chimeric, humanized, or human monoclonal antibody or portion thereof. In certain embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype may be used.

[0250] In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of a human IgG1 or IgG4 isotype. In certain other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody, or antigen-binding portion thereof, contains a S228P mutation, replacing a serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype antibodies. This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 antibodies while maintaining the low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In yet other embodiments, the antibody comprises a light chain constant region that is a human kappa or lambda constant region. In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a mAb or antigen-binding portion thereof. In certain embodiments of any of the methods of treatment described herein that comprise administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in U.S. Patent 8,008,449. In yet other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP-224, or BGB-A317.

[0251] In certain embodiments, the anti-PD-1 antibody is a bispecific antibody that binds to both PD-1 and LAG-3.

[0252] 5. Anti-PD-L1 antibody In some embodiments, the application encompasses the use of anti-PD-L1 antibodies as PD-1 pathway inhibitors. In some embodiments, the anti-PD-L1 antibodies inhibit the binding of PD-L1 receptor, i.e., PD-1 to its ligand PD-L1.

[0253] Anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-PD-L1 antibodies can be used. For example, the human anti-PD-L1 antibodies disclosed in U.S. Patent No. 7,943,743 can be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4.

[0110] In one embodiment, the anti-PD-L1 antibody is atezolizumab (Tecentriq or RG7446) (e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Patent 8,217,149), durvalumab (Imfinzi or MEDI4736) (Khleif (2013) In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802), or avelumab (Bavencio). Other art-recognized anti-PD-L1 antibodies that can be used include those disclosed in, for example, U.S. Patents 7,635,757 and 8,217,149, U.S. Publication No. 2009 / 0317368, and PCT Publication Nos. WO2011 / 066389 and WO2012 / 145493, which are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies or inhibitors of binding to PD-L1 can also be used. Examples of anti-PD-L1 antibodies useful in the methods of the invention include the antibodies disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been shown to exhibit one or more of the following characteristics: (a) a 1×10 binding affinity to human PD-L1 as determined by surface plasmon resonance using a Biacore biosensor system; -7(b) bind with a KD of less than or equal to M; (b) increase T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increase interferon-γ production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and preferably at least five, of the above characteristics.

[0254] In some embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent 7,943,743; WO2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol 31(suppl):3000; U.S. Patent 8,217,149), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802), or MSB0010718C (also known as avelumab; see US2014 / 0341917). In some embodiments, an antibody that cross-competes with the control PD-L1 antibody for binding to human PD-L1 or binds to the same epitope region of human PD-L1 is a mAb. For administration to human subjects, these cross-competing antibodies may be chimeric or humanized or human antibodies. Such chimeric, humanized, or human mAbs may be produced and isolated by methods well known in the art. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (12A4, also known as MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; Tecentriq®; also known as MPDL3280A, RG7446; see U.S. Patent No. 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; Imfinzi) TM, also known as MEDI-4736; see WO2011 / 066389), avelumab (Pfizer; also known as BAVENCIO®, MSB-0010718C; see WO2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO2017 / 034916), and CK-301 (Checkpoint Therapeutics; Gorelik et al., AACR:Abstract 4606 (Apr 2016).

[0255] In one embodiment, the PD-L1 antibody is atezolizumab (Tecentriq®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0256] In one embodiment, the PD-L1 antibody is durvalumab (Imfinzi TM Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0257] In one embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0258] In other embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

[0259] Anti-PD-L1 antibodies useful in the disclosed methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete for binding to human PD-L1 with any of the anti-PD-L1 antibodies disclosed herein, e.g., atezolizumab, durvalumab, and / or avelumab. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, e.g., atezolizumab, durvalumab, and / or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically hinder the binding of another cross-competing antibody to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the control antibody, e.g., atezolizumab and / or avelumab, due to their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0260] In some embodiments, the antibody that cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1 or binds to the same epitope region of a human PD-L1 antibody is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be produced and isolated by methods well known in the art.

[0261] Anti-PD-L1 antibodies useful in the disclosed methods also include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0262] Anti-PD-L1 antibodies suitable for use in the disclosed methods or compositions are those that bind to PD-L1 with high specificity and affinity, block PD-1 binding, and prevent the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to those of whole antibodies in receptor binding inhibition and immune system upregulation. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0263] Anti-PD-L1 antibodies useful in the present invention include those disclosed herein. H and / or V L Anti-PD-L1 antibodies may be engineered using a variety of modifications described above for engineering the modified anti-PD-1 antibodies of the invention, including antibodies engineered starting from an antibody having one or more of the sequences, where the engineered antibody may have modified properties from the starting antibody.

[0264] 6. Anti-CTLA-4 antibody In some embodiments, the present application encompasses the use of an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody binds to and inhibits CTLA-4. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (Yervoy), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, or ATOR-1015.

[0265] 7. Immune Checkpoint Inhibitors In certain embodiments, the present invention relates to methods of using a combination of a PD-1 inhibitor and an immune checkpoint inhibitor in the treatment of malignancies. Any art-recognized immune checkpoint inhibitor may be used.

[0266] In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist, CD80 antagonist, CD86 antagonist, Tim-3 antagonist, TIGIT antagonist, CD20 antagonist, CD96 antagonist, IDO1 antagonist, STING antagonist, GARP antagonist, CD40 antagonist, A2aR antagonist, CEACAM1 (CD66a) antagonist, CEA antagonist, CD47 antagonist, PVRIG antagonist, TDO antagonist, VISTA antagonist, or KIR antagonist.

[0267] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an anti-CTLA-4 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (Yervoy), tremelimumab (ticilimumab; CP-675,206), AGEN-1884 or ATOR-1015.

[0268] In some embodiments, the CTLA-4 antagonist is a soluble CTLA-4 polypeptide. In some embodiments, the soluble CTLA-4 polypeptide is abatacept (Orencia), belatacept (Nulojix), RG2077, or RG-1046. In other embodiments, the CTLA-4 antagonist is a cell-based therapy. In some embodiments, the CTLA-4 antagonist is an anti-CTLA-4 mAb RNA / GITRL RNA-transfected autologous dendritic cell vaccine or an anti-CTLA-4 mAb RNA-transfected autologous dendritic cell vaccine.

[0269] In some embodiments, the immune checkpoint inhibitor is a KIR antagonist. In some embodiments, the KIR antagonist is an anti-KIR antibody or an antigen-binding fragment thereof. In some embodiments, the anti-KIR antibody is lirilumab (1-7F9, BMS-986015, IPH 2101) or IPH4102.

[0270] In some embodiments, the immune checkpoint inhibitor is a TIGIT antagonist.In some embodiments, the TIGIT antagonist is an anti-TIGIT antibody or its antigen-binding fragment.In some embodiments, the anti-TIGIT antibody is BMS-986207, AB 154, COM902 (CGEN-15137) or OMP-313M32.

[0271] In some embodiments, the immune checkpoint inhibitor is a Tim-3 antagonist. In some embodiments, the Tim-3 antagonist is an anti-Tim-3 antibody or its antigen-binding fragment. In some embodiments, the anti-Tim-3 antibody is TSR-022 or LY3321367.

[0272] In some embodiments, the immune checkpoint inhibitor is an IDO1 antagonist. In other embodiments, the IDO1 antagonist is indoximod (NLG8189; 1-methyl- D -TRP), epacadostat (INCB-024360, INCB-24360), KHK2455, PF-06840003, navoximod (RG6078, GDC-0919, NLG919), BMS-986205 (F001287) or pyrrolidine-2,5-dione derivatives.

[0273] In some embodiments, the immune checkpoint inhibitor is a STING antagonist. In some embodiments, the STING antagonist is a 2' or 3'-monofluorosubstituted cyclic dinucleotide; a 2'3'-difluorosubstituted mixed linkage 2',5'-3',5' cyclic dinucleotide; a 2'-fluorosubstituted bis-3',5' cyclic dinucleotide; a 2',2''-diF-Rp,Rp,bis-3',5' cyclic dinucleotide; or a fluorinated cyclic dinucleotide.

[0274] In some embodiments, the immune checkpoint inhibitor is a CD20 antagonist. In some embodiments, the CD20 antagonist is an anti-CD20 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD20 antibody is rituximab (Rituxan; IDEC-102; IDEC-C2B8), ABP 798, ofatumumab or obinutuzumab.

[0275] In some embodiments, the immune checkpoint inhibitor is a CD80 antagonist. In some embodiments, the CD80 antagonist is an anti-CD80 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD80 antibody is galiximab or AV 1142742.

[0276] In some embodiments, the immune checkpoint inhibitor is a GARP antagonist. In some embodiments, the GARP antagonist is an anti-GARP antibody or its antigen-binding fragment. In some embodiments, the anti-GARP antibody is ARGX-115.

[0277] In some embodiments, the immune checkpoint inhibitor is a CD40 antagonist. In some embodiments, the CD40 antagonist is an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD40 antibody is BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9, or dacetuzumab (huS2C6, PRO 64553, RG 3636, SGN 14, SGN-40). In other embodiments, the CD40 antagonist is a soluble CD40 ligand (CD40-L). In some embodiments, the soluble CD40 ligand is a fusion polypeptide. In some embodiments, the soluble CD40 ligand is CD40-L / FC2 or monomeric CD40-L.

[0278] In some embodiments, immune checkpoint inhibitor is A2aR antagonist.In some embodiments, A2aR antagonist is small molecule.In some embodiments, A2aR antagonist is CPI-444, PBF-509, istradefylline (KW-6002), preladenant (SCH420814), tozadenant (SYN115), bipadenant (BIIB014), HTL-1071, ST1535, SCH412348, SCH442416, SCH58261, ZM241385 or AZD4635.

[0279] In some embodiments, the immune checkpoint inhibitor is a CEACAM1 antagonist. In some embodiments, the CEACAM1 antagonist is an anti-CEACAM1 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CEACAM1 antibody is CM-24 (MK-6018).

[0280] In some embodiments, the immune checkpoint inhibitor is a CEA antagonist. In some embodiments, the CEA antagonist is an anti-CEA antibody or its antigen-binding fragment. In some embodiments, the anti-CEA antibody is sergutuzumab amnaleukin (RG7813, RO-6895882) or RG7802 (RO6958688).

[0281] In some embodiments, the immune checkpoint inhibitor is a CD47 antagonist.In some embodiments, the CD47 antagonist is an anti-CD47 antibody or its antigen-binding fragment.In some embodiments, the anti-CD47 antibody is HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM.

[0282] In some embodiments, the immune checkpoint inhibitor is a PVRIG antagonist. In some embodiments, the PVRIG antagonist is an anti-PVRIG antibody or an antigen-binding fragment thereof. In some embodiments, the anti-PVRIG antibody is COM701 (CGEN-15029).

[0283] In some embodiments, immune checkpoint inhibitor is TDO antagonist.In some embodiments, TDO antagonist is 4-(indol-3-yl)-pyrazole derivative, 3-indole substituted derivative or 3-(indol-3-yl)-pyridine derivative.In other embodiments, immune checkpoint inhibitor is dual IDO and TDO antagonist.In some embodiments, dual IDO and TDO antagonist is small molecule.

[0284] In some embodiments, the immune checkpoint inhibitor is a VISTA antagonist. In some embodiments, the VISTA antagonist is CA-170 or JNJ-61610588.

[0285] 8. Pharmaceutical Compositions Pharmaceutical compositions suitable for administration to human patients will generally be formulated so as to be suitable for reconstitution in a liquid carrier, for example, as a solution or suspension for parenteral or intravenous administration.

[0286] In general, such compositions typically contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, additive, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, etc.). Water or saline solutions and aqueous dextrose and glycerol solutions can be used as carriers, particularly for injectable solutions (e.g., containing anti-LAG-3 and / or anti-PD-1 antibodies). Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration for injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. In certain embodiments, the anti-LAG-3 and / or anti-PD-1 antibodies are administered intravenously (e.g., in separate formulations or in a single formulation (either in the same formulation or in separate formulations)).

[0287] 9. Patient population Provided herein are therapeutic methods for treating malignancies (e.g., advanced refractory solid tumors and hematological tumors) in human patients using the immunotherapies disclosed herein, e.g., a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody), a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody), an anti-CTLA-4 antibody, or a combination of a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody). Examples of cancers and / or malignant tumors that can be treated with the methods of the present invention include liver cancer, hepatocellular carcinoma (HCC), bone cancer, pancreatic cancer, skin cancer, oral cancer, head and neck cancer, breast cancer, lung cancer, small cell lung cancer, NSCLC, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck (SCCHN), non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor vasculogenesis Cancers of the present invention include tumors of the spinal axis, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, environmentally induced cancers including those induced by asbestos, hematological malignancies including multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, generalized large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combination of said cancers. The present invention is also applicable to the treatment of metastatic cancers. In certain embodiments, the cancer is renal cell carcinoma (RCC), gastric / esophagogastric junction cancer, non-small cell lung cancer (NSCLC), melanoma, squamous cell carcinoma of the head and neck (SCCHN), hepatocellular carcinoma, or urothelial carcinoma.

[0288] In some embodiments, the melanoma is unresectable or metastatic melanoma. In some embodiments, the patient has previously been treated with an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the tumor is a LAG-3-expressing tumor. In certain embodiments, the tumor is a LAG-3-expressing tumor where LAG-3 expression is ≧1%.

[0289] In certain embodiments, the human patient has unresectable metastatic melanoma and has been previously treated with an anti-PD-1 or anti-PD-L1 metastasis inhibitor. In certain embodiments, the human patient has unresectable metastatic melanoma and has been previously treated with an anti-PD-1 or anti-PD-L1 metastasis inhibitor, and the tumor is a LAG-3-expressing tumor. In certain embodiments, the human patient has unresectable metastatic melanoma and has been previously treated with an anti-PD-1 or anti-PD-L1 metastasis inhibitor, and the tumor is a LAG-3-expressing tumor. In certain embodiments, the human patient has unresectable metastatic melanoma and has been previously treated with an anti-PD-1 or anti-PD-L1 metastasis inhibitor, and the tumor is a LAG-3-expressing tumor, with LAG-3 expression of ≧1%.

[0290] In one embodiment, the human patient has a malignant tumor that is refractory to treatment with an immune checkpoint inhibitor. In another embodiment, the patient has a malignant tumor that is refractory to treatment with a PD-1 inhibitor. In another embodiment, the patient has a malignant tumor that is refractory to treatment with an anti-PD-1 antibody. In another embodiment, the patient has a malignant tumor that is refractory to treatment with an anti-PD-L1 antibody. In one embodiment, the malignant tumor is gastric cancer, renal cancer, HCC, SCCHN, or NSCLC.

[0291] In one embodiment, the human patient has melanoma. In another embodiment, the patient has melanoma that is refractory to treatment with an immune checkpoint inhibitor. In another embodiment, the patient has melanoma that is refractory to treatment with a PD-1 inhibitor. In another embodiment, the patient has melanoma that is refractory to treatment with an anti-PD-1 antibody. In another embodiment, the patient has melanoma that is refractory to treatment with an anti-PD-L1 antibody.

[0292] In some embodiments, the human patient has melanoma, gastric cancer, renal cancer, HCC, SCCHN, or NSCLC. In some embodiments, the human patient has melanoma.

[0293] In some embodiments, the human patient has NSCLC or a virus-associated cancer (e.g., a human papillomavirus (HPV)-associated tumor) or gastric adenocarcinoma. In certain embodiments, the HPV-associated tumor is HPV+ head and neck cancer (HNC). In other specific embodiments, the gastric adenocarcinoma is associated with Epstein-Barr virus (EBV) infection.

[0294] Before, during or after treatment, patients can be tested or selected for one or more of the above clinical characteristics.

[0295] Malignant tumors can be tested to determine LAG-3 expression using the methods described herein. In some embodiments, the malignant tumor treated using the methods disclosed herein is a LAG-3-positive tumor. In some embodiments, the malignant tumor is a LAG-3-positive melanoma. In other embodiments, the malignant tumor is a LAG-3-positive gastric cancer, renal cancer, HCC, SCCHN, or NSCLC.

[0296] In some embodiments, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, or at least about 3% of the total number of cells in a LAG-3-positive melanoma tumor express LAG-3.

[0297] In some embodiments, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% of the total cell number of the malignant tumor expresses LAG-3. In some embodiments, the malignant tumor is melanoma, gastric cancer, renal cancer, HCC, SCCHN, or NSCLC.

[0298] Malignant tumors can be tested to determine LAG-3 and PD-L1 expression using the methods described herein. In some embodiments, the malignant tumor treated using the methods disclosed herein is a LAG-3-positive, PD-L1-positive tumor. In some embodiments, the malignant tumor is a LAG-3-positive, PD-L1-positive melanoma. In other embodiments, the malignant tumor is a LAG-3-positive, PD-L1-positive gastric cancer, renal cancer, HCC, SCCHN, or NSCLC.

[0299] In some embodiments, the malignant tumor treated by the methods disclosed herein is a LAG-3-positive, PD-L1-negative tumor. In some embodiments, the malignant tumor is a LAG-3-positive, PD-L1-negative melanoma. In other embodiments, the malignant tumor is a LAG-3-positive, PD-L1-negative gastric cancer, renal cancer, HCC, SCCHN, or NSCLC.

[0300] 10. Immunotherapy In some embodiments, the immunotherapies provided herein involve the administration of a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and other antibodies that block inhibitory immune receptors (e.g., receptors that inhibit / neutralize activity, such as cytotoxic activity, by binding to a natural ligand), particularly anti-PD-1 or anti-PD-L1 antibodies, to treat a subject with a malignancy (e.g., an advanced, refractory solid tumor or a hematological tumor). In other embodiments, the immunotherapies provided herein involve the administration of an anti-PD-1 or anti-PD-L1 antibody to treat a subject with a malignancy (e.g., an advanced, refractory solid tumor or a hematological tumor). In other embodiments, the immunotherapies provided herein involve the administration of an anti-CTLA-4 antibody to treat a subject with a malignancy (e.g., an advanced, refractory solid tumor or a hematological tumor).

[0301] In certain embodiments, the present invention provides an anti-LAG-3 antibody and an anti-PD-1 antibody in combination with a predetermined clinical dosing regimen for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In certain embodiments, the anti-LAG-3 antibody is BMS-986016. In other embodiments, the anti-PD-1 antibody is BMS-936558. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0302] In another embodiment, the invention provides an anti-LAG-3 antibody and an anti-PD-L1 antibody in combination with a predetermined clinical dosing regimen for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In a particular embodiment, the anti-LAG-3 antibody is BMS-986016. In another embodiment, the anti-PD-L1 antibody is BMS-936559. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0303] In another embodiment, the present invention provides an anti-LAG-3 antibody according to a predetermined clinical dosing regimen for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In a particular embodiment, the anti-LAG-3 antibody is BMS-986016. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0304] In another embodiment, the invention provides an anti-PD-1 antibody with a predetermined clinical dosing regimen for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In a particular embodiment, the anti-PD-1 antibody is BMS-936558. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0305] In another embodiment, the invention provides an anti-PD-L1 antibody, according to a predetermined clinical dosing regimen, for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In a particular embodiment, the anti-PD-L1 antibody is BMS-936559. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0306] In another embodiment, the present invention provides an anti-CTLA-4 antibody with a predetermined clinical dosing regimen for treating a subject with a malignant tumor (e.g., an advanced refractory solid tumor). In a specific embodiment, the anti-CTLA4 antibody is ipilimumab (Yervoy). In a specific embodiment, the anti-CTLA4 antibody is tremelimumab (ticilimumab; CP-675,206), AGEN-1884, or ATOR-1015. In other embodiments, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0307] In other embodiments, the immunotherapy provided herein comprises administration of an anti-PD-1 antibody and an immune checkpoint inhibitor to treat a subject with a malignant tumor (e.g., an advanced refractory solid tumor or a hematological tumor). In one embodiment, the anti-PD-1 antibody is BMS-936558. In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist, CD80 antagonist, CD86 antagonist, Tim-3 antagonist, TIGIT antagonist, CD20 antagonist, CD96 antagonist, IDO1 antagonist, STING antagonist, GARP antagonist, CD40 antagonist, A2aR antagonist, CEACAM1 (CD66a) antagonist, CEA antagonist, CD47 antagonist, PVRIG antagonist, TDO antagonist, VISTA antagonist, or KIR antagonist.

[0308] In other embodiments, the immunotherapy provided herein involves administration of an anti-PD-L1 antibody and an immune checkpoint inhibitor to treat a subject with a malignant tumor (e.g., an advanced refractory solid tumor or a hematological tumor). In one embodiment, the anti-PD-L1 antibody is BMS-936559. In certain embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist, CD80 antagonist, CD86 antagonist, Tim-3 antagonist, TIGIT antagonist, CD20 antagonist, CD96 antagonist, IDO1 antagonist, STING antagonist, GARP antagonist, CD40 antagonist, A2aR antagonist, CEACAM1 (CD66a) antagonist, CEA antagonist, CD47 antagonist, PVRIG antagonist, TDO antagonist, VISTA antagonist, or KIR antagonist.

[0309] As used herein, adjunctive or combined administration (co-administration) includes simultaneous administration of multiple compounds in the same or different dosage forms or separate administration of multiple compounds (e.g., sequential administration). Thus, for example, anti-LAG-3 and anti-PD-1 antibodies can be administered simultaneously in a single formulation. Alternatively, anti-LAG-3 and anti-PD-1 antibodies can be formulated for separate administration and administered simultaneously or sequentially (e.g., one antibody is administered within about 30 minutes before the administration of the second antibody).

[0310] For example, the anti-PD-1 antibody may be administered first, followed (e.g., immediately after) by the anti-LAG-3 antibody, or vice versa. In certain embodiments, the anti-PD-1 antibody is administered before the administration of the anti-LAG-3 antibody. In other embodiments, the anti-PD-1 antibody is administered after the administration of the anti-LAG-3 antibody. In other embodiments, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered simultaneously. Such simultaneous or sequential administration preferably results in the simultaneous presence of both antibodies in the treated patient.

[0311] 11. Treatment Protocol In certain embodiments, a suitable treatment protocol for treating a malignant tumor in a human patient comprises administering to the patient an effective amount of a LAG3 inhibitor (eg, an anti-LAG-3 antibody).

[0312] In certain embodiments, a suitable treatment protocol for treating a malignant tumor in a human patient includes, for example, administering to the patient an effective amount of an anti-LAG-3 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO:3 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:5; wherein the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and wherein, for each of the at least one cycle, the anti-LAG-3 antibody is administered at least four times at a flat dose of about 1 mg, 3 mg, 10 mg, 20 mg, 50 mg, 80 mg, 100 mg, 130 mg, 150 mg, 16 mg, 180 mg, 200 mg, 240 mg, or 280 mg. In other embodiments, the anti-LAG-3 antibody is administered four times at a dose of 0.01 mg / kg, 0.03 mg / kg, 0.25 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, 5 mg / kg, 8 mg / kg, or 10 mg / kg body weight.

[0313] In some embodiments, a suitable treatment protocol for treating a malignant tumor in a human patient comprises administering to the patient an effective amount of a PD1 pathway inhibitor (e.g., an anti-PD-1 antibody). In one embodiment, a suitable treatment protocol for treating a malignant tumor in a human patient comprises, for example, administering to the patient an effective amount of an anti-PD-1 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having SEQ ID NO: 19 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having SEQ ID NO: 21, wherein the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and wherein the anti-PD-1 antibody is administered at least four times at a flat dose of about 50 mg, 80 mg, 100 mg, 130 mg, 150 mg, 180 mg, 200 mg, 240 mg, or 280 mg for each of the at least one cycle. In other embodiments, the anti-PD-1 antibody is administered four times at a dose of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, or 10 mg / kg body weight.

[0314] In certain embodiments, a suitable treatment protocol for treating a malignancy in a human patient comprises administering to the patient an effective amount of an anti-CTLA-4 antibody.

[0315] In one embodiment, a suitable treatment protocol for treating a malignant tumor in a human patient comprises, for example, administering to the patient an effective amount of an anti-CTLA-4 antibody, wherein the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and wherein, for each of the at least one cycle, the anti-CTLA-4 antibody is administered at least four times at a flat dose of about 50 mg, 80 mg, 100 mg, 130 mg, 150 mg, 180 mg, 200 mg, 240 mg, or 280 mg. In another embodiment, the anti-CTLA-4 antibody is administered four times at a dose of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, or 10 mg / kg body weight.

[0316] In some embodiments, a suitable treatment protocol for treating a malignant tumor in a human patient comprises administering to the patient an effective amount of each of a LAG3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody).

[0317] In certain embodiments, a suitable treatment protocol for treating a malignant tumor in a human patient may, for example, comprise administering to the patient an effective amount of: (a) an anti-LAG-3 antibody comprising CDR1, CDR2 and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO: 3 and CDR1, CDR2 and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO: 5; (b) an anti-PD-1 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having SEQ ID NO: 19 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having SEQ ID NO: 21; and administering each of wherein the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and wherein, for each of the at least one cycle, the anti-LAG-3 antibody is administered at least four times in a flat dose of about 1 mg, 3 mg, 10 mg, 20 mg, 50 mg, 80 mg, 100 mg, 130 mg, 150 mg, 160 mg, 180 mg, 200 mg, 240 mg, or 280 mg, and the anti-PD-1 antibody is administered at least four times in a flat dose of about 50 mg, 80 mg, 100 mg, 130 mg, 150 mg, 180 mg, 200 mg, 240 mg, or 280 mg. In other embodiments, the anti-LAG-3 antibody is administered four times at doses of 0.01 mg / kg, 0.03 mg / kg, 0.25 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg, 5 mg / kg, 8 mg / kg, or 10 mg / kg body weight, and the anti-PD-1 antibody is administered four times at doses of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 8 mg / kg, or 10 mg / kg body weight.

[0318] In certain embodiments, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered in the following doses: (a) 3 mg of anti-LAG-3 antibody and 80 mg of anti-PD-1 antibody; (b) 3 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (c) 20 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (d) 80 mg of anti-LAG-3 antibody and 160 mg of anti-PD-1 antibody; (e) 80 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody; (f) 160 mg of an anti-LAG-3 antibody and 240 mg of an anti-PD-1 antibody; or (g) 240 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0319] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 20 mg of anti-LAG-3 antibody and 80 mg of anti-PD-1 antibody. In one embodiment, the tumor is lung cancer.

[0320] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at doses of 20 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0321] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 80 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody. In one embodiment, the tumor is melanoma (e.g., anti-PD1 / PD-L1 antibody-experienced melanoma or first-line melanoma treatment), RCC (e.g., IO-naive RCC), NSCLC (e.g., anti-PD1 / PD-L1 antibody-experienced NSCLC), gastric cancer (e.g., IO-naive gastric cancer), HCC (e.g., IO-naive HCC), NSCLC (e.g., first-line treatment of NSCLC), or SCCHN (e.g., IO-naive SCCHN).

[0322] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 240 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

[0323] In one embodiment, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 160 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody. In one embodiment, the tumor is melanoma (e.g., anti-PD1 / PD-L1 antibody-experienced melanoma or first-line melanoma treatment), RCC (e.g., IO-naive RCC), NSCLC (e.g., anti-PD1 / PD-L1 antibody-experienced NSCLC), gastric cancer (e.g., IO-naive gastric cancer), HCC (e.g., IO-naive HCC), NSCLC (e.g., first-line treatment for NSCLC), or SCCHN (e.g., IO-naive SCCHN). In other embodiments, the tumor is Hodgkin lymphoma (e.g., Hodgkin lymphoma previously treated IO); DLBCL, PD-1 / PD-L1 naive Hodgkin lymphoma, or PD-1 / PD-L1 advanced / refractory Hodgkin lymphoma.

[0324] In other embodiments, the anti-LAG-3 antibody and the anti-PD-1 antibody are administered in the following doses: (a) 0.3 mg / kg anti-LAG-3 antibody and 1 mg / kg anti-PD-1 antibody; (b) 0.3 mg / kg anti-LAG-3 antibody and 3 mg / kg anti-PD-1 antibody; (c) 0.25 mg / kg anti-LAG-3 antibody and 3 mg / kg anti-PD-1 antibody; (d) 1 mg / kg of an anti-LAG-3 antibody and 3 mg / kg of an anti-PD-1 antibody; or (e) 3 mg / kg anti-LAG-3 antibody and 3 mg / kg anti-PD-1 antibody.

[0325] In some embodiments, the dose of the anti-LAG-3 and / or anti-PD-1 antibody is calculated per body weight, e.g., mg / kg body weight. In other embodiments, the dose of the anti-LAG-3 and / or anti-PD-1 antibody is a flat-rate dose. In other embodiments, the dose of the anti-LAG-3 and / or anti-PD-1 antibody varies over time. For example, the anti-LAG-3 antibody and / or anti-PD-1 antibody may be administered initially at a high dose and then decreased over time. In other embodiments, the anti-LAG-3 antibody and / or anti-PD-1 antibody may be administered initially at a low dose and then increased over time.

[0326] In other embodiments, the amount of anti-LAG-3 and / or anti-PD-1 antibody administered remains constant with each administration. In other embodiments, the amount of antibody administered varies with each administration. For example, the maintenance (or continuing) dose of the antibody may be higher than or equal to the loading dose, which is the initial administration. In other embodiments, the maintenance dose of the antibody may be lower than or equal to the loading dose.

[0327] In other embodiments, the anti-LAG-3 and / or anti-PD-1 antibody is formulated for intravenous administration. In one embodiment, the anti-PD-1 antibody is administered on days 1, 15, 29, and 43 of each cycle. In another embodiment, the anti-LAG-3 antibody is administered on days 1, 15, 29, and 43 of each cycle.

[0328] In other embodiments, the anti-LAG-3 and / or anti-PD-1 antibody is administered about once a week, about once every 2 or 3 weeks, about once a month, or as long as clinical benefit is observed, or until complete response, definite disease progression, or unmanageable toxicity.

[0329] In other embodiments, the administration cycle is 8 weeks and can be repeated as needed. In other embodiments, the treatment consists of up to 12 cycles.

[0330] In another embodiment, four anti-PD-1 antibodies are administered per 8-week cycle. In another embodiment, four anti-LAG-3 antibodies are administered per 8-week cycle.

[0331] In other embodiments, the anti-PD-1 antibody and the anti-LAG-3 antibody are administered as a first-line treatment (e.g., initial or initial treatment). In other embodiments, the anti-PD-1 antibody and the anti-LAG-3 antibody are administered as a second-line treatment (e.g., after initial or initial treatment, including after relapse and / or when initial treatment has failed).

[0332] In certain embodiments, the present invention provides a method of treating a human patient with unresectable or metastatic melanoma, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with a PD-1 inhibitor. In certain embodiments, the present invention provides a method of treating a human patient with unresectable or metastatic melanoma, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with a PD-L1 inhibitor. In certain embodiments, the present invention provides a method of treating a human patient with unresectable or metastatic melanoma, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with a PD-1 inhibitor, and the melanoma expresses LAG-3. In certain embodiments, the present invention provides a method of treating a human patient with unresectable or metastatic melanoma, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with a PD-L1 inhibitor, and the melanoma expresses LAG-3. In certain embodiments, the present invention provides a method of treating a human patient with melanoma that has progressed during or after treatment with a PD-1 pathway inhibitor or a PD-L1 pathway inhibitor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with an anti-PD-1 inhibitor. In certain embodiments, the present invention provides a method of treating a human patient with melanoma that has progressed during or after treatment with a PD-1 pathway inhibitor or a PD-L1 pathway inhibitor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with an anti-PD-L1 inhibitor. In certain embodiments, the present invention provides a method of treating a human patient with melanoma that has progressed during or after treatment with a PD-1 pathway inhibitor or a PD-L1 pathway inhibitor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with an anti-PD-1 inhibitor and the melanoma expresses LAG-3.In certain embodiments, the present invention provides a method of treating a human patient with melanoma that has progressed during or after treatment with a PD-1 pathway inhibitor or a PD-L1 pathway inhibitor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has previously been treated with an anti-PD-L1 inhibitor and the melanoma expresses LAG-3. In certain embodiments, the melanoma has LAG-3 expression of ≧1%. In certain embodiments, the PD-1 pathway inhibitor administered is an anti-PD-1 antibody. In certain embodiments, the PD-1 antibody is nivolumab. In certain embodiments, the LAG-3 inhibitor is a LAG-3 antibody. In certain embodiments, the LAG-3 antibody is BMS-986016. In certain embodiments, the PD-1 pathway inhibitor administered is an anti-PD-L1 antibody.

[0333] In one embodiment, the anti-LAG-3 antibody is BMS-986016 and the anti-PD-1 antibody is nivolumab. In one embodiment, the anti-LAG-3 antibody is MK-4280 and the anti-PD-1 antibody is pembrolizumab. In one embodiment, the anti-LAG-3 antibody is REGN3767 and the anti-PD-1 antibody is REGN2810. In one embodiment, the anti-LAG-3 antibody is LAG525 (International Publication No. WO2015 / 138920) and the anti-PD-1 antibody is PDR001.

[0334] In other aspects, the invention relates to any of the above embodiments, in which an anti-PD-1 antibody is replaced with or combined with an anti-PD-L1 or anti-PD-L2 antibody.

[0335] In another aspect, the invention relates to any of the foregoing embodiments, wherein administration of an anti-LAG-3 antibody, or antigen-binding fragment thereof, and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody) activates T cells from the patient. In certain embodiments, administration of an anti-LAG-3 antibody, or antigen-binding fragment thereof, and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody) induces expression of activation markers by the patient's T cells. Expression of activation markers by the patient's T cells can be detected by analysis of a patient sample, e.g., peripheral lymphocytes or tumor-infiltrating lymphocytes, using flow cytometry.

[0336] In another aspect, the invention relates to any of the foregoing embodiments, wherein administration of the anti-LAG-3 antibody or antigen-binding fragment thereof results in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% occupancy of LAG-3 receptors on the patient's T cells. In certain embodiments, the T cells are CD8+ T cells. In certain embodiments, the T cells are tumor-infiltrating T cells.

[0337] In another aspect, the invention relates to any of the aforementioned embodiments, wherein the treatment protocol further comprises administration of at least one additional therapeutic agent. In certain embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the at least one additional therapeutic agent is an immune checkpoint inhibitor.

[0338] 12. Outcomes With respect to target lesions, response to treatment may include: [Table 1]

[0339] For non-target lesions, response to treatment may include: [Table 2]

[0340] Patients treated with the methods disclosed herein preferably experience an improvement in at least one symptom of cancer.In some embodiments, improvement is measured by the reduction in the amount and / or size of measurable tumor lesions.In other embodiments, lesions can be measured by chest x-ray or CT or MRI film.In other embodiments, cytological or histological diagnosis can be used to evaluate response to treatment.

[0341] In certain embodiments, treated patients exhibit a complete response (CR), partial response (PR), stable disease (SD), immune-related complete disease (irCR), immune-related partial response (irPR), or stable immune-related disease (irSD). In other embodiments, treated patients experience tumor shrinkage and / or a slower growth rate, i.e., tumor growth inhibition. In other embodiments, unwanted cell proliferation is reduced or prevented. In still other embodiments, one or more of the following may occur: the number of cancer cells is reduced; tumor size is reduced; cancer cell invasion into peripheral organs is prevented, delayed, slowed, or halted; tumor metastasis is slowed or prevented; tumor growth is prevented; tumor recurrence is prevented or delayed; or one or more symptoms associated with cancer are alleviated to some extent.

[0342] In other embodiments, administration of an effective amount of an anti-LAG-3 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, a combination of an anti-LAG-3 antibody and an anti-PD-1 antibody, or a combination of an anti-PD-1 antibody and an immune checkpoint inhibitor by any of the methods provided herein results in at least one therapeutic effect selected from the group consisting of a decrease in tumor size, a decrease in the number of metastatic lesions appearing over time, a complete response, a partial response, or stable disease.

[0343] In yet another embodiment, the treatment method results in a better clinical benefit rate (CBR = CR + PR + SD > 6 months) than that achieved by a treatment method that does not include the steps of (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof. In other embodiments, the improvement in clinical benefit rate is about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to a treatment method that does not include the steps of (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof.

[0344] In yet other embodiments, the treatment method results in an objective response rate (ORR = CR + PR) of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In some embodiments, the treatment method results in an objective response rate of at least about 15%, wherein the malignant tumor is LAG-3-positive melanoma that is resistant to treatment with an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the median duration of response is ≥ 3 months, ≥ 6 months, ≥ 12 months, or ≥ 18 months. In some embodiments, the median duration of response is ≥ 6 months. In some embodiments, the frequency with which patients experience a response duration of ≥ 6 months is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100%.

[0345] In yet another embodiment, the treatment method results in a better objective response rate (ORR = CR + PR) than that achieved by a treatment method that does not include the steps of: (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof. In other embodiments, the improvement in objective response rate is about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to a treatment method that does not include the steps of: (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof. In some embodiments, the median duration of response is ≧3 months, ≧6 months, ≧12 months, or ≧18 months. In some embodiments, the median duration of response is ≧6 months.

[0346] In yet other embodiments, the treatment method results in a disease control rate (DRR = CR + PR + SD) of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In certain embodiments, the treatment method results in a disease control rate of at least about 70%, wherein the malignant tumor is LAG-3-positive melanoma that is resistant to treatment with an anti-PD-1 or anti-PD-L1 antibody. In certain embodiments, the median duration of response is ≥ 3 months, ≥ 6 months, ≥ 12 months, or ≥ 18 months. In certain embodiments, the median duration of response is ≥ 6 months. In certain embodiments, the frequency with which patients experience a response duration of ≥ 6 months is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100%.

[0347] In yet another embodiment, the treatment method results in a better disease control rate (DRR=CR+PR+SD) than that achieved by a treatment method that does not include the steps of: (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof. In other embodiments, the improvement in disease control rate is about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to a treatment method that does not include the steps of: (i) determining LAG-3 expression levels in tumor samples before treatment, (ii) selecting LAG-3-positive tumors for treatment, (iii) treating tumors identified as LAG-3-positive before treatment, or (iv) any combination thereof. In some embodiments, the median duration of response is ≧3 months, ≧6 months, ≧12 months, or ≧18 months. In some embodiments, the median duration of response is ≧6 months.

[0348] 13. Kits and Unit Dosage Forms Also within the scope of the present invention is a diagnostic kit comprising an anti-LAG-3 antibody for assaying LAG-3 expression as a biomarker for screening patients for immunotherapy or for predicting the efficacy of immunotherapy. The kit generally includes a label indicating the intended use and instructions for use of the contents of the kit. The term "label" includes any writing or recorded medium that is on the kit, with the kit, or otherwise accompanies the kit. In some embodiments of the diagnostic kit, a first anti-LAG-3 antibody for assaying, detecting, and / or quantifying LAG-3 expression is co-packaged with at least one therapeutic antibody for treating LAG-3-positive tumors (e.g., a second anti-LAG-3 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody). In some embodiments, the kit further includes an anti-PD-L1 antibody for assaying, detecting, and / or quantifying PD-L1 expression as a biomarker for predicting the efficacy of immunotherapy. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody) and a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody). In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody. In certain embodiments, the immunotherapy comprises administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) and an immune checkpoint inhibitor.

[0349] In some embodiments, the diagnostic kit comprises an anti-human LAG-3 monoclonal antibody for assaying, detecting, and / or quantifying LAG-3 expression. See, e.g., J. Matsuzaki, et al.; PNAS 107, 7875 (2010).

[0350] Also provided herein is a therapeutic kit comprising a pharmaceutical composition comprising an anti-LAG-3 antibody, such as BMS-986016, and an anti-PD-1 antibody, such as nivolumab, in therapeutically effective amounts suitable for use in the above methods, and a pharmaceutically acceptable carrier. In some embodiments of the therapeutic kit, the anti-LAG-3 antibody is co-packaged with the anti-PD-1 antibody in a unit dosage form. The kit may also optionally include instructions, including, for example, an administration schedule, to enable a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient with cancer (e.g., a solid tumor). The kit may also include a syringe.

[0351] Optionally, the diagnostic and / or therapeutic kit includes multiple packages of single-dose pharmaceutical compositions, each containing an effective amount of an anti-LAG-3 or anti-PD-1 antibody for single administration according to the methods provided above. Apparatus or devices necessary for administering the pharmaceutical compositions may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing an effective amount of an anti-LAG-3 or anti-PD-1 antibody.

[0352] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) a dose of an anti-LAG-3 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO:3 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:5; (b) a dose of an anti-PD-1 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having SEQ ID NO: 19 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having SEQ ID NO: 21; and (c) Instructions for using the anti-LAG-3 antibody and the anti-PD-1 antibody in the methods described herein. Includes.

[0353] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) a dose of an anti-LAG-3 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO:3 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:5; and (b) Instructions for using anti-LAG-3 antibodies in the methods described herein. Includes.

[0354] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) a dose of an anti-PD-1 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having SEQ ID NO: 19 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having SEQ ID NO: 21; and (b) Instructions for using anti-PD-1 antibodies in the methods described herein. Includes.

[0355] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) one dose of an anti-PD-L1 antibody, such as BMS-936559; and (b) Instructions for using the anti-PD-L1 antibodies in the methods described herein Includes.

[0356] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) one dose of an anti-CTLA-4 antibody, such as ipilimumab (Yervoy); and (b) Instructions for using anti-CTLA-4 antibodies in the methods described herein. Includes.

[0357] In certain embodiments, the present invention provides a kit for treating a patient with a malignant tumor, the kit comprising, e.g., (a) a dose of an anti-LAG-3 antibody comprising the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO:3 and the CDR1, CDR2, and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:5; (b) one dose of an immune checkpoint inhibitor; and (c) Instructions for using the anti-PD-1 antibodies and immune checkpoint inhibitors in the methods described herein. Includes.

[0358] In some embodiments, the malignant tumor is a LAG-3 positive tumor. In some embodiments, the malignant tumor is a LAG-3 / PD-L1 positive tumor. In some embodiments, the malignant tumor is a LAG-3 positive / PD-L1 negative tumor.

[0359] In some embodiments, the malignant tumor is melanoma.

[0360] This invention is further illustrated by the following examples which should not be construed as further limiting. [Example]

[0361] Example 1 Optimization and validation of an assay for automated detection of LAG3 (mouse clone 17B4) by single-stain immunohistochemistry with DAB chromogen and evaluation by image analysis in formalin-fixed, paraffin-embedded human tissues The objective of this study was to validate an immunohistochemical assay for lymphocyte activation gene-3 (LAG3) using a commercially available antibody (mouse clone 17B4) from LS Biosciences for use in formalin-fixed, paraffin-embedded (FFPE) human tissue.

[0362] Immunohistochemistry (IHC) refers to a method for localizing proteins or other molecules in cells of tissue sections. Immunohistochemical staining is widely used in cancer diagnosis and has recently been used to aid in predicting whether a patient is likely to respond to certain targeted chemotherapy agents. Contrary to many other analytical techniques, such as Western blot or ELISA, IHC preserves the spatial localization of protein expression within a tissue specimen. The technique involves the use of an antibody (primary antibody) that specifically binds to a target within a cellular context, followed by the use of the bound antibody to deposit a pigment in the target area.

[0363] Test System. FFPE validation was performed on remnant, de-identified, or anonymous human samples. Tissues used for sensitivity testing and analysis included 40 bladder urothelial carcinomas, 41 gastric / GEJ carcinomas, 41 HNSCCs, 41 melanomas, 41 NSCLCs, and 43 RCCs. The positive and negative controls selected for LAG3 IHC were tonsil tissues. Tonsil tissues contain cellular signatures that are both positive and negative for LAG3.

[0364] Test Articles. LAG3 mouse clone 17B4 antibody was purchased from LS Biosciences (Seattle, WA) and stored at -20°C. Mouse IgG isotype control antibody was purchased from BD Pharmingen (San Jose, CA) and stored at 2-8°C.

[0365] Immunohistochemistry. Immunohistochemistry was performed by standard laboratory techniques.

[0366] Pretreatment. The procedure for IHC analysis of LAG3 (mouse clone 17B4) was performed using commercially available reagents and automated detection on a Leica Bond Rx (Leica Biosystems, Buffalo Grove, IL) at room temperature (RT). Specimens were sectioned offline at 4 micron thickness, mounted on positively charged glass slides, dried, baked, deparaffinized, and rehydrated. Tissues were then loaded onto an autostainer and pretreated with Epitope Retrieval Solution 1 (Catalog # AR9961, Leica) for 20 minutes at 100°C, followed by rinsing with Bond Wash Buffer (Catalog # AR9590, Leica) at RT.

[0367] DAB Chromogen Assay: Tissues were incubated with Peroxide Block (Catalog# DS9800, Leica) for 5 minutes, followed by rinsing three times with Bond Wash Buffer. Tissues were incubated with Protein Block, Serum Free (Catalog# X0909, Dako, Carpinteria, CA) for 5 minutes, followed by 30 minutes of incubation with primary antibody or isotype negative control reagent diluted in Bond Primary Antibody Diluent (Catalog# AR9352, Leica) and rinsing three times with Bond Wash Buffer. Tissues were incubated with Post Primary (Bond Polymer Refine Detection Kit, Catalog# DS9800, Leica) for 8 minutes, followed by rinsing three times with Bond Wash Buffer for 2 minutes each. Tissues were incubated with Polymer (Bond Polymer Refine Detection Kit) for 8 minutes, followed by rinsing three times with Bond Wash Buffer for 2 minutes each and rinsing twice with distilled water. The tissue was incubated with DAB (Bond Polymer Refine Detection Kit) for 10 minutes, followed by rinsing four times with distilled water.

[0368] The red chromogen assay tissues were then incubated with 3% hydrogen peroxide for 5 minutes, followed by rinsing three times with Bond Wash Buffer. The tissues were incubated with Protein Block, Serum Free for 5 minutes, followed by 30 minutes with primary antibody or isotype negative control reagent diluted in Bond Primary Antibody Diluent, followed by rinsing three times with Bond Wash Buffer. The tissues were incubated with Post Primary AP (Catalog# DS9390, Bond Polymer Refine Red Detection Kit, Leica) for 20 minutes, followed by rinsing three times with Bond Wash Buffer for 2 minutes each. The tissues were incubated with Polymer AP (Bond Polymer Refine Red Detection Kit) for 30 minutes, followed by rinsing three times with Bond Wash Buffer for 2 minutes each and rinsing twice with distilled water. The tissues were incubated with Red Refine (Bond Polymer Refine Red Detection Kit) for 10 minutes, followed by rinsing four times with distilled water.

[0369] After processing, tissues were incubated with hematoxylin (Bond Polymer Refine Detection Kit) for 5 min, followed by one rinse with distilled water and one rinse with Bond Wash Buffer. Coverslipping was performed offline using an automated coverslipper (Leica) according to standard procedures.

[0370] Slides were scanned using an Aperio Turbo AT system (Aperio, Vista, CA) to obtain whole-slide images. 20x JPEG images of each stain are provided in this report.

[0371] Image analysis: Tissues stained with LAG3 (mouse clone 17B4) using DAB or red chromogen were evaluated by image analysis using the Nuclear v9 algorithm from Aperio. The ROI included areas of tumor tissue with intervening stroma. Areas excluded from analysis included normal tissue, large stromal areas, necrotic tissue, tar (if possible), and staining artifacts.

[0372] The nuclear algorithm was chosen because intense cytoplasmic staining in small cells, such as immune cells, often obscures nuclear hematoxylin. The cytoplasmic and membrane algorithms required visualization of nuclear hematoxylin for cell quantification. The nuclear algorithm has a feature called "hole-filling," which, if hematoxylin is present, fills in the central portion of a lymphocyte and records it as a single cell.

[0373] A subset of samples within the dynamic range were also scored by the pathologist during image analysis QC. The purpose of the pathologist's visual immunoscore was to provide a backup result when the image analysis score was deemed inaccurate by a board-certified pathologist. Reasons for image analysis failure could include, but are not limited to: 1) weak counterstaining; 2) tissue crushing; 3) the presence of tar in NSCLC tissue; 4) hemosiderin staining; or 5) the presence of melanin, which interferes with evaluation. The pathologist's visual immunoscore is the percentage of positive immune cells within the annotated area (due to algorithmic mimicry).

[0374] LAG3 IHC Assay Validation - Sensitivity A sensitivity analysis was performed using the optimized LAG3 (murine clone 17B4) IHC assay on 247 FFPE human tissues (40 bladder urothelial carcinomas, 41 gastric / GEJ carcinomas, 41 HNSCCs, 41 melanomas, 41 NSCLCs, and 43 RCCs) to demonstrate the dynamic range of the assay within six indications. All specimens were evaluated by image analysis of one ROI (tumor + intervening stroma), and a subset of tissues (10 each within the six indications) was also evaluated by visual immunoscore by a pathologist.

[0375] On average, LAG-3 (murine clone 17B4) expression was highest in melanoma (3.54%), followed by bladder urothelial carcinoma (2.58%), NSCLC (1.68%), HNSCC (1.47%), gastric / GEJ carcinoma (1.27%), and RCC (1.24%). Positivity ranged from 0.01% to 25.57%, with a mean of 1.95% and a median of 0.84%. Using a 2% threshold, we found 192 negative and 55 positive tissues (12 bladder urothelial, 6 gastric / GEJ carcinoma, 7 HNSCC, 18 melanoma, 8 NSCLC, and 4 RCC).

[0376] Figure 1 shows the anti-LAG-3 staining patterns observed in tumor samples using monoplex IHC. The observed staining patterns included partial membrane / cytoplasmic localization, dot-like localization, and complete membrane / cytoplasmic localization.

[0377] FIG. 2 shows the frequency distribution of LAG-3 positive cells as a proportion of total tumor cells across different tumors, as detected by monoplex LAG-3 IHC.

[0378] Example 2 Early efficacy of anti-lymphocyte-activation gene-3 (anti-LAG-3; BMS-986016) in combination with nivolumab in patients with melanoma previously treated with anti-PD1 / PD-L1 therapy Simultaneous blockade of the negative T cell regulators LAG-3 and PD-1 may function synergistically to restore T cell activation and enhance antitumor immunity. A phase 1 / 2a trial of BMS-986016 (a fully human IgG4 mAb targeting LAG-3) plus nivolumab (a fully human IgG4 mAb targeting PD-1) demonstrated that the combination was well tolerated and showed promising antitumor activity in patients with melanoma that was refractory to or relapsed during prior anti-PD-1 / PD-L1 therapy (NCT01968109; Ascierto et al. J Clin Oncol. 2017;35(suppl) [abstract 9520]). Efficacy data are presented below in patients with advanced melanoma who had progressed on prior anti-PD-1 / PD-L1 therapy.

[0379] This was a Phase I / IIa, open-label, dose-escalation and cohort expansion study evaluating the safety, tolerability, and efficacy of BMS-986016 administered alone or in combination with nivolumab in patients with advanced solid tumors. Patients received the investigational treatment intravenously every 2 weeks for up to 12 8-week treatment cycles. The combination dose for expansion was BMS-986016 80 mg + nivolumab 240 mg.

[0380] The study design and endpoints are shown in Figures 3 and 17.

[0381] Key eligibility criteria for patients in melanoma before the IO expansion cohort are shown in Figure 3.

[0382] Results. As of data cutoff on April 7, 2017, 212 patients had been treated, including 55 patients with melanoma that had progressed on prior anti-PD1 / PD-L1 therapy (mel prior IO). Of the 212 patients, 61% were still on treatment at the time of data cutoff. Of the 83 patients who discontinued treatment, the primary reason was disease progression (86%). In the mel prior IO cohort, 67% of patients had M1C disease without brain metastases, 15% had lactate dehydrogenase (LDH) ≥ 2× the upper limit of normal (ULN), and 20% had liver metastases. Figure 4.

[0383] Patients in the mel prior IO cohort were heavily pretreated (Figure 5). Of the 55 patients, 76% had received ≥2 prior therapies; 40% of patients had progressive disease (PD) as their best response to prior anti-PD1 / PD-L1 therapy.

[0384] Figure 6 shows the LAG-3 expression status of the first 40 IO-experienced melanoma samples. 40% (16 / 40) of the samples were scored as LAG-3 positive using a ≥1% cutoff in the monoplex IHC assay.

[0385] Efficacy in melanoma in the prior IO cohort. The median follow-up for all efficacy-evaluable patients (n=48; all progressed on prior anti-PD-1 / PD-L1 therapy) was 14 weeks (range, 4.1–41 weeks). Investigator-assessed responses are shown in Figure 7. The overall response rate (ORR) was 13%, with six patients experiencing PR (two of whom experienced PD as their best response to prior anti-PD-1 / PD-L1 therapy). Fifteen patients experienced a reduction in tumor burden from baseline; a reduction of >30% was seen in seven patients (Figure 8). As shown in Figure 8, LAG-3 expression enriched for response. Figure 9 shows the depth and duration of response for patients with LAG-3 ≥1%, LAG-3 <1%, and LAG-3 unknown.

[0386] Figure 10 shows progression-free survival. Of 48 evaluable patients, 46% (22 / 48) of patients remained on treatment without progression at the time of data cutoff.

[0387] There was an approximately three-fold increase in ORR in patients with LAG-3 expression ≥ 1% (20%) versus LAG-3 expression < 1% (7.1%), as shown in Figure 11. PD-L1 expression did not appear to enrich for response.

[0388] Updated results from the clinical trial are shown in Figures 16-23. As of August 2017, 262 patients had been treated, including 68 patients with melanoma that had progressed on prior anti-PD1 / PD-L1 therapy (mel prior IO). Updated baseline demographic and disease characteristics are shown in Figure 17. In the mel prior IO cohort, 68% of patients had M1C disease without brain metastases, 13% had lactate dehydrogenase (LDH) ≥ 2x the upper limit of normal (ULN), and 25% had liver metastases.

[0389] Figure 18 shows the updated prior treatment history of the mel prior IO cohort. Of the 68 patients, 77% had received ≥2 prior therapies; 46% of patients had progressive disease (PD) as their best response to prior anti-PD1 / PD-L1 therapy. Most patients (57%) also received prior anti-CTLA-4 therapy. 46% of patients had PD as their best response to prior anti-PD-1 / PD-L1 therapy.

[0390] Figure 19 shows updated efficacy data for the mel prior IO cohort. The ORR was 11.5% and the DCR was 49%. LAG-3 expression (≥1%) appeared to enrich for response. The median duration of response was not reached (range, 0.1+ to 39.3+).

[0391] Figure 20 shows baseline characteristics and response by LAG-3 expression observed in the mel prior IO cohort. LAG-3 expression (≧1%) enriches for response independent of PD-L1 expression.

[0392] Figures 21 and 22 show the best change in target lesion size by LAG-3 and PD-L1 expression, and the depth and duration of response by LAG-3 and PD-L1 expression, respectively, in the mel prior IO cohort. Responses were more likely in patients with LAG-3 expression ≥ 1%. PD-L1 expression did not appear to enrich for response.

[0393] Figure 23 shows progression-free survival. Of the 61 evaluable patients, 34% (21 / 61) of patients had not progressed at the time of data cutoff. Of the 33 evaluable patients with LAG-3 ≥ 1%, 55% (18 / 33) of patients had not progressed at the time of data cutoff. Of the 20 evaluable patients with LAG-3 < 1%, 5% (1 / 20) of patients had not progressed at the time of data cutoff.

[0394] Example 3 Preliminary efficacy and biomarker enrichment across several advanced solid tumor types in a phase 1 / 2a trial of a combination anti-LAG-3 and anti-PD-1 monoclonal antibody LAG-3 is a transmembrane receptor that negatively regulates T cell activation. Signaling through LAG-3 and other T cell inhibitory receptors, including programmed death-1 (PD-1), can lead to T cell exhaustion, a mechanism of tumor immune evasion. Simultaneous blockade of LAG-3 and PD-1 may function synergistically to restore T cell activation and enhance antitumor immunity. In a phase 1 / 2a clinical trial, BMS-986016 (an IgG4 mAb targeting LAG-3) plus nivolumab (an IgG4 mAb targeting PD-1) demonstrated tolerability, peripheral T cell activation, and preliminary clinical activity (NCT01968109; Lipson et al. J Immunother Cancer. 2016;4(suppl):173 [abstract P232]). The efficacy of BMS-986016 plus nivolumab across several advanced solid tumor expansion cohorts was evaluated in both the overall population and biomarker-enriched populations.

[0395] All patients (n=204 as of April 7, 2017) were treated with BMS-986016 80 mg plus nivolumab 240 mg Q2W in 56-day cycles until disease progression, confirmed complete response, completion of 12 cycles, or toxicity not permitted. The majority of cohorts focused on immunotherapy-naïve patients who had progressed on or after at least one other prior therapy, including patients with advanced gastric / gastroesophageal junction cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, and NSCLC. One other cohort included patients with NSCLC who had progressed on or after prior anti-PD-1 / PD-L1 therapy as their most recent treatment. Biomarker-defined patient subsets were represented based on PD-L1 and LAG-3 immunohistochemical scoring in tumor biopsies.

[0396] Figure 12 shows the LAG-3 expression status of cancer immunotherapy-naive gastric tumor samples. 48% (10 / 21) of the samples were scored as LAG-3 positive using a ≥ 1% cutoff in the monoplex IHC assay.

[0397] Figure 13 shows the change in target lesion size in cancer immunotherapy-naïve gastric cancer patients in response to treatment with a combination of anti-LAG-3 and anti-PD-1 antibodies. LAG-3-positive tumors were enriched in patients who responded to treatment. Tumor response was determined according to RECIST. The patient group shown had not been previously exposed to anti-PD-1 / PD-L1 treatment.

[0398] FIG. 14 shows the LAG-3 expression status of cancer immunotherapy-naive SCCHN, renal cancer, HCC, and NSCLC tumor samples as determined by monoplex IHC assay.

[0399] Example 4 Multitumor profiling of LAG-3 and correlation with immune cell phenotypes LAG-3 negatively regulates T cell activation. Sierro S et al. Expert Opin Ther Targets. 15:91-101 (2011); Grosso JF et al. J Clin Invest. 117:3383-3392 (2007). LAG-3 and programmed death-1 (PD-1) receptors are overexpressed and coexpressed on tumor-infiltrating lymphocytes (TILs). Goding SR et al. J Immunol. 190:4899-4909 (2013). LAG-3 and PD-1 overexpression limit the treatment response to anti-PD-1 therapy and may lead to tumor progression. Ascierto P et al. Poster 9520 presented at the 53rd Annual Meeting of the American Society of Clinical Oncology; June 2-6, 2017; Chicago, IL; Wherry, Nat Immunol. 12(6):492-9 (2011); Woo SR et al. Cancer Res. 72:917-927 (2012); Huang CT et al. Immunity. 21:503-513 (2004). BMS-986016 is a fully human IgG4 antibody that targets LAG-3 and blocks binding to its ligand, major histocompatibility complex class II (MHC II) (Figure 24). Huard B et al. Proc Natl Acad Sci US A. 94:5744-5749 (1997). The combination of BMS-986016 and nivolumab (anti-PD-1) can restore T cell activation and tumor responses in patients whose disease has progressed on anti-PD-1 monotherapy. Ascierto P et al. Poster 9520 presented at the 53rd Annual Meeting of the American Society of Clinical Oncology; June 2-6, 2017; Chicago, IL. This dual blockade may also enhance the durability of responses in patients not previously treated with anti-PD-1 therapy.Simultaneous blockade of LAG-3 and PD-1 with BMS-986016 and nivolumab, respectively, resulted in peripheral T cell activation and demonstrated clinical activity and manageable safety in patients with advanced solid tumors. Ascierto P et al. Poster 9520 presented at the 53rd Annual Meeting of the American Society of Clinical Oncology; June 2-6, 2017; Chicago, IL; Lipson E et al. J Immunother Cancer. 4(suppl 1):173 (2016). To further understand the relationship between LAG-3 and markers of resistance in tumors, we are conducting comprehensive profiling of commercially available tumor samples to investigate and characterize LAG-3 and MHC II expression in the context of inflammatory biomarkers.

[0400] method Quantitative immunohistochemistry (IHC). Solid tumor specimens were profiled from patients with renal cell carcinoma (RCC), gastric cancer, non-small cell lung cancer (NSCLC), melanoma, squamous cell carcinoma of the head and neck (SCCHN), and urothelial carcinoma. Slide sections were stained by IHC for LAG-3, CD8, FOXP3, CD68, CD163, PD-L1, and MHC II using the Leica Bond Rx or Dako Link 48 platform. Percent positivity for immune cell markers (LAG-3, CD8, FOXP3, CD68, CD163) was determined by defining the ratio of total nucleated cells expressing the biomarker in the tumor microenvironment using Aperio image analysis software. MHC II and PD-L1 expression by IHC on tumor cells was manually scored. Unsupervised clustering (Ward's method) was performed on the IHC data to identify correlations between LAG-3 and other immune biomarkers. To determine MHC II+ and LAG-3+ colocalization, MHC II-high (>70% MHC II+) or MHC II-low (<10% MHC II+) tumor cell areas were assessed for the number of LAG-3-stained cells (average of three 20x fields each for positive and negative areas).

[0401] mRNA analysis. In patients with RCC and melanoma, changes in LAG-3 mRNA levels were determined by differential gene expression analysis of Affymetrix (RCC) or RNA sequencing (melanoma) data from tumor biopsy samples collected at screening and 2–4 weeks after initiation of immunotherapy.

[0402] Statistical Analysis. Correlations between LAG-3 expression and other immune biomarkers were assessed by Spearman correlation, r. A Mann-Whitney test was performed to assess statistical significance. Differential gene expression analysis was performed using a generalized linear model including treatment group and time as factors.

[0403] result LAG-3 expression in tumors. For tumor samples analyzed across six different solid tumor types (n=245: RCC, 43; gastric, 41; NSCLC, 41; melanoma, 40; SCCHN, 40; urothelial, 40), a range of low to high LAG-3 expression was observed (0.01% to 33% of total nucleated cells). LAG-3 expression could be localized to the perinuclear, membrane, or cytoplasmic regions of lymphocytes, as shown by IHC staining (Figure 25).

[0404] Correlation of LAG-3 with immune and inflammatory biomarkers. Moderate correlations were observed between LAG-3 expression and CD8, FOXP3, CD163, and CD68 (n=237: RCC, 43; gastric, 39; NSCLC, 39; melanoma, 39; SCCHN, 40; urothelial, 37) (Figure 26A-D, r=0.49-0.65); no correlations were observed between LAG-3 and PD-L1 or MHC II tumor expression (Figures 26E and 26F, r=0.28-0.30). MHC II expression (≥1%) in tumor cells was frequently observed, ranging from a low of 55% (melanoma) to a high of 82% (gastric cancer).

[0405] Tumors with MHC II expression in ≥1% tumor cells showed a significantly increased frequency of LAG-3+ TILs (Figure 27, n=241: RCC, 43; gastric, 40; NSCLC, 40; melanoma, 38; SCCHN, 40; urothelial, 40).

[0406] Unsupervised clustering of samples by tumor type revealed clusters of tumors with inflammation ranging from low to high across the six tumor types analyzed (examples in Figure 28A, urothelial carcinoma, n=37; and 28B, gastric carcinoma, n=39).

[0407] Increased MHC II tumor expression was often observed in tumors with high inflammation, but also in tumors with low levels of inflammation (example in Figure 28A, urothelial carcinoma). Among those specimens that stained positive for tumor cell MHC II expression, MHC II expression levels correlated with LAG-3+ TIL levels in some tumor types (examples in Figures 28A and 298, urothelial and gastric carcinomas). The majority of tumors with high MHC II expression had low PD-L1 expression (Figure 28C, n=229: RCC, 43; gastric, 39; NSCLC, 38; melanoma, 33; SCCHN, 39; urothelial, 37).

[0408] Heterogeneous MHC II tumor cell expression and LAG-3+ TILs. Heterogeneous MHC II tumor cell expression ranging from low (<10%) to high (>70%) was observed in a subset of tumor specimens examined (n=6) (Figure 29A; urothelial carcinoma, n=4; gastric carcinoma, n=2). In this subset, a significant increase in the number of LAG-3+ TILs was observed in tumor areas with high MHC II expression relative to low MHC II expression (Figure 29A-C).

[0409] LAG-3 mRNA Level Changes During Anti-PD-1 Monotherapy. In analyses of tissue samples from patients with metastatic melanoma (NCT01621490 / CheckMate 038) or metastatic RCC (NCT01358721 / CheckMate 009), a significant increase in LAG-3 mRNA levels was observed at screening and weeks 2–4 of nivolumab treatment (Figure 30).

[0410] As demonstrated by IHC, LAG-3 expression correlated with cellular inflammation in the tumor microenvironment. MHC II tumor cell expression was frequently observed across the six tumor types analyzed; LAG-3 expression on immune cells was enriched in tumors with MHC II expression on tumor cells. A high frequency of LAG-3+ TILs was observed in MHC II-high / positive tumor areas relative to MHC II-low / negative tumor areas within individual tumor specimens, raising the possibility that colocalization of LAG-3 and MHC II expression on tumor cells may be a mechanism of LAG-3 checkpoint activation in certain tumors. These findings and the observation that nivolumab can induce LAG-3 expression support the use of LAG-3 as a predictive biomarker for BMS-986016 treatment in patients whose disease progresses after treatment with anti-PD-1 therapy.

[0411] array SEQ ID NO: 1: Heavy chain amino acid sequence; anti-LAG-3 mAb (BMS-986016) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0412] SEQ ID NO: 2: Light chain amino acid sequence; anti-LAG-3 mAb (BMS-986016) EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0413] SEQ ID NO: 3: Heavy chain variable region (VH) amino acid sequence; anti-LAG-3 mAb (BMS-986016) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSS

[0414] SEQ ID NO: 4: Heavy chain variable region (VH) nucleotide sequence; anti-LAG-3 mAb (BMS-986016) caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtgattactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaagcaccaactccaac ccgtccctcaagagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggtctgtgaccgccgcggacacggctgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggccagggaaccctggtcaccgtctcctca

[0415] SEQ ID NO: 5: Light chain variable region (VL) amino acid sequence; anti-LAG-3 mAb (BMS-986016) EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK

[0416] SEQ ID NO: 6: Light chain variable region (VL) nucleotide sequence; anti-LAG-3 mAb (BMS-986016) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaaca gggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaa

[0417] SEQ ID NO: 7: Heavy chain CDR1 amino acid sequence; anti-LAG-3 mAb (BMS-986016) DYYWN

[0418] SEQ ID NO: 8: Heavy chain CDR2 amino acid sequence; anti-LAG-3 mAb (BMS-986016) EINHRGSTNSNPSLKS

[0419] SEQ ID NO: 9: Heavy chain CDR3 amino acid sequence; anti-LAG-3 mAb (BMS-986016) GYSDYEYNWFDP

[0420] SEQ ID NO: 10 Light chain CDR1 amino acid sequence; anti-LAG-3 mAb (BMS-986016) RASQSISSYLA

[0421] SEQ ID NO: 11 Light chain CDR2 amino acid sequence; anti-LAG-3 mAb (BMS-986016) DASNRAT

[0422] SEQ ID NO: 12 Light chain CDR3 amino acid sequence; anti-LAG-3 mAb (BMS-986016) QQRSNWPLT

[0423] SEQ ID NO: 13 Human LAG-3 amino acid sequence MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGD FSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGL EPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSF SGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLTLGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEQL

[0424] SEQ ID NO: 14 LAG-3 epitope PGHPLAPG

[0425] SEQ ID NO: 15 LAG-3 epitope HPAAPSSW

[0426] SEQ ID NO: 16 LAG-3 epitope PAAPSSWG

[0427] SEQ ID NO: 17 Heavy chain amino acid sequence; anti-PD-1 mAb (BMS936558) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0428] SEQ ID NO: 18 Light chain amino acid sequence; anti-PD-1 mAb (BMS936558) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0429] SEQ ID NO: 19: Heavy chain variable region (VH) amino acid sequence; anti-PD-1 mAb (BMS936558) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

[0430] SEQ ID NO: 20 Heavy chain variable region (VH) nucleotide sequence; anti-PD-1 mAb (BMS936558) caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactcgactgtaaagcgtctggaatcaccttcagtaactctggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatttggtatgatggaagta aaagatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgacaaacgacgactactggggccagggaaccctggtcaccgtctcctca

[0431] SEQ ID NO: 21: Light chain variable region (VL) amino acid sequence; anti-PD-1 mAb (BMS936558) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK

[0432] SEQ ID NO: 22: Light chain variable region (VL) nucleotide sequence; anti-PD-1 mAb (BMS936558) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagtagttacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaaca gggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagagtagcaactggcctcggacgttcggccaagggaccaaggtggaaatcaaa

[0433] SEQ ID NO: 23: Heavy chain CDR1 amino acid sequence; anti-PD-1 mAb (BMS936558) NSGMH

[0434] SEQ ID NO: 24: Heavy chain CDR2 amino acid sequence; anti-PD-1 mAb (BMS936558) VIWYDGSKRYYADSVKG

[0435] SEQ ID NO: 25: Heavy chain CDR3 amino acid sequence; anti-PD-1 mAb (BMS936558) NDDY

[0436] SEQ ID NO: 26 Light chain CDR1 amino acid sequence; anti-PD-1 mAb (BMS936558) RASQSVSSYLA

[0437] SEQ ID NO: 27 Light chain CDR2 amino acid sequence; anti-PD-1 mAb (BMS936558) DASNRAT

[0438] SEQ ID NO: 28 Light chain CDR3 amino acid sequence; anti-PD-1 mAb (BMS936558) QQSSNWPRT

[0439] SEQ ID NO: 29 Complete Homo sapiens PD-1 sequence

[0440] SEQ ID NO: 30 Heavy chain nucleotide sequence; anti-LAG-3 mAb (BMS-986016)

[0441] Accession No. 31 Light chain nucleotide sequence; anti-LAG-3 mAb (BMS-986016) gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtattagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

[0442] Accession No. 32 Motif MYPPPY

Claims

1. 1. A method for selecting a malignant tumor in a human patient for immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, selecting the tumor for immunotherapy; A method comprising:

2. 1. A method for identifying a malignant tumor in a human patient as eligible for immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, identifying the tumor as eligible for immunotherapy; A method comprising:

3. 1. A method for identifying malignant tumors in a human patient that may be responsive to immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, identifying the tumor as potentially responsive to treatment; A method comprising:

4. 1. A method for classifying a malignant tumor in a human patient as likely to be responsive to immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, classifying the tumor as likely to be responsive to immunotherapy; A method comprising:

5. The method of any one of claims 1 to 4, further comprising determining the PD-L1 expression level in the tumor sample.

6. 6. The method of any of claims 1-5, wherein the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor.

7. The method of any one of claims 1 to 5, wherein the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor.

8. 6. The method of any one of claims 1 to 5, wherein the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor.

9. The method of any one of claims 1 to 5, wherein the immunotherapy comprises contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody.

10. 6. The method of any of claims 1-5, wherein the immunotherapy comprises contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

11. 6. The method of any of claims 1-5, comprising contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor.

12. 6. The method of any of claims 1 to 5, comprising contacting the tumor with a therapeutically effective amount of a LAG-3 inhibitor.

13. 6. The method of any of claims 1-5, comprising contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor.

14. The method of any one of claims 1 to 5, comprising contacting the tumor with a therapeutically effective amount of an anti-CTLA-4 antibody.

15. 6. The method of any of claims 1-5, comprising contacting the tumor with a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

16. The method of any of claims 1 to 5, comprising administering to a patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor.

17. The method of any one of claims 1 to 5, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor.

18. The method of any of claims 1 to 5, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor.

19. The method of any one of claims 1 to 5, comprising administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody.

20. The method of any one of claims 1 to 5, comprising administering to a patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

21. 1. A method for identifying a patient having a malignant tumor that is likely to respond to immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, the patient is identified as likely to respond to treatment. A method comprising:

22. 1. A method for selecting a patient having a malignant tumor for immunotherapy, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) Selecting patients for immunotherapy if the tumor is a LAG-3 positive tumor A method comprising:

23. 23. The method of claim 21 or claim 22, further comprising determining the level of PD-L1 expression in the tumor sample.

24. 24. The method of any of claims 21-23, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor.

25. 24. The method of any of claims 21 to 23, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor.

26. 24. The method of any of claims 21-23, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor.

27. The method of any of claims 21 to 23, comprising administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody.

28. 24. The method of any of claims 21 to 23, comprising administering to a patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor.

29. A method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is predicted to respond to treatment with the LAG-3 inhibitor and the PD-1 pathway inhibitor based on LAG-3 expression in a tumor sample from the patient.

30. A method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor, wherein the patient is predicted to respond to treatment with the LAG-3 inhibitor based on LAG-3 expression in a tumor sample from the patient.

31. 1. A method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient is predicted to respond to treatment with the PD-1 pathway inhibitor based on LAG-3 expression in a tumor sample from the patient.

32. 1. A method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody, wherein the patient is predicted to respond to treatment with the anti-CTLA-4 antibody based on LAG-3 expression in a tumor sample from the patient.

33. 1. A method of treating a malignant tumor in a human patient, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is predicted to respond to treatment with the PD-1 pathway inhibitor and the immune checkpoint inhibitor based on LAG-3 expression in a tumor sample from the patient.

34. 34. The method of any of claims 29 to 33, wherein the patient is predicted to respond to the treatment based on LAG-3 and PD-L1 expression in a tumor sample from the patient.

35. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor. A method comprising:

36. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, administering to the patient a therapeutically effective amount of a LAG-3 inhibitor; A method comprising:

37. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor; A method comprising:

38. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody; A method comprising:

39. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) determining the LAG-3 expression level in a tumor sample; and (b) if the tumor is a LAG-3 positive tumor, administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. A method comprising:

40. 40. The method of any of claims 35 to 39, further comprising determining the level of PD-L1 expression in the tumor sample.

41. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor prior to administration.

42. A method of treating a malignant tumor in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of a LAG-3 inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor prior to administration.

43. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor prior to administration.

44. A method of treating a malignant tumor in a human patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody, wherein the patient has been identified as having a LAG-3-positive malignant tumor prior to administration.

45. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising administering to the patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient has been identified as having a LAG-3-positive malignant tumor prior to administration.

46. 46. ​​The method of any of claims 41-45, wherein the patient is identified as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

47. 46. ​​The method of any of claims 41-45, wherein the patient is identified as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

48. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor. A method comprising:

49. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of a LAG-3 inhibitor. A method comprising:

50. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor. A method comprising:

51. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody. A method comprising:

52. 1. A method of treating a malignant tumor in a human patient in need thereof, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor. A method comprising:

53. 53. The method of any of claims 48-52, further comprising identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy.

54. 53. The method of any of claims 48-52, further comprising identifying the patient as having a LAG-3 positive, PD-L1 negative malignancy.

55. 1. A method for extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to the administration, and wherein the patient exhibits progression-free survival beyond 12 months.

56. A method for extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient an LAG-3 inhibitor, wherein the patient is identified as having an LAG-3-positive malignant tumor prior to administration, and wherein the patient exhibits progression-free survival beyond 12 months.

57. A method for extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering a PD-1 pathway inhibitor to the patient, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the patient exhibits progression-free survival beyond 12 months.

58. A method for extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient an anti-CTLA-4 antibody, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the patient exhibits progression-free survival beyond 12 months.

59. 1. A method for extending progression-free survival beyond 12 months in a human patient having a malignant tumor, comprising administering to the patient a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the patient exhibits progression-free survival beyond 12 months.

60. 60. The method of any of claims 55-59, wherein the patient is identified as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

61. 60. The method of any of claims 55-59, wherein the patient is identified as having a LAG-3 positive, PD-L1 negative malignancy prior to administration.

62. 62. The method of any of claims 55-61, wherein the progression free survival of the patient is extended for more than about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration.

63. 63. The method of claim 62, wherein the progression-free survival of the patient is extended to more than 14 months.

64. 1. A method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

65. A method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient a therapeutically effective amount of an LAG-3 inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

66. 1. A method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient a therapeutically effective amount of a PD-1 pathway inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

67. 1. A method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient a therapeutically effective amount of an anti-CTLA-4 antibody, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

68. 1. A method for reducing tumor size by at least 10% in a human patient having a malignant tumor, comprising administering to the patient therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein the patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the administration reduces the tumor size by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to administration.

69. 69. The method of any of claims 64-68, wherein the patient is identified as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

70. 69. The method of any of claims 64-68, wherein the patient is identified as having a LAG-3 positive, PD-L1 negative malignancy prior to administration.

71. 71. The method of any of claims 55-70, further comprising identifying the patient as having a LAG-3 positive malignancy prior to administration.

72. 72. The method of any of claims 55-71, further comprising identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

73. 72. The method of any of claims 55-71, further comprising identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

74. 74. The method of any of claims 55-73, wherein the patient experiences (i) an extended progression-free survival of greater than 12 months, (ii) at least about a 10%, about 20%, about 30%, about 40%, or about 50% reduction in tumor size compared to the tumor size before administration, or (iii) both.

75. A method for increasing the objective response rate to a cancer treatment to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

76. A method for increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of a LAG-3 inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

77. A method for increasing the objective response rate to a cancer treatment to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

78. A method for increasing the objective response rate to cancer treatment to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of an anti-CTLA-4 antibody, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

79. A method for increasing the objective response rate to a cancer treatment to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

80. A method for increasing a disease control rate to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

81. A method for increasing a disease control rate to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of a LAG-3 inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

82. A method for increasing a disease control rate to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

83. A method for increasing the disease control rate to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients a therapeutically effective amount of an anti-CTLA-4 antibody, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

84. A method for increasing a disease control rate to greater than 50% in a population of human patients, each having a malignant tumor, comprising administering to the patients therapeutically effective amounts of a PD-1 pathway inhibitor and an immune checkpoint inhibitor, wherein each patient is identified as having a LAG-3-positive malignant tumor prior to administration, and wherein the objective response rate is greater than about 55%, about 60%, about 65%, about 70%, or about 75%.

85. 85. The method of any of claims 75-84, wherein each patient is identified as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

86. 85. The method of any of claims 75-84, wherein each patient is identified as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

87. 87. The method of any of claims 75-86, wherein the median duration of response is > 3 months, > 6 months, > 12 months or > 18 months.

88. 88. The method of any of claims 75-87, further comprising identifying each patient in the patient population as having a LAG-3 positive malignancy prior to administration.

89. 89. The method of any of claims 75-88, further comprising identifying each patient in the patient population as having a LAG-3-positive, PD-L1-positive malignancy prior to administration.

90. 89. The method of any of claims 75-88, further comprising identifying each patient in the patient population as having a LAG-3-positive, PD-L1-negative malignancy prior to administration.

91. 91. The method of any of claims 75-90, wherein each patient in the patient population is further characterized by (i) an extended progression-free survival of greater than 12 months, (ii) at least about a 10%, about 20%, about 30%, about 40%, or about 50% reduction in tumor size compared to tumor size prior to administration, or (iii) both.

92. 92. The method of any of claims 75-91, wherein the patient population comprises at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 patients with a LAG-3 positive malignancy.

93. 1. A method for selecting a human patient suitable for combination therapy, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) instructing a healthcare professional to administer to the patient a therapeutically effective amount of a LAG-3 inhibitor and a PD-1 pathway inhibitor; A method comprising:

94. 1. A method for selecting a human patient suitable for combination therapy, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) instructing a healthcare professional to administer a therapeutically effective amount of a LAG-3 inhibitor to the patient; A method comprising:

95. 1. A method for selecting a human patient suitable for combination therapy, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) instructing a healthcare professional to administer a therapeutically effective amount of a PD-1 pathway inhibitor to the patient; A method comprising:

96. 1. A method for selecting a human patient suitable for combination therapy, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) instructing a healthcare professional to administer a therapeutically effective amount of an anti-CTLA-4 antibody to the patient; A method comprising:

97. 1. A method for selecting a human patient suitable for combination therapy, comprising: (a) identifying a patient as having a LAG-3 positive malignant tumor; and (b) instructing a healthcare professional to administer a therapeutically effective amount of a PD-1 pathway inhibitor and an immune checkpoint inhibitor to the patient; A method comprising:

98. 98. The method of any of claims 93-97, further comprising identifying the patient as having a LAG-3 positive, PD-L1 positive malignancy.

99. 98. The method of any of claims 93-97, further comprising identifying the patient as having a LAG-3 positive, PD-L1 negative malignancy.

100. 100. The method of any of claims 93-99, wherein the administration treats a malignant tumor.

101. 101. The method of any of claims 93-100, wherein identifying the patient as having a LAG-3 positive malignancy comprises determining LAG-3 expression in the malignancy.

102. 102. The method of any of claims 93-101, wherein identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy comprises determining PD-L1 expression in the malignancy.

103. 102. The method of any of claims 93-101, wherein identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy comprises determining PD-L1 expression in the malignancy.

104. 104. The method of any of claims 93 to 103, wherein LAG-3 expression is determined by examining the results of an assay capable of determining LAG-3 expression.

105. 104. The method of any of claims 93 to 103, wherein LAG-3 expression is determined by examining the results of an immunohistochemical assay capable of detecting LAG-3 expression.

106. 106. The method of any of claims 93 to 105, wherein PD-L1 expression is determined by examining the results of an assay capable of determining PD-L1 expression.

107. 106. The method of any of claims 93 to 105, wherein PD-L1 expression is determined by examining the results of an immunohistochemical assay capable of detecting PD-L1 expression.

108. The method of any of claims 1 to 107, wherein the LAG-3 positive tumor contains at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100% LAG-3 expressing cells.

109. 108. The method of any of claims 1-107, wherein the LAG-3 positive tumor comprises at least about 1% LAG-3 expressing cells.

110. 108. The method of any of claims 1-107, wherein the LAG-3 positive tumor comprises at least about 5% LAG-3 expressing cells.

111. The method of any of claims 108 to 110, wherein the LAG-3 expressing cells comprise tumor-infiltrating lymphocytes.

112. The method of any of claims 109 to 110, wherein the LAG-3 expressing cells are the total number of cells.

113. The method of any one of claims 109 to 112, wherein the cells express LAG-3 on the cell surface.

114. malignant tumors include liver cancer, bone cancer, pancreatic cancer, skin cancer, oral cancer, head and neck cancer, breast cancer, lung cancer including small cell and non-small cell lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, 114. The method of any of claims 1 to 113, wherein the cancer is selected from the group consisting of environmentally induced cancers including those induced by asbestos, for example hematological malignancies including multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, generalized large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphoblastic lymphoma, and any combination thereof.

115. 114. The method of any of claims 1 to 113, wherein the malignant tumor is selected from melanoma, non-small cell lung cancer (NSCLC), human papillomavirus (HPV)-associated tumors, and gastric adenocarcinoma.

116. The method of any of claims 1 to 113, wherein the malignant tumor is non-small cell lung cancer (NSCLC), a virus-associated cancer-associated tumor, or gastric adenocarcinoma.

117. 114. The method of any of claims 1 to 113, wherein the malignant tumor is melanoma, gastric cancer, gastroesophageal junction cancer, non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, or renal cell carcinoma.

118. The method of any one of claims 1 to 113, wherein the malignant tumor is lung cancer, melanoma, head and neck squamous cell carcinoma, renal cancer, gastric cancer, or hepatocellular carcinoma.

119. The method of any one of claims 1 to 113, wherein the LAG-3 positive malignant tumor is a melanoma tumor containing about 1% or more LAG-3 expressing cells.

120. The method of any one of claims 1 to 113, wherein the LAG-3 positive malignant tumor is a gastric cancer tumor containing about 1% or more LAG-3 expressing cells.

121. The method of any one of claims 1 to 120, wherein the malignant tumor is refractory to treatment with an immune checkpoint inhibitor.

122. The method of any one of claims 1 to 121, wherein the malignant tumor is refractory to treatment with an anti-PD-1 antibody.

123. The method of any one of claims 1 to 122, wherein the malignant tumor is refractory to treatment with an anti-PD-L1 antibody.

124. 1. A method of treating melanoma in a human patient, comprising: (a) identifying a patient as having LAG-3 positive melanoma; and (b) administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor. A method comprising:

125. 125. The method of claim 124, wherein identifying the patient as having LAG-3 positive melanoma comprises determining LAG-3 expression in the melanoma tumor.

126. 126. The method of claim 125, wherein LAG-3 expression is determined by examining the results of an assay capable of determining LAG-3 expression.

127. 126. The method of claim 125, wherein LAG-3 expression is determined by an immunohistochemical assay capable of detecting LAG-3 expression.

128. 128. The method of any of claims 124-127, further comprising identifying the patient as having a LAG-3-positive, PD-L1-positive malignancy.

129. 128. The method of any of claims 124-127, further comprising identifying the patient as having a LAG-3-positive, PD-L1-negative malignancy.

130. A method for treating melanoma in a human patient in need thereof, comprising administering to the patient therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient has been identified as having LAG-3-positive melanoma prior to the administration.

131. 1. A method of extending progression-free survival beyond 12 months in a human patient with melanoma, comprising administering to the patient a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein the patient is identified as having LAG-3-positive melanoma prior to the administration, and wherein the patient exhibits progression-free survival beyond 12 months.

132. The method of claim 130 or claim 131, wherein the patient is identified as having LAG-3 positive, PD-L1 positive melanoma prior to administration.

133. The method of claim 130 or claim 131, wherein the patient is identified as having LAG-3 positive, PD-L1 negative melanoma prior to administration.

134. A method for increasing the objective response rate to a cancer treatment to greater than 15% in a population of human patients, each having melanoma, comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each patient is identified as having a LAG-3-positive malignancy prior to administration, and wherein the objective response rate is greater than 15%.

135. 1. A method for increasing the disease control rate for a cancer treatment to greater than 70% in a population of human patients, each of whom has melanoma, comprising administering to the patients therapeutically effective amounts of a LAG-3 inhibitor and a PD-1 pathway inhibitor, wherein each of the patients is identified as having LAG-3-positive melanoma prior to administration, and wherein the objective response rate is greater than 70%.

136. 136. The method of claim 134 or claim 135, further comprising identifying each patient in the patient population as having LAG-3 positive melanoma prior to administration.

137. 137. The method of any of claims 134-136, wherein the median duration of response is > 3 months, > 6 months, > 12 months or > 18 months.

138. The method of any of claims 134-137, wherein each patient is identified as having LAG-3-positive, PD-L1-positive melanoma prior to administration.

139. The method of any of claims 134-137, wherein each patient is identified as having LAG-3-positive, PD-L1-negative melanoma prior to administration.

140. 140. The method of any of claims 124-139, wherein the melanoma is refractory to treatment with an immune checkpoint inhibitor.

141. The method of any of claims 124 to 140, wherein the melanoma is refractory to treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.

142. 142. The method of any of claims 1-141, wherein determining LAG-3 and / or PD-L1 expression levels comprises providing a test tissue sample from the patient, wherein the test tissue sample comprises tumor cells and / or tumor-infiltrating immune cells.

143. 143. The method of claim 142, wherein the test tissue sample is a tumor biopsy.

144. The method of claim 142 or claim 143, wherein the test tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample.

145. 145. The method of any of claims 1 to 144, wherein determining comprises detecting LAG-3 and / or PD-L1 protein or RNA expression in the test tissue sample.

146. 146. The method of claim 145, wherein LAG-3 and / or PD-L1 expression is detected by an assay capable of detecting LAG-3 and / or PD-L1 protein levels, respectively, in a test tissue sample.

147. The method of claim 142, wherein LAG-3 and / or PD-L1 expression is detected by immunohistochemistry assay.

148. The method of claim 147, wherein the immunohistochemical assay is a monoplex assay.

149. The method of claim 147, wherein the immunohistochemical assay is a multiplex assay.

150. 150. The method of any of claims 147 to 149, wherein the immunohistochemical assay comprises contacting the tumor sample with 17B4, SP346, 11E3, 874501 or EPR4392(2) anti-human LAG-3 monoclonal antibody.

151. 150. The method of any of claims 147-149, wherein the immunohistochemistry assay comprises contacting the tumor sample with an anti-LAG-3 antibody comprising heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 3 and 5, respectively.

152. 152. The method of any of claims 145 to 151, wherein the immunohistochemical assay uses a black or brown chromogen.

153. 152. The method of any of claims 145 to 151, wherein the immunohistochemical assay uses a red chromogen.

154. 152. The method of any of claims 145 to 151, wherein the immunohistochemical assay uses a blue chromogen.

155. 152. The method of any of claims 145 to 151, wherein the immunohistochemical assay uses a green chromogen.

156. 152. The method of any of claims 145 to 151, wherein the immunohistochemical assay uses a purple chromogen.

157. 157. The method of any of claims 145 to 156, wherein the immunohistochemical assay is scored at low magnification.

158. 146. The method of claim 145, wherein the low magnification is about 20x.

159. 157. The method of any of claims 145 to 156, wherein the immunohistochemical assay is scored at high magnification.

160. 160. The method of claim 159, wherein the high magnification is about 40x.

161. 161. The method of any of claims 145 to 160, wherein the immunohistochemical assay is scored by image analysis software.

162. 161. The method of any of claims 145 to 160, wherein the immunohistochemical assay is scored by a pathologist visual immunoscore.

163. 161. The method of any of claims 145 to 160, wherein the immunohistochemical assay is scored manually.

164. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of cells in the test tissue sample that express LAG-3 and / or assessing the proportion of cells in the test tissue sample that express PD-L1.

165. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of tumor cells in the test tissue sample that express LAG-3 and / or assessing the proportion of tumor cells in the test tissue sample that express PD-L1.

166. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of immune cells in the test tissue sample that express LAG-3 and / or assessing the proportion of immune cells in the test tissue sample that express PD-L1.

167. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of T cells in the test tissue sample that express PD-L1.

168. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of CD8+ T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of CD8+ T cells in the test tissue sample that express PD-L1.

169. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of CD4+ T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of CD4+ T cells in the test tissue sample that express PD-L1.

170. 164. The method of any of claims 145 to 163, wherein scoring the immunohistochemistry assay comprises assessing the proportion of FOXP3+ T cells in the test tissue sample that express LAG-3 and / or assessing the proportion of FOXP3+ T cells in the test tissue sample that express PD-L1.

171. 171. The method of any of claims 145 to 170, wherein cells with partial membrane / cytoplasmic LAG-3 localization are scored as LAG-3 expressing cells.

172. 171. The method of any of claims 145 to 170, wherein cells with dot-like LAG-3 localization are scored as LAG-3 expressing cells.

173. 171. The method of any of claims 145 to 170, wherein cells with complete membrane / cytoplasmic LAG-3 localization are scored as LAG-3 expressing cells.

174. The method of any of claims 145 to 170, wherein cells having any LAG-3 localization pattern are scored as LAG-3 expressing cells.

175. The method of any of claims 145 to 174, wherein the immunohistochemical assay is a multiplex assay further comprising detection of MHC class II expression by tumor cells.

176. 176. The method of any of claims 145 to 175, wherein scoring the immunohistochemical assay comprises assessing the proportion of cells in the test tissue sample that express MHC class II.

177. 176. The method of any of claims 145 to 175, wherein scoring the immunohistochemical assay comprises assessing the proportion of non-immune cells in the test tissue sample that express MHC class II.

178. 145. The method of claim 144, wherein LAG-3 and / or PD-L1 protein expression is detected by flow cytometry.

179. 179. The method of claim 178, wherein the test tissue sample obtained from the patient comprises tumor-infiltrating immune cells.

180. 180. The method of claim 179, wherein the malignant tumor is a hematopoietic tumor and the tissue sample comprises circulating lymphocytes.

181. The method of any of claims 178 to 180, wherein the flow cytometry is a multiplex assay.

182. 182. The method of claim 181, wherein the flow cytometry comprises detecting expression of markers including LAG-3, PD-L1, CD4, CD8, FOXP3, and any combination thereof.

183. 183. The method of claim 182, wherein the flow cytometric scoring comprises assessing the proportion of T cells in the test tissue sample that express LAG-3.

184. 183. The method of claim 182, wherein the flow cytometric scoring comprises assessing the proportion of CD8+ T cells in the test tissue sample that express LAG-3.

185. 183. The method of claim 182, wherein flow cytometric scoring comprises assessment of the proportion of CD4+ T cells in the test tissue sample that express LAG-3.

186. 183. The method of claim 182, wherein the flow cytometric scoring comprises assessment of the proportion of FOXP3+ T cells that express LAG-3 in the test tissue sample.

187. 144. The method of claim 143, wherein LAG-3 and / or PD-L1 expression is detected by an assay capable of detecting the level of LAG-3 and / or PD-L1 RNA, respectively, in a tumor sample.

188. 188. The method of claim 187, wherein LAG-3 and / or PD-L1 expression is detected by an RT-PCR-based assay.

189. 135. The method of claim 134, wherein scoring the RT-PCR-based assay comprises assessing LAG-3 and / or PD-L1 RNA expression levels in the test tissue sample relative to a predetermined level.

190. The method of any of claims 1 to 189, wherein the LAG-3 inhibitor is an anti-LAG-3 antibody or an antigen-binding fragment thereof.

191. 191. The method of claim 190, wherein the anti-LAG-3 antibody is a bispecific antibody.

192. The method of claim 190 or 191, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:7; (b) a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:8; (c) a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:9; (d) a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:10; (e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:11; and (f) a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:

12.

193. 193. The method of any of claims 190-192, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 3 and 5, respectively.

194. The method of claim 190, wherein the anti-LAG-3 antibody is MK-4280 (28G-10), REGN3767, GSK2837781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-525), IMP321, FS-118, Sym022, TSR-033, MGD013, FS118, or GSK2831781.

195. 195. The method of any of claims 1-194, wherein the PD-1 pathway inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.

196. The method of claim 195, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:23; (b) a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:24; (c) a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:25; (d) a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO:26; (e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO:27; and (f) a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO:

28.

197. 200. The method of claim 196, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 19 and 21, respectively.

198. 200. The method of claim 197, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain comprising the sequences set forth in SEQ ID NOs: 17 and 18, respectively.

199. The method of claim 195, wherein the anti-PD-1 antibody is pembrolizumab (Keytruda; MK-3475), pidilizumab (CT-011) or nivolumab (Opdivo; BMS-936558).

200. The method of any of claims 1 to 194, wherein the PD-1 pathway inhibitor is an anti-PD-L1 antibody or an antigen-binding fragment thereof.

201. The method of claim 200, wherein the anti-PD-L1 antibody is atezolizumab (Tecentriq or RG7446), durvalumab (Imfinzi or MEDI4736), avelumab (Bavencio) or BMS-936559.

202. The method of any of claims 1 to 194, wherein the PD-1 pathway inhibitor is an anti-PD-L2 antibody or an antigen-binding fragment thereof.

203. 195. The method of any of claims 1-194, wherein the anti-CTLA-4 antibody is ipilimumab or an antigen-binding fragment thereof.

204. The method of any of claims 1 to 194, wherein the immune checkpoint inhibitor is a CTLA-4 antagonist, CD80 antagonist, CD86 antagonist, Tim-3 antagonist, TIGIT antagonist, CD20 antagonist, CD96 antagonist, IDO1 antagonist, STING antagonist, GARP antagonist, CD40 antagonist, A2aR antagonist, CEACAM1 (CD66a) antagonist, CEA antagonist, CD47 antagonist, PVRIG antagonist, TDO antagonist, VISTA antagonist, or KIR antagonist.

205. 205. The method of any of claims 190-204, comprising at least one administration cycle, said cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-LAG-3 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg or 240 mg.

206. 206. The method of any of claims 190-205, comprising at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-PD-1 antibody is administered four times at a dose of 80 mg or 240 mg.

207. 206. The method of any of claims 190-205, comprising at least one administration cycle, the cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-PD-L1 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg, or 240 mg.

208. 208. The method of any of claims 190-207, comprising at least one administration cycle, said cycle being 8 weeks in duration, and for each of the at least one cycle, the anti-CTLA-4 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg or 240 mg.

209. 209. The method of any of claims 190-208, wherein the method comprises at least one administration cycle, the cycle being 8 weeks in duration, and wherein, for each of the at least one cycle, the anti-LAG-3 antibody is administered four times at a dose of 3 mg, 20 mg, 80 mg, 160 mg, or 240 mg, and the anti-PD-1 antibody is administered four times at a dose of 80 mg or 240 mg.

210. The method of any of claims 190 to 209, wherein the anti-LAG-3 antibody and the anti-PD-1 antibody are administered in the following doses: (a) 3 mg of the anti-LAG-3 antibody and 80 mg of the anti-PD-1 antibody; (b) 3 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody; (c) 20 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody; (d) 80 mg of the anti-LAG-3 antibody and 160 mg of the anti-PD-1 antibody; (e) 80 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody; (f) 160 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody; or (g) 240 mg of the anti-LAG-3 antibody and 240 mg of the anti-PD-1 antibody.

211. 211. The method of claim 210, wherein the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 80 mg of anti-LAG-3 antibody and 160 mg of anti-PD-1 antibody.

212. 211. The method of claim 210, wherein the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at a dose of 80 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

213. 211. The method of claim 210, wherein the anti-LAG-3 antibody and the anti-PD-1 antibody are administered at doses of 160 mg of anti-LAG-3 antibody and 240 mg of anti-PD-1 antibody.

214. The method of any of claims 190-213, wherein the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated for intravenous administration.

215. The method of any of claims 190-214, wherein the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated together.

216. The method of any of claims 190-214, wherein the anti-PD-1 and anti-LAG-3 antibodies or antigen-binding fragments thereof are formulated separately.

217. 217. The method of any of claims 205-216, wherein the treatment consists of a maximum of 12 cycles.

218. 218. The method of any of claims 205-217, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered on days 1, 15, 29, and 43 of each cycle.

219. The method of any of claims 205-217, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof is administered on days 1, 15, 29, and 43 of each cycle.

220. The method of any of claims 205-217, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof is administered on days 1, 15, 29, and 43 of each cycle.

221. The method of any of claims 205-217, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered prior to administration of the anti-LAG-3 antibody or antigen-binding fragment thereof.

222. 222. The method of any of claims 190-221, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof is administered within 30 minutes prior to administration of the anti-PD-1 antibody or antigen-binding fragment thereof.

223. The method of any of claims 190 to 222, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered after administration of the anti-LAG-3 antibody or antigen-binding fragment thereof.

224. The method of any of claims 190 to 222, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered prior to administration of the anti-LAG-3 antibody or antigen-binding fragment thereof.

225. The method of any of claims 190 to 222, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is administered simultaneously with the anti-LAG-3 antibody or antigen-binding fragment thereof.

226. The method of any of claims 190-225, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof and the PD-1 pathway inhibitor are administered as a first line treatment.

227. 226. The method of any of claims 190-225, wherein the anti-LAG-3 antibody or antigen-binding fragment thereof and the PD-1 pathway inhibitor are administered as a second line treatment.

228. 228. The method of any of claims 190-227, further comprising administration of at least one additional therapeutic agent.

229. The method of claim 228, wherein at least one additional therapeutic agent is a chemotherapeutic agent.

230. 230. The method of claim 229, wherein at least one additional therapeutic agent is an immune checkpoint inhibitor.

231. 231. The method of any of claims 1-230, wherein the method results in at least one therapeutic effect selected from a reduction in tumor size, a reduction in the number of metastatic lesions over time, a complete response, a partial response, and stable disease.

232. 232. The method of any of claims 1-231, wherein administration of an anti-LAG-3 antibody or antigen-binding fragment thereof and a PD-1 pathway inhibitor activates T cells in the patient.

233. 233. The method of any of claims 1-232, wherein administration of an anti-LAG-3 antibody or antigen-binding fragment thereof and a PD-1 pathway inhibitor induces expression of activation markers by T cells in the patient.

234. The method of any of claims 1-233, wherein administration of the anti-LAG-3 antibody or antigen-binding fragment thereof results in at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% occupancy of LAG-3 receptors on the patient's T cells.

235. The method of claim 234, wherein the T cells are CD8+ T cells.

236. 235. The method of claim 234, wherein the T cells are tumor-infiltrating T cells.

237. 225. The method of any of claims 219-224, wherein the PD-1 pathway inhibitor comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

238. 1. A kit for treating a patient with a malignant tumor, comprising: (a) a dose of an anti-LAG-3 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; (b) a dose of an anti-PD-1 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; and (c) Instructions for use of an anti-LAG-3 antibody and an anti-PD-1 antibody, or antigen-binding fragment thereof, in the method of any one of claims 1 to 237. Includes a kit.

239. 1. A kit for treating a patient with a malignant tumor, comprising: (a) a dose of an anti-PD-1 antibody in the range of about 0.1 to about 10 mg / kg body weight; (b) a dose of an immune checkpoint inhibitor; and (c) Instructions for use of an anti-PD-1 antibody or antigen-binding fragment thereof and an immune checkpoint inhibitor in a method according to any one of claims 1 to 237. Includes a kit.

240. 1. A kit for treating a patient with a malignant tumor, comprising: (a) a dose of an anti-LAG-3 antibody or antigen-binding fragment thereof in the range of about 0.1 to about 10 mg / kg body weight; and (b) Instructions for use of an anti-LAG-3 antibody or antigen-binding fragment thereof in the method of any of claims 1 to 237. Includes a kit.

241. 1. A kit for treating a patient with a malignant tumor, comprising: (a) one dose of an anti-PD-1 antibody or antigen-binding fragment thereof in the range of 0.1 to 10 mg / kg body weight; and (b) Instructions for use of the anti-PD-1 antibody or antigen-binding fragment thereof in the method of any of claims 1 to 237. Includes a kit.

242. 1. A method for identifying a patient who is refractory to treatment with a PD-1 antagonist, comprising determining an LAG-3 expression level, wherein an increase in LAG-3 expression level after treatment with the PD-1 antagonist relative to the LAG-3 expression level before treatment with the PD-1 antagonist indicates that the patient is refractory to PD-1 antagonist therapy.

243. 1. A method for identifying a patient at risk of becoming refractory to treatment with a PD-1 antagonist, comprising determining an LAG-3 expression level, wherein an increase in LAG-3 expression level after treatment with the PD-1 antagonist relative to the LAG-3 expression level before treatment with the PD-1 antagonist indicates that the patient is at risk of becoming refractory to treatment with the PD-1 antagonist.

244. 1. A method for identifying a patient likely to respond to LAG-3 therapy, comprising determining the LAG-3 expression level in the patient, wherein an increase in the LAG-3 expression level after treatment with a PD-1 antagonist relative to the LAG-3 expression level before treatment with the PD-1 antagonist indicates that the patient is likely to respond to LAG-3 therapy.

245. 1. A method of selecting a patient for treatment with LAG-3 therapy, comprising determining the LAG-3 expression level in the patient, wherein an increase in the LAG-3 expression level after treatment with a PD-1 antagonist relative to the LAG-3 expression level before treatment with the PD-1 antagonist indicates that the patient is likely to respond to LAG-3 therapy.

246. The method of any of claims 242 to 245, wherein the PD-1 antagonist is a PD-1 inhibitor.

247. The method of any of claims 242-245, wherein the PD-1 antagonist is a PD-1 antibody or an antigen-binding fragment thereof.

248. 246. The method of any of claims 244-245, wherein the LAG-3 treatment is a LAG-3 inhibitor.

249. 249. The method of any of claims 244, 245 or 248, wherein the LAG-3 therapy is an anti-LAG-3 antibody or antigen-binding fragment thereof.

250. 246. The method of any of claims 244 or 245, wherein the LAG-3 treatment is a combination treatment.

251. 251. The method of claim 250, wherein the LAG-3 combination therapy is a combination of an anti-LAG-3 antibody, or antigen-binding fragment thereof, and an anti-PD-1 antibody, or antigen-binding fragment thereof.

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