Anti-LAG-3 antibodies to treat hematological malignancies

Abstract

To provide methods for clinical treatment of hematological malignancies, such as relapsed or refractory chronic lymphocytic leukemia or lymphoma.SOLUTION: The present invention provides a method comprising administering an effective amount of an anti-LAG-3 antibody to a patient, wherein the method comprises at least one administration cycle, wherein the cycle is a period of eight weeks, wherein for each of the at least one cycles, the anti-LAG-3 antibody is administered as follows: (a) four doses of 20 mg anti-LAG-3 antibody; (b) four doses of 80 mg anti-LAG-3 antibody; (c) four doses of 240 mg anti-LAG-3 antibody; or (d) four doses of 800 mg anti-LAG-3 antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 932,589, filed Jan. 28, 2014, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] Lymphocyte activation gene-3 (LAG-3; CD223) is a marker for activated CD4 + and CD8 + It is a type I transmembrane protein expressed on the cell surface of T cells and a subset 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, a coreceptor for the activation of helper T cells. Both molecules have four extracellular Ig-like domains and require binding to their ligand, major histocompatibility complex (MHC) class II, for their 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 is also found as a soluble protein, but does not bind to MHC class II and its function is unknown.

[0003] 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 have elevated LAG-3 expression, which is required for Tregs to exert their full suppressive function (Camisaschi C, et al., J. Immunol. 2010; 184:6545-6551 and Huang CT, et al., Immunity. 2004; 21:503-513). In addition, CD4 + Ectopic expression of LAG-3 on effector T cells reduced their proliferation capacity and conferred them regulatory ability on third party T cells (Huang CT, et al., Immunity. 2004; 21:503-513). Also, a recent study showed that exhausted lymphocytic choriomeningitis virus (LCMV)-specific CD8 + High LAG-3 expression on T cells contributes to their unresponsive state, and CD8 + It has also been shown to limit 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). Indeed, LAG-3 inhibited the expression of CD8 + Direct effects on T cells maintained tolerance to self and tumor antigens (Grosso JF, et al., J. Clin. Invest. 2007; 117:3383-3392).

[0004] Epstein-Barr virus infection is yet another factor to be considered for the possible induction of T cell exhaustion in hematological malignancies. It is known that EBV-associated CLL, Richter's syndrome, and lymphoma cases are usually more aggressive than their EBV(-) counterparts (Tsimberidou AM, et al., Leuk Lymphoma 2006;47:827; Ansell SM, et al., Am J Hematol 1999;60:99.; Dolcetti R, et al., Infectious Agents and Cancer 2010;5:22; Kanakry JA, et al., Blood 2013;121:3547). Interestingly, expression of PD-L1 and checkpoint inhibitors such as LAG-3 has also been shown in EBV-associated malignancies (Green MR, et al., Clin Cancer Res 2012;18:1611; Monti S, et al., Blood 2005;105:1851). High expression of LAG-3 has indeed been shown in chronic viral infections, and its blockade with anti-LAG-3 antibodies can reduce viral titers and checkpoint inhibitor expression in mouse models (Blackburn SD, et al., Nat. Immunol. 2009;10:29-37). Furthermore, LAG-3 expression, alone or in combination with other markers, has been evaluated as a prognostic or predictive marker in CLL and Hodgkin's lymphoma (Zhang J, et al., BMC Bioinformatics 2010;11(Suppl 9):S5; Kotaskova J, et al., J Mol Diagn 2010;12(3):328-334). Emerging data indicate that LAG-3 expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mediates T cell exhaustion in hematological malignancies (Dickinson JD, et al., Leuk Lymphoma 2006;47(2):231-44).Furthermore, LAG-3 blockade with specific antibodies shows antitumor activity in leukemia (Berrien-Elliott, M, et al., Cancer Research 2013; 73(2):605-616) and solid tumor models (Woo, SR, et al., Cancer Research 2011; 72(4):917-927; Goding, SR, et al., Journal of Immunology, Baltimore, Md. 1950; 190(9):4899-909). Thus, LAG-3 is a therapeutic target in hematological malignancies. Its blockade with anti-LAG-3 antibodies alone or in combination with standard therapies (e.g., ibrutinib, lenalidomide) or other checkpoint inhibitors (e.g., nivolumab) merits further testing in clinical trials.

[0005] It is therefore an object of the present invention to provide improved methods for treating subjects with hematological malignancies using anti-LAG-3 immunotherapy. Summary of the Invention

[0006] Provided herein are methods for treating malignancies, particularly hematological malignancies (e.g., malignancies derived from myeloid or lymphoid cell lineages, e.g., leukemias, lymphomas, and myelomas) in a human patient, comprising administering to the patient an anti-LAG-3 antibody, where the antibody is administered (or is for administration) according to a particular clinical dosing regimen (i.e., at a particular dose, according to a particular dosing schedule). In one embodiment, the human patient suffers from relapsed or refractory chronic lymphocytic leukemia or lymphoma, such as chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), or non-Hodgkin's lymphoma (NHL).

[0007] An exemplary anti-LAG-3 antibody is BMS-986016, or antigen-binding fragments and variants thereof, comprising heavy and light chains having the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively (see, e.g., WO2014 / 008218). In other embodiments, the antibody comprises the heavy and light chain complementarity determining regions (CDRs) or variable regions (VRs) of BMS-986016. Thus, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the heavy chain variable (VH) region of BMS-986016 having the sequence set forth in SEQ ID NO:3, and the CDR1, CDR2 and CDR3 domains of the light chain variable (VL) region of BMS-986016 having the sequence set forth in SEQ ID NO:5. In another embodiment, the antibody comprises the CDR1, CDR2 and CDR3 heavy chain sequences set forth in SEQ ID NO:7, 8 and 9, respectively, and the CDR1, CDR2 and CDR3 light chain sequences set forth in SEQ ID NO:10, 11 and 12, respectively. In another embodiment, the antibody has a VH and / or VL region comprising the amino acid sequence set forth in SEQ ID NO:3 and / or SEQ ID NO:5, respectively. In another embodiment, the antibody comprises a VH and / or VL region encoded by the nucleic acid sequence set forth in SEQ ID NO:4 and / or SEQ ID NO:6, respectively. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on LAG-3 as the above antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies (e.g., at least about 90%, 95% or 99% variable region identity with SEQ ID NO:3 or SEQ ID NO:5).

[0008] Thus, in one embodiment, a method for treating relapsed or refractory chronic lymphocytic leukemia and lymphoma (e.g., CLL, HL, or NHL) in a human patient is provided, comprising 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, comprising at least one administration cycle, the cycle being 8 weeks, and for each of at least one cycle, four doses of the anti-LAG-3 antibody are administered at a dose of 20, 80, 240, or 800 mg. In another embodiment, the four doses of the anti-LAG-3 antibody are administered at a dose of about 0.03, 0.25, 1, or 3 mg / kg body weight.

[0009] In one embodiment, the anti-LAG-3 antibody is (a) 20 mg of anti-LAG-3 antibody; (b) 80 mg of anti-LAG-3 antibody; (c) 240 mg of anti-LAG-3 antibody; or (d) 800 mg of anti-LAG-3 antibody is administered at a dose of

[0010] In another embodiment, the anti-LAG-3 antibody is (a) 0.03 mg / kg anti-LAG-3 antibody; (b) 0.03 mg / kg anti-LAG-3 antibody; (c) 0.25 mg / kg of anti-LAG-3 antibody; (d) 1 mg / kg of an anti-LAG-3 antibody; or (e) 3 mg / kg anti-LAG-3 antibody is administered at a dose of

[0011] In one embodiment, the dose of anti-LAG-3 antibody is calculated per mg / kg body weight. In another embodiment, the dose of anti-LAG-3 antibody is a flat fixed dose. In another embodiment, an intermediate dose of LAG-3 antibody is used. For example, anti-LAG-3 antibody can be administered at 0.4 mg / kg. In another embodiment, the dosing regimen is adjusted to provide the optimal desired response (e.g., an effective response).

[0012] In another embodiment, the anti-LAG-3 antibody is administered on days 1, 15, 29, and 43 of each cycle. In another embodiment, the treatment consists of up to 12 cycles.

[0013] In one embodiment, the anti-LAG-3 antibody is administered as a first-line ("front-line") treatment (e.g., initial or first treatment). In another embodiment, the anti-LAG-3 antibody is administered as a second-line treatment (e.g., after initial treatment with the same or different therapeutic agent, including after relapse and / or if the first treatment has failed). The anti-LAG-3 antibody can be administered to the subject by any suitable means. In one embodiment, the antibody is formulated for intravenous administration.

[0014] The efficacy of the treatment methods provided herein can be assessed using any suitable means. In one embodiment, the treatment results in at least one therapeutic effect, such as a reduction in the number of malignant cells over time, a complete response, a partial response, and stable disease.

[0015] Also provided are kits that contain a pharmaceutical composition comprising a therapeutically effective amount of an anti-LAG-3 antibody, such as BMS-986016, adapted for use in the methods described herein, and a pharma- ceutically acceptable carrier. (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 the anti-LAG-3 antibody in the methods of the invention. Includes.

[0016] In another aspect, an anti-LAG-3 antibody is provided that comprises 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, for administration in at least one cycle, wherein for each cycle, four doses of the anti-LAG-3 antibody are administered at a dose of 20, 80, 240 or 800 mg. In another embodiment, the four doses of the anti-LAG-3 antibody are administered at a dose of 0.03, 0.25, 1 or 3 mg / kg body weight. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a diagram illustrating parts of a Phase I clinical trial. [Diagram 2] FIG. 2 is a schematic illustrating the screening, treatment, clinical follow-up, and survival follow-up phases of the clinical trial. [Diagram 3] FIG. 3 is a table illustrating the biomarker sampling schedule. [Figure 4] FIG. 4 shows the results of IHC analysis of LAG-3 in NSCLC cells. [Diagram 5] FIG. 5 shows the results of IHC analysis of LAG-3 in gastric cancer cells. [Figure 6] FIG. 6 is a graph comparing the percentage of LAG-3 positive cells in melanoma cells versus all other cell types in tumor sections. [Figure 7] FIG. 7 is a graph comparing the percentage of LAG-3 positive cells in NSCLC cells versus all other cell types in tumor sections. [Figure 8] FIG. 8 is a graph comparing the percentage of LAG-3 positive cells in renal cell carcinoma (RCC) cells versus all other cell types in tumor sections. [Figure 9] FIG. 9 is a graph comparing the percentage of LAG-3 positive cells in gastric cancer cells versus all other cell types in tumor sections. [Figure 10] FIG. 10 is a graph comparing the percentage of LAG-3 positive cells in head and neck squamous cell carcinoma cells versus all other cell types in tumor sections. [Figure 11] FIG. 11 is a table summarizing the percentage of LAG-3 positive cells in lymphoid cells (tumor cells and TILS) versus all other cell types in tumor sections based on LAG-3 light microscopy analysis of non-Hodgkin's lymphoma cells. [Figure 12] FIG. 12 shows the results of IHC analysis of LAG-3 in NHL and DBLCL cells; (A) low magnification view and (B) high magnification view. [Figure 13] FIG. 13 shows the results of IHC analysis of LAG-3 in NHL and FL cells; (A) low magnification view and (B) high magnification view. [Figure 14] FIG. 14 shows the results of IHC analysis of LAG-3 in NHL, TMA, and CLL cells; (A) low magnification view and (B) high magnification view. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description I. definition As used herein, the term "subject" or "patient" refers to a human cancer patient (eg, a patient with an advanced solid tumor, such as an advanced refractory solid tumor).

[0019] As used herein, "hematological malignancies" refers to types of cancer that affect the blood, bone marrow, and / or lymph nodes. Such malignancies are characterized by malignant or cancerous cells and originate from either of the two major blood cell lineages: myeloid (producing granulocytes, red blood cells, platelets, macrophages, and mast cells) or lymphoid (producing B, T, NK, and plasma cells). These cancers include all types of leukemias, lymphomas, and myelomas, including acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; at cell maturation), promyeloma, my ... Myeloid leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmocytic lymphoma, and lymphoplasmocytic lymphoma. lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), Burkitt lymphoma, diffuse histiocytic lymphoma (DHL), cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenström's macroglobulinemia; myeloma, e.g., IgG myeloma, light chain myeloma (light These include secretory myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma.Particular cancers that can be treated using the methods of the present invention include, for example, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), or non-Hodgkin's lymphoma (NHL).

[0020] As used herein, "effective treatment" refers to a treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder. A beneficial effect may take the form of an improvement compared to a baseline, i.e., an improvement compared to a measurement or observation made before the initiation of therapy according to the method. A beneficial effect may also take the form of a halt, slowing, delay, or stabilization of the adverse progression of a marker of a hematological malignancy. Effective treatment may refer to a reduction in at least one symptom of a hematological malignancy. Such an effective treatment may, for example, reduce the patient's pain, reduce the size and / or number of malignant cells, reduce or prevent metastasis of malignant cells, and / or slow down malignant cell proliferation.

[0021] The term "effective amount" refers to an amount of an agent that provides a desired biological, therapeutic, and / or prophylactic result. The result may be a decrease, amelioration, alleviation, reduction, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. With reference to hematological malignancies, an effective amount includes an amount sufficient to reduce the rate of proliferation of malignant cells (such as inhibiting the progression of the malignancy) or prevent or delay other undesirable malignant cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the development of malignant cells. In some embodiments, an effective amount is an amount sufficient to prevent or delay the recurrence of malignant cells. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of malignant cells; (ii) inhibit, delay, slow down, and stop malignant cell invasion to some extent; (iii) inhibit (i.e., slow down and stop) malignant cell metastasis; (iv) prevent or delay the appearance and / or recurrence of malignant cells; and / or (vii) alleviate to some extent one or more symptoms associated with malignant tumors. In one example, an "effective amount" is an amount of anti-LAG-3 antibody that has been clinically proven to affect a significant reduction in malignant tumors or the progression of malignant tumors, e.g., slow down the increase in the number of malignant cells. 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 thus provided as an absolute amount of agent (e.g., anti-LAG-3 antibody) rather than as a mg / kg dose.

[0022] As used herein, "body surface area (BSA) based dose" refers to a dose (e.g., of an anti-LAG-3 antibody) that is adjusted for the body surface area (BSA) of an individual patient. A BSA based dose can be provided as mg / kg body weight. Various calculations for arriving at BSA without direct measurement have been published, the most widely used being the Du Bois formula (Du Bois, EF, Arch. Intern. Medicine 1916; 17:863-871; and Verbraecken J, et al., Metabolism - Clinical and Experimental 2006;55(4): 515-24). Other exemplary BSA formulas include Mosteller's formula (Mosteller, et al., N. Engl. J. Med. 1987; 317:1098), Haycock's formula (Haycock, et al., J. Pediatr. 1978; 93:62-66), Gehan and George's formula (Gehan, et al., Cancer Chemother. Rep. 1970, 54:225-235), Boyd's formula (Current JD, The Internet Journal of Anesthesiology 1998, 2(2); and Boyd, Edith (1935), University of Minnesota. The Institute of Child Welfare, Monograph Series, No. x. London: Oxford University Press), Fujimoto's formula (Fujimoto S, et al., Nippon Eiseigaku Zasshi 1968;5:443-50), Takahira's formula (Fujimoto S, et al., Nippon Eiseigaku Zasshi 1968;5:443-50), and Schlich's formula (Schlich E, et al., Ernaehrung Umschau 2010;57:178-183).

[0023] The term "antibody" describes a polypeptide that comprises at least one antibody-derived antigen-binding site (e.g., VH / VL region or Fv, or CDR). Antibodies include known forms of antibodies. For example, an antibody may be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. An antibody may also be a Fab, Fab'2, ScFv, SMIP, Affibody®, nanobody, or domain antibody. An antibody may also be of any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. An antibody may be a naturally occurring antibody or an altered antibody (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, an antibody may contain one or more amino acid variants (compared to a naturally occurring antibody) that alter the properties (e.g., functional properties) of the antibody. For example, several such changes are known in the art and affect, for example, the half-life, effector functions, and / or immune response of the antibody in a patient. The term antibody also includes artificial polypeptide constructs that contain at least one antibody-derived antigen-binding site.

[0024] The term "LAG-3" refers to lymphocyte activation gene-3. The term "LAG-3" includes variants, isoforms, homologs, orthologs and paralogs. For example, an antibody specific for human LAG-3 protein may in certain cases cross-react with LAG-3 protein from a 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 show species or other type cross-reactivity, or may cross-react with LAG-3 from certain other species but not with LAG-3 from all other species (e.g., cross-react with monkey LAG-3 but not with mouse LAG-3). The term "human LAG-3" refers to a human LAG-3 sequence, 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 a mouse LAG-3 sequence, such as the complete amino acid sequence of mouse LAG-3 with 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 with Genbank Accession No. NP_002277, for example, by having a conserved mutation or a mutation in a non-conserved region, and LAG-3 has substantially the same biological function as human LAG-3 with Genbank Accession No. NP_002277. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody of the present disclosure, or the biological function of human LAG-3 is to bind to an MHC class II molecule.

[0025] The term "monkey LAG-3" is intended to encompass LAG-3 proteins expressed by Old World and New World monkeys, including, but not limited to, cynomolgus monkey LAG-3 and rhesus monkey LAG-3. A representative amino acid sequence of monkey LAG-3 is the rhesus monkey LAG-3 amino acid sequence also deposited under Genbank Accession No. XM_001108923. Another representative amino acid sequence of monkey LAG-3 is the alternative rhesus monkey sequence of clone pa23-5 described in US2011 / 0150892A1. This alternative rhesus monkey sequence shows a single amino acid difference at position 419 compared to the sequence deposited in Genbank.

[0026] A particular human LAG-3 sequence is generally at least 90% identical in amino acid sequence to 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 to the LAG-3 amino acid sequence of other species (e.g., mouse). In certain cases, the human LAG-3 may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the LAG-3 of Genbank Accession No. NP_002277. In certain embodiments, the human LAG-3 sequence may show 10 or fewer amino acid differences from the LAG-3 of Genbank Accession No. NP_002277. In certain embodiments, the human LAG-3 may show 5 or fewer, or even 4, 3, 2 or fewer, or 1 amino acid differences from the LAG-3 of Genbank Accession No. NP_002277. Percent identity can be determined as described herein.

[0027] II. Anti-LAG-3 antibody 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. For example, the anti-human LAG-3 antibody called monoclonal antibody 25F7 (also known as "25F7" and "LAG3.1") described in US2011 / 0150892A1 (the teachings of which are incorporated herein by reference) can be used. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 described in US2011 / 007023 (the teachings of which are also incorporated herein by reference).

[0028] Antibodies that compete with any of the above-referenced art-recognized antibodies for binding to LAG-3 can also be used.

[0029] An exemplary anti-LAG-3 antibody is BMS-986016, comprising heavy and light chains comprising the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively, as described in WO2014 / 008218 (the teachings of which are incorporated herein by reference), or antigen-binding fragments and variants thereof.

[0030] In other embodiments, the antibody has the heavy and light chain CDRs or variable regions of BMS-986016. Thus, in one embodiment, 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 another embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains comprising the sequences set forth in SEQ ID NOs:7, 8 and 9, respectively, and the CDR1, CDR2 and CDR3 domains comprising the sequences set forth in SEQ ID NOs:10, 11 and 12, respectively. In another embodiment, the antibody comprises the 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 another embodiment, the antibody comprises the 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 another embodiment, the antibody competes for binding with and / or binds to the same epitope on LAG-3 as the antibodies described above. In another embodiment, the antibody binds to an epitope on human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 14). In another embodiment, the antibody binds to an epitope on human LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 15) or PAAPSSWG (SEQ ID NO: 16).

[0031] In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with an antibody described above (eg, at least about 90%, 95% or 99% variable region identity with SEQ ID NO:3 or SEQ ID NO:5).

[0032] III. Pharmaceutical Compositions Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier or suitable for reconstitution in a liquid solution or suspension for intravenous administration.

[0033] In general, such compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutical acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol silanolate, and the like. Water or aqueous solutions, saline and aqueous dextrose and glycerol solutions can be used as carriers, particularly for injectable solutions (e.g., containing anti-LAG-3 antibodies). Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. In one embodiment, the anti-LAG-3 antibody is administered intravenously.

[0034] IV. Patient population Provided herein are clinical methods for treating hematological malignancies (eg, relapsed or refractory chronic lymphocytic leukemia or lymphoma) in human patients with anti-LAG-3 antibodies.

[0035] Examples of cancers that can be treated using the methods of the present invention include hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lineage (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lineage (producing B, T, NK, and plasma cells). These cancers include all types of leukemias, lymphomas, and myelomas, including acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; at cell maturation), promyeloma, my ... Myeloid leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmocytic lymphoma, and lymphoplasmocytic lymphoma. lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), Burkitt lymphoma, diffuse histiocytic lymphoma (DHL), cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenström's macroglobulinemia; myeloma, e.g., IgG myeloma, light chain myeloma (light Myelomas include secretory myeloma, non-secretory myeloma, smoldering myeloma (also called asymptomatic myeloma), solitary plasmacytoma, and multiple myeloma. Particular cancers that may be treated using the methods of the present invention include, for example, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), or non-Hodgkin's lymphoma (NHL).

[0036] In one embodiment, the human patient suffers from relapsed or refractory chronic lymphocytic leukemia or lymphoma. In a particular embodiment, the human patient suffers from chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), or non-Hodgkin's lymphoma (NHL).

[0037] Before, during, or after treatment, patients can be tested or selected for one or more of the above clinical attributes.

[0038] V. Treatment Protocol Suitable treatment protocols for treating hematological malignancies in human patients include, for example: 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; to the patient, The method includes at least one administration cycle, the cycle lasting 8 weeks, and for each of the at least one cycle, at least four doses of anti-LAG-3 antibody are administered at a flat dose of about 1, 3, 10, 20, 50, 80, 100, 130, 150, 180, 200, 240, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 mg. In another embodiment, the four doses of anti-LAG-3 antibody are administered at a dose of 0.01, 0.03, 0.25, 0.1, 0.3, 1 or 3, 5, 8, or 10 mg / kg body weight.

[0039] In one embodiment, the anti-LAG-3 antibody is (a) 20 mg of anti-LAG-3 antibody; (b) 80 mg of anti-LAG-3 antibody; (c) 240 mg of anti-LAG-3 antibody; or (d) 800 mg of anti-LAG-3 antibody is administered at a dose of

[0040] In another embodiment, the anti-LAG-3 antibody is (a) 0.3 mg / kg anti-LAG-3 antibody; (b) 0.25 mg / kg anti-LAG-3 antibody; (c) 1 mg / kg of an anti-LAG-3 antibody; or (d) 3 mg / kg of anti-LAG-3 antibody is administered at a dose of

[0041] In one embodiment, the dose of anti-LAG-3 antibody is calculated per body weight, e.g., mg / kg body weight. In another embodiment, the dose of anti-LAG-3 antibody is a flat fixed dose. In another embodiment, the dose of anti-LAG-3 antibody varies over time. For example, anti-LAG-3 antibody can be initially administered at a high dose and decreased over time. In another embodiment, anti-LAG-3 antibody is initially administered at a low dose and increased over time.

[0042] In another embodiment, the amount of anti-LAG-3 antibody administered is constant for each dose. In another embodiment, the amount of antibody administered varies with each dose. For example, the maintenance (or continuing) dose of the antibody may be higher than or the same as the loading dose administered initially. In another embodiment, the maintenance dose of the antibody may be lower than or the same as the loading dose.

[0043] In another embodiment, the anti-LAG-3 antibody is formulated for intravenous administration. In one embodiment, the anti-LAG-3 antibody is administered on days 1, 15, 29, and 43 of each cycle.

[0044] In other embodiments, the anti-LAG-3 antibody is administered once a week, once every two weeks, or once every three weeks, once a month, or as long as clinical benefit is observed, or until there is a complete response, confirmed progressive disease, or unmanageable toxicity.

[0045] In another embodiment, the cycle of administration is 8 weeks and can be repeated as necessary, hi another embodiment, the treatment consists of up to 12 cycles.

[0046] In another embodiment, 4 doses of anti-LAG-3 antibody are administered per 8 week cycle.

[0047] In another embodiment, the anti-LAG-3 antibody is administered as a first line treatment (e.g., initial or initial treatment). In another embodiment, the anti-LAG-3 antibody is administered as a second line treatment (e.g., after initial or initial treatment, such as after relapse and / or if the first treatment has failed).

[0048] VI. Outcome Response to therapy may include the following criteria: 2007 INTERNATIONAL WORKING GROUP (IWG) RESPONSE CRITERIA FOR MALIGNANT LYMPHOMA (Cheson, BD, et al. J Clin Oncol. 2007; 25:579) [Table A] Key: CR = complete response, CT = computed tomography; FDG = [18F]fluorodeoxyglucose; IWG = International Working Group; NA = not applicable; PD = progressive disease; PET = positron emission tomography; PR = partial response; SD = stable disease; SPD = sum of diameter products.

[0049] 2008 INTERNATIONAL WORKING GROUP (IWG) RESPONSE CRITERIA FOR CLL WITH MODIFICATIONS (Hallek M, et al., Blood. 2008;111(12):5446-5456) [Table B] [Table C]

[0050] Patients treated according to the methods disclosed herein preferably experience an improvement in at least one symptom of malignancy. In one embodiment, the improvement is measured by a reduction in the number of malignant cells. In another embodiment, complete blood count and / or blood smear can be used to assess responsiveness to therapy. In another embodiment, biopsy from lymph nodes and / or bone marrow biopsy can be used to assess responsiveness to therapy.

[0051] In one embodiment, the treated patient shows a complete response (CR), partial response (PR), stable disease (SD), immune-related complete disease (irCR), immune-related partial response (irPR), or immune-related stable disease (irSD). In another embodiment, the treated patient experiences a decrease in the rate of proliferation of malignant cells, i.e., suppression of malignant cell proliferation. In another embodiment, recurrence of malignant cells can be prevented or delayed; one or more symptoms associated with cancer can be alleviated to some extent.

[0052] In other embodiments, administration of an effective amount of an anti-LAG-3 antibody by any of the methods provided herein results in at least one therapeutic effect selected from the group consisting of a reduction in tumor size, a reduction in the number of malignant cells appearing over time, complete remission, partial remission, or stable disease. In yet other embodiments, the treatment method results in a substantial clinical benefit rate (CBR=CR+PR+SD≧6 months) that is better than that achieved by the anti-LAG-3 antibody compared to another therapeutic regimen. In other embodiments, the improvement in clinical benefit rate is about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to another therapeutic regimen.

[0053] VII. Kits and Unit Dosage Forms Also provided are kits that contain a pharmaceutical composition containing an anti-LAG-3 antibody, such as BMS-986016, in a therapeutically effective amount adapted for use in the methods, and a pharma- ceutically acceptable carrier. The kits may also contain instructions, including, as appropriate, an administration schedule, so that a practitioner (e.g., a doctor, a nurse, or a patient) can administer the composition contained in the kit to a patient with cancer (e.g., a solid tumor). The kits may also contain a syringe.

[0054] The kit may include multiple packages of single-dose pharmaceutical compositions, each containing an effective amount of anti-LAG-3 antibody for a single administration according to the methods provided above. The equipment or device required for administering the pharmaceutical composition may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing a quantity of anti-LAG-3 antibody.

[0055] In one embodiment, the present invention provides a kit for treating a hematological malignancy in a human patient, comprising: (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 the anti-LAG-3 antibody in any of the methods and clinical dosing regimens described herein; The kit further comprises:

[0056] Since numerous modifications and equivalents will become apparent to those of skill in the art upon reading this disclosure, the following examples are merely illustrative and should not be construed in any way as limiting the scope of the disclosure.

[0057] The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

[0058] Example 1: Preclinical Pharmacology of Anti-LAG-3 Antibody (BMS-986016) Anti-LAG-3 antibody, BMS-986016 BMS-986016 is a fully human antibody specific for human LAG-3 isolated from immunized transgenic mice expressing human immunoglobulin genes. BMS-986016 is expressed as an IgG4 isotype antibody that contains a stabilizing hinge mutation (S228P) that attenuates Fc receptor binding to reduce or eliminate antibody or complement mediated killing of target cells. The heavy and light chain amino acid sequences of BMS-986016 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0059] The ability of BMS-986016 to bind recombinant human LAG-3 antigen was determined using Biacore and enzyme-linked immunosorbent assay (ELISA). Binding to human and primate LAG-3+ transfectants and activated human or primate T cells was measured using flow cytometry and Scatchard analysis. BMS-986016 binds with high affinity (K D =0.12-0.5 nM) and inhibits LAG-3 binding to cells expressing its ligand, MHC class II (IC50, 0.67 nM). BMS-986016 binds to cynomolgus LAG-3 on transfected CHO cells and activated cynomolgus T cells with lower affinity (EC50, 21.5-34.3 nM) than to activated human T cells. In the absence of a second costimulation, high concentrations of BMS-986016 do not elicit a measurable cytokine response from cultured human peripheral blood cells, nor does the drug mediate measurable antibody- or complement-dependent killing of target cells. BMS-986016 promotes activation of antigen-specific mouse T cell hybridomas expressing human LAG-3 in cocultures with MHC class II-positive antigen-presenting cells. Furthermore, BMS-986016 enhances the activation of human T cells in a superantigen stimulation assay when added alone or in combination with nivolumab (an anti-PD-1 antibody).

[0060] Example 2: Toxicity of anti-LAG-3 antibody (BMS-986016) The following preclinical toxicology studies were performed:

[0061] A. A GLP-Compliant Combination Toxicity Study of BMS-986016 and Nivolumab in Cynomolgus Monkeys by Intravenous Administration for 4 Weeks with 6 Weeks Recovery Results for single-agent treatment with BMS-986016 were as follows:

[0062] 1. Single-agent BMS-986016 administered at doses up to 100 mg / kg / week produced no adverse changes.

[0063] 2. The NOAEL for single-agent BMS-986016 was considered to be 100 mg / kg / week (mean AUC[0-168h] = 474,000 μg h / mL). The dose administered (100 mg / kg BMS-986016) is more than 10-fold higher than the maximum dose proposed for this study.

[0064] B. GLP-Compliant Tissue Cross-Reactivity Study in Human and Selected Cynomolgus Monkey Tissues with BMS-986016 The cross-reactivity results were as follows:

[0065] 1. Positive staining with BMS-986016-FITC was observed in the following human tissues: mononuclear leukocytes of the bladder, blood cells, colon-large intestine, eye, esophagus, small intestine, stomach, kidney, lung, lymph node, placenta, salivary gland, skin, spleen, thymus, tonsil, cervix, and endometrium; and cell membrane or cell membrane granules of hematopoietic cells of bone marrow. In addition, staining with BMS-986016-FITC was observed in the cytoplasm of human pituitary endocrine cell epithelium. Within the limited panel of cynomolgus monkey tissues evaluated, staining with BMS-986016-FITC was observed in the plasma membrane or plasma membrane granules of mononuclear leukocytes of the spleen.

[0066] 2. Staining of mononuclear leukocytes and hematopoietic cells by BMS-986016-FITC in this study (in human and cynomolgus tissues) was expected with regard to the scientific report of LAG-3 expressing cells with lymphocyte morphology and distribution in germinal centers and interfollicular T cell areas of normal human lymphoid tissues (lymph nodes, tonsils, spleen, thymus, bone marrow and mucosa-associated lymphoid tissues) (Huard, et al., Immunogenetics 1994; 39(3):213-217).

[0067] 3. Given that LAG-3 mRNA is expressed in the human pituitary gland and LAG3.1-G4P-FITC staining was observed in the anterior lobe of the human pituitary gland in a pilot tissue cross-reactivity study, BMS-986016-FITC staining of human pituitary endocrine cell epithelial cytoplasm and cytoplasmic granules was also expected. Although BMS-986016 is not expected to have access to the cytoplasmic compartment in vivo and repeated dose toxicity studies in monkeys showed no effects on the pituitary gland, these findings may be clinically relevant and can be monitored.

[0068] C. Evaluation of BMS-986016 in vitro cytokine release and lymphocyte activation using human peripheral blood mononuclear cells The results for the assessment of in vitro cytokine release and lymphocyte activation were as follows:

[0069] 1. BMS-986016 did not induce cytokine release when presented to human peripheral blood mononuclear cells (PBMCs) regardless of concentration, donor, or incubation time. Observed cytokine levels were at or near the lower assay limit of quantification, with no evidence of dose-dependency or pattern across donors (IL-1β, IL-2, IL-5, IL-10, IL-12p70, and IFN-γ), or generally overlapped with cytokine levels from PBMCs incubated with negative controls (IL-6, IL-8, TNF-α).

[0070] 2. Consistent with the lack of cytokine release, there was no evidence that BMS-986016 induced T or NK cell activation, as measured by surface expression of CD25 and CD69. Expression levels of these markers on T and NK cells following stimulation with BMS-986016 were similar to those observed upon stimulation with the negative control.

[0071] Overall, these data indicate that BMS-986016 has no agonistic capacity to induce T or NK cell activation or cytokine release.

[0072] Example 3: Preclinical metabolism and pharmacokinetics of an anti-LAG-3 antibody (BMS-986016) In accordance with regulatory guidelines for biotechnology-derived pharmaceuticals (ICH Harmonised Tripartite Guideline, S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. International Conference on Harmonisation, 2011), no metabolism studies were performed in animals with BMS-986016. The expected in vivo degradation of monoclonal antibodies (mAbs) is to small peptides and amino acids by biochemical pathways that are independent of cytochrome P450 enzymes.

[0073] BMS-986016 demonstrated favorable pharmacokinetic (PK) properties in cynomolgus monkeys. From both single-dose and multiple-dose IV PK studies, BMS-986016 decayed biexponentially, with exposure being approximately dose proportional. Total body clearance (CLTp) was 0.12-0.22 mL / h / kg, and terminal half-life (T-HALF) was 133-414 hours. Volume of distribution at steady state (Vss) was 62-72 mL / kg, suggesting limited distribution outside of plasma. Anti-BMS-986016 antibodies were detected in some monkeys, but the presence of anti-BMS-986016 antibodies did not appear to have an effect on BMS-986016 exposure.

[0074] Example 4: Phase 1 Study in Patients with Relapsed or Refractory CLL and Lymphoma A Phase 1 trial of an anti-LAG-3 antibody (BMS-986016) will be conducted in patients with relapsed or refractory CLL and lymphoma to demonstrate the efficacy of administering BMS-986016 as a treatment.

[0075] This is a Phase 1, open-label study of BMS-986016 administered as a single agent to subjects with relapsed or refractory CLL and lymphoma. The study will be conducted in two parts. Part A will consist of a 3+3+3 dose escalation design in subjects with relapsed or refractory CLL, HL and NHL. Part B will consist of cohort expansion in four disease-limited populations of approximately 12 subjects each (Figure 1). Treatment in Part B will begin when the MTD (or MAD if the MTD is not established) for Part A has been determined.

[0076] Subjects will complete up to three periods of the study: screening (up to 28 days), treatment (up to 12 8-week cycles of study therapy), and clinical follow-up (135 days after the last dose of study drug; longer follow-up periods may be considered in selected cases if efficacy signals are evident). The WOCBP will have a further follow-up assessment over 165 days for pregnancy determination by home test.

[0077] The treatment period will consist of up to 12 8-week treatment cycles. Each treatment cycle will contain 4 doses of BMS-986016 administered on days 1, 15, 29, and 43. Subjects will continue on study therapy until the first occurrence of either: (1) meeting criteria for discontinuation, (2) completion of a maximum of 12 8-week cycles, (3) confirmation of progressive disease (PD), or (4) clinical deterioration. Subjects who discontinue treatment will enter a 135-day clinical follow-up period (Figure 2).

[0078] Physical examination, vital sign measurements, 12-lead electrocardiogram (ECG), pulse oximetry, and clinical laboratory assessments will be performed at selected times throughout the dosing interval. Subjects will be closely monitored for AEs throughout the study. Blood will be collected after the start of study drug administration for pharmacokinetic (PK) analysis.

[0079] Subjects will continue therapy for up to 12 8-week cycles until confirmation of PD or meet the discontinuation criteria described herein.Subjects may be on study for a total of up to about 2.3 years, including a 28-day screening period, up to 12 8-week cycles of treatment, and a 135-day clinical follow-up period.The total duration of study is expected to be about 4.3 years from the first visit of the first subject to the required survival follow-up of the last subject enrolled.

[0080] Part A: Dose Escalation In Part A, a 3+3+3 design will be used to evaluate the safety of BMS-986016. The dose escalation doses are shown in Figure 1 and Table 1 (described below). Three subjects will be treated initially in each dose cohort; in dose cohort 1, the first three subjects will be designated as monitored subjects and will begin treatment at least 5 days apart. Subjects in subsequent cohorts will not be required to be observed at 5-day intervals from the treatment initiation date.

[0081] Dose escalation, as determined by the medical monitor and the investigator, is based on the number of DLTs experienced during the DLT assessment interval, which begins on the first day of treatment and continues for 8 weeks, or 56 days, of the first cycle.

[0082] Dose escalation in Part A will proceed as follows: - If 0 of the first 3 evaluable subjects experience a DLT within the DLT assessment interval in a dose cohort, then the next 3 subjects will be treated in the next higher dose cohort. - If one of the first 3 evaluable subjects in a cohort experiences a DLT within the DLT assessment interval, 3 additional subjects will be treated in that dose cohort. - If 1 or less of the first 6 evaluable subjects experience a DLT during the DLT assessment interval, the next 3 subjects will be enrolled in the next higher dose cohort. -If 2 of the first 6 evaluable subjects experience a DLT within a cohort, expand the cohort to 9 evaluable subjects. - If 2 or more of the first 3 evaluable subjects, 3 or more of the first 6 evaluable subjects, or 3 or more of the first 9 evaluable subjects experience a DLT during the DLT assessment interval within a cohort, that dose level exceeds the MTD and a dose escalation decision is made.

[0083] [Table 1]

[0084] Part B: Cohort expansion The purpose of the cohort expansion is to accumulate additional safety, tolerability, preliminary efficacy, PK, and pharmacodynamic information on BMS-986016. The dose selected for Part B may not exceed the MTD in Part A (or MAD if the MTD is not determined), may be intermediate between the doses evaluated in Part A, and may include evaluation of other data, including delayed toxicity and PK and pharmacodynamic data from Parts A and B. Modeling may be used to help inform the selection of the dose evaluated in Part B.

[0085] The four expansion cohorts are limited to the tumor types listed in Figure 1 and Table 2 (shown below). Serial assessment of toxicity events will be assessed throughout enrollment in the expansion cohorts. If at any time the pooled rate of treatment-related toxicity meeting DLT criteria exceeds 33% across all subjects treated in the Part B cohort expansion, the findings will be discussed with the medical monitor and the investigator; further enrollment may be halted. Depending on the nature and grade of toxicity and after assessing the risk:benefit ratio, the dose for the entire cohort or for selected cohorts may be reduced.

[0086] [Table 2] a All subjects in Part B are naive to immune cell modulating antibody regimens (ICMAR), except for anti-CD20, alemtuzumab, or brentuximab antibody treatment, such as, but not limited to, anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-KIR, anti-CD137, and / or anti-OX40 antibodies.

[0087] Dose-limiting toxicity For the purposes of guiding decisions regarding dose escalation in Part A, dose-limiting toxicities (DLTs) will be determined based on the incidence, intensity, and duration of adverse events (AEs) deemed related to study treatment. DLT assessment intervals will begin on the first day of treatment and continue for 56 days (i.e., 8 weeks) of the first cycle. The severity of adverse events will be graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0. For the purposes of subject management, AEs that meet DLT criteria will lead to dose interruptions, regardless of the cycle in which they occur.

[0088] Dose escalation is based on the number of DLTs experienced during the DLT assessment interval, as determined by the medical monitor and the investigator. Intrasubject dose escalation is not permitted. Subjects who receive at least one dose of study drug during the 8-week assessment interval are considered evaluable for DLT determination. Subjects who withdraw from the study during the DLT assessment interval for reasons other than DLT and / or whose safety data are not available for the entire DLT assessment interval may be replaced at the same dose level. If an infusion cannot be administered at the scheduled visit during the DLT assessment interval, it must be administered as soon as possible. If the delay is 1-7 days, the procedure at the original scheduled visit should be performed and the subject is considered evaluable for DLT determination. If the delay is more than 7 days, it is considered not administered and is not replaced. Subjects with a delay of more than 7 days are not considered evaluable for DLT determination. Non-evaluable subjects may be replaced at the same dose level.

[0089] Test period Subjects will continue therapy for up to 12 8-week cycles until confirmation of PD or meeting discontinuation criteria. Subjects may be on study for a total of up to about 2.3 years, including a 28-day screening period, up to 12 8-week cycles of treatment, 135 days of clinical follow-up, and for WOCBP, an additional 165 days of home pregnancy test. The total duration of the study is expected to be about 4.3 years from the first visit of the first subject to the required survival follow-up of the last subject enrolled.

[0090] Number of targets Approximately 84 subjects may be administered (approximately 36 subjects during dose escalation and up to 48 subjects in cohort expansion).

[0091] Study population Male and female subjects with histological or cytological confirmation of CLL, Hodgkin lymphoma or non-Hodgkin lymphoma (including T-cell and B-cell lymphoma), who have relapsed after or are refractory to previous treatment and meet all inclusion criteria, are eligible to participate.

[0092] Dose Escalation (Part A) Adult subjects with relapsed or refractory non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or chronic lymphocytic leukemia (CLL) were eligible.

[0093] Cohort Expansion (Part B) Four disease-specific groups will be allowed during cohort expansion per Table 2 (above)

[0094] Parts A and B Neutrophil count must be greater than 750 / μL and platelet count greater than 50,000 / μL. Subjects with primary cutaneous lymphoma, lymphoproliferative disorders associated with primary immunodeficiency, and lymphomas associated with human immunodeficiency virus (HIV) infection are excluded. Subjects with autoimmune diseases are also excluded.

[0095] Women cannot be lactating or pregnant. WOCBP must have a negative pregnancy test within 24 hours prior to receiving the first dose of study medication. WOCBP must agree to follow instructions regarding contraceptive methods for a total of 24 weeks (30 days plus the time required to receive 5 half-lives of study medication (i.e., a total of 165 days or 24 weeks) after the last dose of study medication.

[0096] WOCBP and sexually active men must agree to follow instructions regarding contraception based on the information in Appendix 3 for a total of 33 weeks (90 days plus the time required to receive 5 half-lives of study drug (i.e., a total of 225 days or 33 weeks) after the last dose of study drug.

[0097] Hepatic, non-hematological, and hematological DLTs are defined separately as outlined below.

[0098] 1. Liver DLT Any of the following drug-related events are considered hepatic DLTs: Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >8xULN for any duration Failure to return to grade 1 or lower within 2 weeks despite medical intervention, ALT or AST >5x and ≤8xULN Total bilirubin >5×ULN ALT or AST >3xULN and concurrent total bilirubin >2xULN.

[0099] 2. Non-hematological DLT Any of the following drug-related events are considered non-hematologic DLTs: Grade 2 eye pain or loss of vision requiring systemic treatment Grade 2 eye pain or decreased vision that is not responsive to topical therapy and does not improve to Grade 1 within 2 weeks of starting topical therapy Grade 3 or greater non-hepatic or non-hematologic toxicity, except as specified below.

[0100] The following grade 3 non-hematologic events are not considered DLTs: Grade 3 electrolyte abnormalities that last less than 72 hours, are not clinically worsening, and resolve spontaneously or respond to conventional medical intervention Grade 3 increase in amylase or lipase not associated with symptoms, clinical symptoms, or radiographic evidence of pancreatitis Grade 3 nausea or vomiting that lasts less than 48 hours and resolves spontaneously or with conventional medical intervention to Grade 1 or less Grade 3 fever lasting less than 72 hours and not associated with hemodynamic compromise (including hypotension, or clinical or laboratory evidence of end-organ perfusion impairment) Grade 3 tumor flare (defined as pain, irritation, or rash localized to the site of a known or suspected tumor) Grade 3 fatigue for less than 7 days.

[0101] 3. Definition of hematological DLT Any of the following drug-related events are considered hematological DLTs: Grade 4 anemia Grade 4 febrile neutropenia of any duration Grade 4 neutropenia that does not resolve to grade 3 or less within 5 days of initiating granulocyte colony-stimulating factor (G-CSF) Platelet transfusion or platelet count <10,000 / μL Grade 3 thrombocytopenia associated with clinically significant bleeding Grade 3 hemolysis Grade 3 anemia in subjects with ≤ Grade 1 anemia at baseline.

[0102] Subjects who receive at least one dose of study drug during the 8-week assessment interval are considered evaluable for DLT determination. If study drug cannot be administered at the scheduled visit during the DLT assessment interval, it must be administered as soon as possible. If the delay is 1-7 days, the procedure at the original scheduled visit should be performed and the subject is considered evaluable for DLT determination. If the delay is more than 7 days, the subject is considered not dosed and is not replaced. Subjects with a delay of more than 7 days are not considered evaluable for DLT determination. Non-evaluable subjects may be replaced at the same dose level. Subjects who miss a dose during the DLT assessment period may continue treatment if they do not otherwise meet the criteria for permanent subject discontinuation.

[0103] Study Inclusion Criteria 1. Signed informed consent Subjects must sign and date an IRB / IEC-approved informed consent document prior to the administration of any study-related procedures that are not considered part of the standard of care.

[0104] Consent for biopsy samples: (i) The subject must consent to allow a pre-treatment tumor biopsy (e.g., lymph node) to be performed (all subjects). When a pre-treatment tumor biopsy is not clinically feasible, the subject must consent to allow for the procurement of an archived tumor sample (e.g., primary tumor, lymph node, etc.). Subjects unable to provide a fresh pre-treatment tumor biopsy or an archived tumor sample are not eligible. Subjects with insufficient quantity or quality of tissue obtained by pre-treatment biopsy are not ineligible on this basis alone.

[0105] (ii) Subjects must agree to allow a unilateral bone marrow biopsy and / or aspirate to be performed (all subjects) and, when clinically indicated, pre-treatment at treatment for complete response (CR), partial response (PR), or progressive disease (PD). Subjects who have had a bone marrow biopsy and / or aspirate due to completion of their last treatment may not use those results in place of the required baseline bone marrow biopsy.

[0106] 2. Target population (a) Subjects must have histologically or cytologically confirmed chronic lymphocytic leukemia, Hodgkin's lymphoma, or non-Hodgkin's lymphoma and must have relapsed after previous treatment or be refractory to previous treatment. (b) Part A Dose Escalation: (i) Chronic lymphocytic lymphoma (ii) Hodgkin's lymphoma (iii) non-Hodgkin's lymphoma (c) Part B Cohort Expansion (i) Chronic lymphocytic leukemia (ii) Diffuse large B-cell lymphoma (iii) Mantle cell lymphoma (iv) Hodgkin's lymphoma (v) Multiple myeloma

[0107] (d) Subjects must have at least one measurable lesion larger than 1.5 cm as defined by lymphoma (Cheson et al., J. Clin. Oncol. 2007;25(5):579-586) and CLL (Hallek et al., Blood 2008;111(12)5446-5456) response criteria. Tumor sites considered measurable cannot have received prior radiation therapy.

[0108] (e) Only subjects with no prior exposure to immune cell modulatory antibody regimens (ICMAR) such as anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-KIR, anti-CD137, or anti-OX40 antibodies are permitted. Prior anti-CD20, alemtuzumab, or brentuximab antibody treatment is permitted.

[0109] (f) Subjects must have progressed or be refractory to at least one prior standard of care, including radiation, immunotherapy, cytotoxic chemotherapy, and selective antibody (anti-CD20, alemtuzumab, or brentuximab) therapy. The following are not considered alternative lines of treatment: addition of a compound to an ongoing regimen, reinitiating the same regimen after a drug holiday, or switching from IV to oral therapy.

[0110] (g) The subject is not eligible for transplantation or any standard treatment known to be life-prolonging or life-saving. (A subject who is eligible for transplantation or any standard treatment known to be life-prolonging or life-saving and who declines transplantation or any standard treatment known to be life-prolonging or life-saving is eligible for the study.

[0111] (h) Subjects must be >100 days post-autologous transplant. (i) Eastern Cooperative Oncology Group (ECOG) status of 0 or 1 (j) Life expectancy of 10 weeks or more at the time of informed consent as assessed by the investigator

[0112] (k) Adequate organ function as defined by: (i) ≥ 500 neutrophils / μL (stable without growth factors within 1 week of first study drug administration) (ii) 50x10 3 / μL or more (transfusions to achieve this level will not be permitted within 2 weeks of the first study drug administration) (iii) Hemoglobin ≥ 8.5 g / dL (transfusions to achieve this level are not permitted within 2 weeks of first study drug administration) (iv) Creatinine less than 1.5xULN or creatinine clearance greater than 40mL / min (Cockcroft-Gault formula) (v) ALT and AST below 3xULN (vi) Total bilirubin less than or equal to 1.5xULN (except for subjects with Gilbert's syndrome, who must have normal direct bilirubin) (vii) Normal thyroid function as assessed by the investigator or stable to hormone replacement

[0113] (l) Ability to comply with treatment, PK, and pharmacodynamic sampling and required study follow-up. (m) Subject Re-enrollment: This study will allow re-enrollment of subjects who discontinued the study as a pre-treatment failure (i.e., non-randomized / non-treated subjects). If re-enrolled, subjects must consent again.

[0114] 3. Age and reproductive status (a) Men and women aged 18 years or older at the time of informed consent (b) Women of childbearing potential (WOCBP) must have a negative serum or urine pregnancy test within 24 hours prior to starting study drug (urine pregnancy test: minimum sensitivity 25 IU / L or equivalent units of human chorionic gonadotropin [hCG]). (c) The woman must not be breastfeeding. (d) WOCBP must agree to the following instructions for contraceptive methods for the duration of treatment with BMS-986016 + 5 half-lives of BMS-986016 (135 days) + 30 days (duration of the ovulation cycle), for a total of 165 days (24 weeks) after completion of treatment. (e) WOCBP and sexually active males must agree to the following instructions for contraceptive methods for the duration of treatment with BMS-986016 + 5 half-lives of BMS-986016 (135 days) + 90 days (duration of sperm turnover), for a total of 225 days (33 weeks) after completion of treatment.

[0115] Investigators should advise WOCBP and male subjects who are sexually active with WOCBP about the importance of pregnancy prevention and the implications of unintended pregnancy. Investigators should advise WOCBP and male subjects who are sexually active with WOCBP about the use of highly effective methods of contraception. Highly effective methods of contraception have a failure rate of less than 1% per year when used consistently and correctly.

[0116] At a minimum, subjects must agree to the use of two methods of contraception, one method being highly effective and the other method being either highly effective or less effective.

[0117] (f) Azoospermic men and WOCBP who are not continuously heterosexually active are exempt from the need for contraception. However, WOCBP who are continuously abstaining from heterosexual activity must undergo pregnancy testing as described in this protocol.

[0118] Exclusion criteria 1. Exceptions to Target Disease (a) Subjects with primary cutaneous lymphoma, lymphoproliferative disorders associated with primary immunodeficiencies, and lymphomas associated with human immunodeficiency virus (HIV) infection are excluded. (b) Subjects with known or suspected central nervous system (CNS) metastases or CNS as the only site of active disease are excluded: (i) Subjects with controlled brain metastases will be allowed to enroll. Controlled brain metastases are defined as no radiographic progression for at least 4 weeks following irradiation or surgical treatment at the time of consent. Subjects must be off steroids for at least 2 weeks prior to informed consent and must have no new or progressive neurological signs or symptoms. (ii) Subjects with signs or symptoms of brain metastases are not eligible unless brain metastases are excluded by computed tomography (CT) or magnetic resonance imaging (MRI).

[0119] 2. Medical history and concurrent illnesses (a) Subjects with a previous malignancy, except for adequately treated basal or squamous cell skin cancer, cervical carcinoma in situ or bladder cancer, or ductal or lobular carcinoma in situ of the breast, are excluded. Subjects with other previous malignancies diagnosed more than 2 years prior (at the time of informed consent), who have received therapy with curative intent, have had no evidence of disease during the interval, and are considered by the investigator to be at low risk of recurrence are eligible.

[0120] (b) Subjects with any active autoimmune disease or a history of known or suspected autoimmune disease, except for subjects with isolated vitiligo, resolved childhood asthma / atopy, controlled hypoadrenalism or hypopituitarism, and euthyroid patients with a history of Graves' disease (subjects with controlled hyperthyroidism must be negative for thyroglobulin and thyroid peroxidase antibodies and thyroid-stimulating immunoglobulin prior to study drug administration).

[0121] (c) the subject has autoimmune hemolytic anemia (AIHA) or autoimmune thrombocytopenia (ITP) requiring therapeutic doses of systemic steroids

[0122] (d) the subject has received any allogeneic transplant

[0123] (e) Any known or underlying medical condition that, in the opinion of the Investigator or Guarantor, would render administration of the study drug unsafe for the subject or that may adversely affect the subject's ability to comply with or tolerate the study procedures and / or study therapy, or that would confound the ability to interpret the tolerability of BMS-986016 in the subject being treated.

[0124] (f) Requirement for daily supplemental oxygen.

[0125] (g) Uncontrolled or significant cardiovascular disease, including but not limited to any of the following: (i) Myocardial infarction or stroke / transient ischemic attack (TIA) within 6 months prior to consent (ii) Uncontrolled angina pectoris within 3 months prior to consent (iii) History of clinically significant arrhythmia (ventricular tachycardia, ventricular fibrillation, or ventricular arrhythmia) (iv) QTc prolongation of more than 480 msec (v) History of other clinically significant cardiac disease (i.e., cardiomyopathy, congestive heart disease with New York Heart Association (NYHA) functional class III-IV, pericarditis, significant pericardial effusion)

[0126] (h) A positive blood screen for HIV or known acquired immune deficiency syndrome (AIDS).

[0127] (i) Any history of chronic hepatitis as evidenced by: (i) A positive test for Hepatitis A antibody (HepA IgM) (Note: a history of resolved Hepatitis A virus infection is not an exclusion criterion) (ii) A positive test for hepatitis B surface antigen (HBsAg) and / or hepatitis B core antigen (iii) A positive test for qualitative hepatitis C viral load (by PCR)

[0128] (j) evidence of active infection requiring systemic antibacterial, antiviral, or antifungal therapy within 7 days prior to initiation of study medication

[0129] (k) Any other significant acute or chronic medical illness.

[0130] (l) Subjects unable to undergo venipuncture and / or unable to tolerate venous access

[0131] (m) Any other valid medical, psychiatric, and / or social reasons as determined by the Investigator.

[0132] (n) Any of the following procedures or medications: (i) Within two weeks prior to the time of informed consent: (1) Systemic or topical corticosteroids in immunosuppressive doses (≥7.5 mg / day prednisone or equivalent) (2) Medicinal herbal preparations (ii) Within 4 weeks prior to study drug administration: (1) Any study drug or placebo (2) Any anti-cancer therapy (chemotherapy, anti-tumor monoclonal antibodies [e.g., anti-CD20, anti-CD30, or alemtuzumab antibody therapy], therapeutic vaccines, radiation therapy, or hormonal treatments). (3) Non-tumor vaccines containing live viruses (4) Allergen desensitization therapy (5)Major surgery (iii) Within 6 weeks prior to study drug administration: (1) Nitrosoureas, Fludarabine (iv) Within 10 weeks prior to study drug administration: (1) radioactive or toxic immunoconjugates (e.g., brentuximab)

[0133] 3. Allergies and Adverse Drug Reactions (a) History of allergy to any component of BMS-986016 (e.g., history of severe hypersensitivity reaction to drugs formulated with polysorbate 80)

[0134] 4. Other exclusion criteria (a) Prisoners or involuntarily imprisoned subjects; (b) Subjects who are involuntarily detained for the treatment of psychiatric or physical (e.g., infectious) illnesses; (c) Failure to Comply with Restrictions and Prohibited Activities / Procedures

[0135] Safety assessment Adverse events will be assessed continuously throughout the study and for 135 days after the last treatment. Adverse events will be assessed according to the NCI CTCAE version 4.0. Adverse events will be coded using the latest version of the Medical Dictionary for Regulatory Activities (MedDRA) and reviewed for potential significance and importance.

[0136] Efficacy evaluation Efficacy assessment is performed and reported in eCRF using the appropriate efficacy assessment based on tumor type.Subjects with NHL or HL are evaluated using the revised response criteria for malignant lymphoma (Cheson et al., J. Clin. Oncol. 2007;25(5):579-586).Subjects with CLL are evaluated using the guidelines for the diagnosis and treatment of chronic lymphocytic leukemia (Hallek et al., Blood 2008;111(12):5446-56).

[0137] Secondary efficacy evaluation Disease assessments will occur on days 50 to 56 of each treatment cycle (up to 12 8-week treatment cycles) and at the follow-up visit on day 30. Tumor response will be assessed by the investigator for subjects with sufficient data as defined by the following efficacy criteria: For NHL and HL: Revised Response Criteria for Malignant Lymphoma For CLL: Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia

[0138] Pharmacokinetic Sample Analysis Serum samples are analyzed for BMS-986016 by a validated immunoassay. In addition, for technical testing purposes, selected serum samples may be analyzed by an exploratory analytical method that measures BMS-986016; exploratory data will not be reported.

[0139] Exploratory Biomarker Evaluation Tumor tissue, bone marrow, and / or aspirates will be collected prior to therapy and at selected time points during treatment in all subjects in Parts A and B. Peripheral blood will be collected prior to therapy and at selected time points during treatment in the first three subjects enrolled at each dose level in Part A and all subjects in Part B. When biomarker samples are drawn but study drug is not administered, samples will be retained. A schedule of pharmacokinetic assessments is provided in Figure 3.

[0140] Soluble Biomarkers Soluble factors, such as cytokines, chemokines, soluble receptors, and antibodies against tumor antigens are characterized and quantified by serum immunoassay. Analytes include, but are not limited to, soluble CD25, soluble PD-1, soluble LAG-3, and CXCL-9. Collected serum samples are also used to evaluate the tumor antigen-specific response caused after treatment with monotherapy to explore whether anti-tumor antibodies are significantly associated with clinical response. Antibody levels against cancer test antigens are evaluated by multiplex assay and ELISA.

[0141] Immunophenotyping of PBMCs and bone marrow aspirates The proportion of specific lymphocyte subsets and expression levels of T cell costimulatory markers in PBMC preparations are quantified by flow cytometry.Analysis may include, but is not limited to, the proportion of T, B and NK cells, the proportion of memory and effector T cell subsets, and the expression levels of LAG-3, PD-1, PD-L1, PD-L2, ICOS and Ki67.

[0142] Ex vivo functional assays To explore whether nivolumab restores T cell activation and function, PBMCs are isolated and cryopreserved. Assays of effector T cell functional status are performed, including but not limited to assays for interferon-gamma (IFN-γ) and CD107.

[0143] Peripheral blood gene expression The expression levels of genes associated with response to BMS-986016 are quantified using molecular methods such as Affymetrix microarray and / or quantitative RT-PCR analysis in whole blood samples.The analysis may include, but is not necessarily limited to, genes associated with immune-related pathways such as T cell activation and antigen processing and presentation.

[0144] BMS-986016 Receptor Occupancy The percentage of BMS-986016 bound to immune cells as well as the mean fluorescence intensity (MFI) of bound drug are quantified by a flow cytometry-based receptor occupancy assay. This assay is performed on peripheral blood and bone marrow aspirates when available. The receptor occupancy assay provides data to determine the amount and type of T cells with which the drug is interacting in different biological compartments. Samples are taken before treatment to define a baseline and at the time of bone marrow aspirate collection. Peripheral blood and bone marrow aspirate analysis are performed on fresh samples. It is therefore important that samples are shipped immediately after collection.

[0145] T cell repertoire analysis Low diversity in the peripheral T cell compartment has been shown to correlate with poor overall survival in metastatic breast cancer (Manuel et al., Oncoimmunology 2012;1(4):432-440). A well-established theory in immuno-oncology suggests that a diverse and activated immune environment is more adept at eradicating tumors compared to a skewed repertoire of naive and tolerized T cells. To explore whether a diverse T cell repertoire predicts response to therapy, next-generation high-throughput DNA sequencing is performed on DNA isolated from peripheral blood and tumor tissues to quantify the composition of the T cell repertoire before and during monotherapy.

[0146] SNP analysis Selected genes will be evaluated for single nucleotide polymorphisms (SNPs) to identify potential polymorphisms associated with the safety and efficacy of BMS-986016. Analysis will be limited to sequence polymorphisms linked to genes and pathways associated with PD-1 / PD-L1, LAG-3, and the activated T-cell phenotype, including PD-1, PD-L1, PD-L2, LAG-3, MHC II, and CTLA-4.

[0147] Tumor-Based Biomarker Measurements Tumor biopsy specimens (eg, primary tumor, lymph node) will be obtained before and after treatment with BMS-986016 to characterize immune cell populations and expression of selected tumor markers.

[0148] Pre-treatment tumor biopsies (e.g., primary tumor, lymph node) will be collected in all consenting adults. When a pre-treatment tumor biopsy is clinically not feasible, either archived tumor tissue samples (e.g., primary tumor, lymph node, etc.), formalin-fixed paraffin-embedded (FFPE) blocks or unstained slides must be provided for performance of correlative testing. Pathology reports should be attached with archived or fresh biopsy samples. When both fresh biopsy and archived samples are available, both samples should be sent.

[0149] Tumor biopsies during treatment are optional and will be collected in subjects who had a biopsy at baseline. Biopsies may be obtained during cycle 1, days 50 to 56 (or earlier if clinically indicated) and again at PD. Biopsies may be coordinated with protocol-specific diagnostic imaging.

[0150] Unilateral bone marrow biopsies and / or aspirates will be performed in all subjects at baseline or up to 28 days prior to the first dose of study drug. Subjects who have had bone marrow aspirates and biopsy results due to completion of their last treatment may not use those bone marrow results in place of the baseline bone marrow required for this study. On-treatment bone marrow biopsies and / or aspirates samples may be obtained in CR, PR, or PD when clinically indicated and coordinated with protocol-specific diagnostic imaging.

[0151] Biopsies and bone marrow samples may be used to assess:

[0152] Characterization of tumor infiltrating lymphocytes (TIL), tumor antigens, and virus positivity Immunohistochemistry analyses (IHC) are used to assess the number and composition of immune infiltrates to define immune cell subsets present within FFPE tumor tissue before and after exposure to therapy. These IHC analyses include, but are not limited to, the following markers: CD4, CD8, FOXp3, PD-1, LAG-3, and MHC II. The Epstein-Barr Virus (EBV) status of the tumor may also be assessed by assessment of EBV-encoding RNA, LMP-1 expression, or similar assays. For example, IHC analyses for human LAG-3 were performed in human tonsil and NHL cells according to the following protocol, and the results are shown in Figure 4-14B:

[0153] Equipment and materials: Leica autostainer BioGenex i6000 Autostainer BioCare Medical Decloaking Chamber TM Plus

[0154] reagent: Xylene® (EM Science, Cat # UN1307) Ethanol (AAPER alcohol and Chemical Co.) AR10 (BioGenex®, Cat# HK057-5K) Wash Buffer (DAKO®, Cat# S3006) Peroxidase Block (Leica® Cat#RE7101-CE) Protein block (Leica® Cat#RE7102-CE) Comment: Antibody Diluent (BioGenex®, Cat# HK156-5K) DAB (Leica(R) Cat#RE7190-CE) Hematoxylin (Dako®, Cat# S3309) antibody: [Table 3] Novolink Max Polymer Detection System(R)(Leica(R), Cat# RE7260-CE)

[0155] procedure: 1. Deparaffinization and rehydration were performed using the following solutions: xylene (2 times, 5 min each), 100% ethanol (2 times, 2 min each), 95% ethanol (2 times, 2 min each), 70% ethanol (2 times, 2 min each), and distilled H 2 O(dH 2 O) (2 min) was performed using a Leica autostainer; 2. Antigen retrieval was accomplished using a Biocare Medical Decloaking Chamber Plus®. The AR10 solution was heated to 105°C (P1) for 20 minutes and then transferred to the next step (P2 FAN ON, 95°C; FAN OFF, 90°C); 3. The slides were cooled at room temperature for 15 minutes and rinsed with water (approximately 1 minute); 4. The IHC reagents were placed on the i6000 automated stainer; 5. Using a pap pen to define the tissue area, the slide was placed in the i6000 Autostainer; and 6. IHC was performed on a BioGenex i6000 autostainer using research mode as follows.

[0156] IHC steps for i6000 (NovoLink Kit): 1. Apply "Peroxidase Block" (NovoLink Kit) for 10 minutes, then rinse with IHC wash buffer (3 times); 2. Protein block (NovoLink Kit) was applied to the slides and incubated for 10 min at room temperature, then washed with buffer (3 times); 3. Antibody was applied to the slides, incubated for 1 hour at room temperature, and washed with buffer (3 times); 4. "Post-primary block" (NovoLink Kit) was added to the slides, incubated for 30 minutes, and washed with buffer (3 times); 5. "NovoLink Polymer" (NovoLink Kit) was added to the slide and incubated for 45 minutes; 6. Rinse slides with IHC wash buffer (3 times); 7. Add DAB chromogenic substrate (NovoLink Kit) and develop for 5 minutes; 8. Wash the slides five times in dHO at room temperature. 2 Washed by rinsing with O; 9. Slides were counterstained with hematoxylin (Dako) 1:1 for 1 min at room temperature; 10. Next, place the slide in dH 2 The mixture was washed by rinsing with O (5 times at room temperature).

[0157] Dehydration and coverslipping: 1. Slides were removed from the i6000 autostainer and placed into the Leica autostainer; 2. Slides were dehydrated in 70% ethanol, 95% ethanol, 2X100% ethanol (2 min each); 3. The slides were washed in xylene (2 times for 2 min each); and 4. Sections were coverslipped with Cytoseal Mounting Medium in a Leica CM5030 coverslipper.

[0158] Serum viral load Epstein-Barr virus (EBV)- and cytomegalovirus (CMV)-viral load status is assessed in serum by PCR at different time points. Viral load is then correlated with clinical outcome and expression of markers such as CD4, CD8, FOXp3, PD-1, LAG-3, and MHC II in tumor tissue.

[0159] Characterization of the T cell repertoire As above, DNA sequencing will be performed on pre- and post-treatment tumor tissue to assess the composition of the T cell repertoire. DNA will be isolated from either FFPE tumor blocks or RNAlater or equivalent preparations.

[0160] Gene expression profiling Tumor biopsies and bone marrow samples taken in RNAlater or an equivalent fixative are examined for mRNA gene expression by Affymetrix gene array technology and / or RT-PCR to detect expression of selected immune-related genes.

[0161] Cytogenetics, Mutations and Tumor Markers The following analyses should be performed at baseline if not previously performed: Subjects with Hodgkin lymphoma: Amplification 9p24.1, CD30 Subjects with CLL: del 11q, del 13q, del 17p, IGHV status, CD38 and β2 microglobulin Subjects with DLBCL: phenotype ABC, GCB, or unclassifiable, and β2 microglobulin. The results of these clinical markers are associated with both clinical outcome and an exhausted phenotype in T cells.

[0162] Tumor sample collection 1. Biopsy (primary tumor and / or lymph node) A minimum of 1 FFPE tumor tissue block (preferred) or a minimum of 10 FFPE unstained sections are required for assessment of LAG-3 status, EBER-ISH and other biomarker evaluation. When FFPE is not available, tumor biopsies in formalin are also acceptable.

[0163] Biopsy samples should be excision, incision or core needle. Fine needle aspirates or other cytology samples will only be permitted after discussion with the guarantor's medical monitor.

[0164] It is recommended that samples be fixed in 10% neutral buffered formalin for 24 to 48 hours.

[0165] When slides are presented, the recommended tissue section thickness is 4 microns and the slides should be positively charged. Slides should be transported chilled at 2-8°C.

[0166] When taking fresh biopsies, a maximum of four core biopsies are recommended. Assessment of biopsy quality by a pathologist is strongly encouraged at the time of processing. Tumor tissue from these biopsies will be split equally into FFPE samples and RNAlater.

[0167] In consultation with the investigator and radiologist, the degree of risk associated with the procedure must be determined and tolerated. Biopsies may be performed using local anesthesia or conscious sedation. Institutional guidelines regarding the safe performance of biopsies should be followed. Excisional biopsies may be performed to obtain tumor biopsy samples. Invasive procedures requiring general anesthesia should not be performed to obtain biopsy specimens. However, when performing surgical procedures for clinical indications, excess tumor tissue may be used for research purposes with the subject's consent.

[0168] 2. Bone marrow and / or aspirate Bone marrow biopsies and / or aspirates taken at baseline, in CR or PR, and at progression will be utilized to assess the phenotype and functional status of immune and tumor cells.

[0169] Bone marrow biopsies and / or aspirates are obtained using institutional standards for these procedures. Detailed instructions for bone marrow biopsy and aspirate collection are provided in the Laboratory Procedures Manual. Briefly, a minimum of 1 FFPE tumor tissue block (preferred) or a minimum of 10 FFPE unstained sections of bone marrow are required, and approximately 10 ml of aspirate is collected in a sodium heparin tube and transported ambiently. A pathology report should be attached with the biopsy sample.

[0170] Adverse events An adverse event (AE) is defined as any new untoward medical occurrence or worsening of a pre-existing medical condition in a clinical subject receiving an investigational drug and that does not necessarily have a causal relationship to this treatment. Thus, an AE is any untoward and unintended sign (such as an abnormal laboratory finding), symptom, or disease that is temporally associated with the use of an investigational drug, regardless of whether it is considered related to the investigational drug.

[0171] The causal relationship to study drug will be determined by the investigator and will be used to evaluate all adverse events (AEs). A causal relationship may be one of the following:

[0172] Related: A reasonable causal relationship exists between administration of study medication and the AE. Unrelated: There is no reasonable causal relationship between the administration of study drug and the AE.

[0173] 1. Serious adverse events Serious adverse events (SAEs) occurred at either dose: Causes death · Life-threatening (defined as an event in which the subject was at risk of death at the time of the event; it does not refer to an event that is hypothesized to have caused death if it had been more severe) Requiring hospitalization or causing a prolongation of hospitalization that has already begun Causes persistent or significant disability / incapacity - Congenital / birth defects Any adverse medical occurrence that is a major medical event (defined as a medical event that is not immediately life-threatening or does not result in death or hospitalization, but that, based on appropriate medical and scientific judgment, may endanger the subject or require intervention (e.g., medical, surgical) to prevent one of the other serious outcomes listed in the definition above). Examples of such events include, but are not limited to, emergency room or intensive treatment at home for allergic bronchospasm; blood dyscrasias or seizures that do not result in hospitalization. Potential drug-induced liver injury (DILI) is also considered a major medical event.

[0174] Suspected transmission of an infectious agent (e.g., pathogenic or nonpathogenic) by the study drug is an SAE. Pregnancy, overdose, cancer, and potential drug-induced liver injury (DILI) are not always serious by regulatory definition, but these events must be treated as SAEs. Any component of the study endpoint considered related to study therapy (e.g., if death is an endpoint and death occurs due to anaphylaxis, anaphylaxis must be reported) is reported as an SAE.

[0175] The following hospitalizations are not considered SAEs: - A visit to an emergency room or other medical unit for less than 24 hours that does not result in hospitalization (unless considered a significant medical event or a life-threatening event). - Elective surgery planned prior to signing consent. - Protocol hospitalization for planned medical / surgical procedures. - Routine health assessment requiring hospitalization for baseline / trend health status (e.g., routine colonoscopy). - Medical / surgical procedures other than to correct an underlying condition and planned prior to enrollment in the study. Appropriate documentation is required in these cases. - Hospitalization due to other living circumstances (e.g., lack of housing, financial insufficiency, caregiver withdrawal, family circumstances, administrative reasons) that did not affect health status and did not require medical / surgical intervention.

[0176] After written consent of subjects to enter the study, all SAEs will be collected, including those considered to be related to protocol-specific procedures, whether or not related to study drug. All SAEs occurring during the screening period and within 135 days of discontinuation of medication will be collected. If applicable, SAEs associated with any subsequent protocol-specific procedures (e.g., follow-up skin biopsies) will be collected. All SAEs will be followed until resolution or stabilization.

[0177] 2.Non-serious adverse events Non-serious adverse events are AEs not classified as serious. Collection of non-serious AE information begins at the initiation of study drug and continues for 135 days after discontinuation of dosing. Non-serious AEs will be tracked until resolution or stabilization or reported as SAEs if they become serious. Tracking is required for non-serious AEs that cause interruption or discontinuation of study drug and those present at the end of study treatment, if appropriate. All identified non-serious AEs will be recorded and documented on the non-serious AEs page of the CRF (paper or electronic).

[0178] Any AEs and / or laboratory abnormalities reported / identified during the course of the study will require completion of additional CRFs.

[0179] Sample size determination 1. Part A: Dose Escalation Sample size at each dose will depend on toxicity observed and cannot be precisely determined. Part A will have 3-9 subjects in each cohort.

[0180] 2. Part B: Cohort expansion A sample size of about 12 subjects per cohort allows for better estimation of toxicity rates and provides better precision for approximating preliminary efficacy estimates. If 3 of 12 subjects (i.e., less than 30%) experience toxicity in a cohort, there is at least 90% confidence (based on Clopper-Pearson direct binomial one-sided 90% confidence interval) that the true toxicity rate is 48% or less. In addition, for safety signals explored across cohorts, there is at least 90% confidence that the true rate of toxicity will not exceed 40% when 14 of 48 subjects (i.e., less than 30%) experience toxicity in the extension portion of the study.

[0181] A sample size of approximately 12 subjects per cohort also allows for estimation of the proportion of subjects within the cohort who have an objective response (i.e., complete response [CR] + partial response [PR]), such that the two-sided 90% confidence interval for the objective response rate is 7% to 53% when 3 subjects (25%) have a response and 12% to 61% when 4 subjects (33%) have a response. In addition, given a true response rate of 15% or greater, there is a greater than 86% chance of seeing at least one response in a cohort of 12 subjects.

[0182] Population for analysis · All Enrolled Subjects: All subjects who signed informed consent for the study (including those who failed screening). · All Treated Subjects: All subjects who receive at least one full or partial dose of BMS-986016. Response-evaluable subjects: All subjects with a baseline assessment of evaluable disease and one of the following: (1) at least one evaluable on-treatment disease assessment, (2) clinical progression, or (3) death prior to the first on-treatment disease assessment. Pharmacokinetic Analysis Set: Include in the summary table all treated subjects with sufficient PK data to define at least one valid PK parameter. List all available serum concentration data from treated subjects. Immunogenicity Analysis Set: All subjects who received one full or partial dose of BMS-986016 and had a baseline and at least one available post-baseline immunogenicity sample will be included in the summary table. All available data from treated subjects will be listed. Pharmacodynamic Analysis Set: All subjects treated with at least one valid measurement for a particular biomarker are included in the summary and table for that biomarker.

[0183] Evaluation items 1. Primary endpoint The primary endpoint of this Phase 1 study is safety, measured at the study level by rates of AEs, SAEs, deaths, and laboratory abnormalities assessed during treatment and for up to 135 days after the last treatment. All subjects receiving at least one dose of BMS-986016 or nivolumab will be analyzed for safety.

[0184] 2. Secondary endpoints Pharmacokinetics The PK of BMS-986016 will be evaluated as a secondary objective using the following endpoints derived from serum concentration versus time data at various time points:

[0185] PK parameters evaluated include: Cmax maximum blood concentration Tmax Time to reach maximum blood concentration Ctrough Trough blood concentration Ceoinf Concentration at the end of infusion Ctau - concentration at the end of the dosing interval (e.g., concentration at 336 hours) Css,avg Average concentration over the dosing interval ([AUC(TAU) / tau]) AUC(TAU) Area under the concentration-time curve during one dosing interval CLT Total Body Clearance Vss Distributed capacitance in steady state T-HALFeff AUC The effective elimination half-life that explains the observed AUC accumulation T-HALFeff Cmax The effective elimination half-life that explains the observed Cmax accumulation AI_AUC Accumulation index; the ratio of AUC(TAU) at steady state to AUC(TAU) after the first dose AI_Cmax Cmax accumulation index; ratio of Cmax at steady state to Cmax after first dose AI_Ctau Ctau accumulation index; the ratio of Ctau at steady state to Ctau after the first dose AI_Ceoinf Ceoinf accumulation index; ratio of Ceoinf at steady state to Ceoinf after first dose DF Variability or Variability Index ([Cmax-Ctau] / Css,avg)

[0186] Individual subject PK parameter values ​​are derived by non-compartmental methods with a validated PK analysis program. Actual times are used for the analysis.

[0187] b.Efficacy The following secondary endpoints will be evaluated to explore the preliminary antitumor activity of BMS-986016: Objective response rate (ORR), i.e., the proportion of subjects with a best overall response of CR or PR Duration of response (DOR) based on current criteria relevant to each disease type Landmark progression-free survival (PFSR) rates at 4, 6, 8 and 10 months on treatment and 30 days after the last dose will be evaluated as exploratory endpoints.

[0188] 3. Exploratory endpoints / biomarkers Biomarker endpoints from peripheral blood may include measurements such as levels of soluble factors, and T cell subsets characterized by immunophenotyping at each scheduled time point. Biomarker endpoints from tumor biopsies may include, but are not limited to, measurements such as lymphocyte functional status and location and lymphocyte activation gene 3 (LAG-3), major histocompatibility complex (MHC) class II, programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1) expression. Measurements of receptor occupancy characterized in peripheral blood, bone marrow, and lymph nodes (when available) may also be provided.

[0189] analysis Unless otherwise noted, safety data from Parts A and B are summarized 1) overall across and by dose levels, and also 2) both across and by dose levels within each tumor type. Efficacy data are summarized by dose level within each tumor type.

[0190] 1. Demographic data and baseline characteristics Frequency distributions for gender and race will be tabulated. Summary statistics for age, weight, height and body mass index (BMI) will be tabulated.

[0191] 2. Efficacy analysis In subjects with HL and NHL, the Revised Response Criteria for Malignant Lymphomas (Appendix 1) are used to assess efficacy. For subjects with CLL, the Guidelines for the Diagnosis and Treatment of Chronic Lymphocytic Leukemia are used.

[0192] Individual BOR, ORR, DOR and PFSR at selected time points are determined. Distribution tables are used to summarize BOR results. ORR, breakthrough PFSR (4, 6, 8 and 10 months on treatment and 30 days after last dose) and corresponding confidence intervals are calculated for Part B and may be calculated for Part A when supported by data. DOR and PFSR are estimated by tumor type using Kaplan-Meier methodology. Further exploratory presentation of efficacy may include subjects in both dose escalation and cohort expansion, categorized by tumor type, treatment, prior exposure to immunotherapy, or baseline tumor markers. Plots of individual changes in burden of disease over time are also generated. Individual changes in tumor markers over time may be graphed by dose level within selected disease types. Depending on the purpose of the analysis, efficacy may be reported for either all treated subjects or response-evaluable subjects.

[0193] 3.Safety analysis All subjects receiving study medication will be included in the analysis of safety endpoints. All recorded AEs will be listed and tabulated by system organ class, preferred duration, relationship to study drug, and treatment. Coding will be done with the latest version of MedDRA. Vital signs and selected clinical laboratory test results will be listed and summarized by treatment. Any significant physical examination findings and results of clinical laboratory tests will be listed. ECG abnormalities identified by the investigator will be tabulated.

[0194] 4. Pharmacokinetic Analysis The PK parameters of BMS-986016 are calculated using non-compartmental analysis. Summary statistics are tabulated for the PK parameters of BMS-986016 by treatment and study day / week. To describe the dose dependence of BMS-986016, scatter plots of Cmax and AUC(TAU) versus dose can be provided for each day measured. Dose proportionality of BMS-986016 can also be evaluated based on the power model.

[0195] 5. Exploratory biomarker analysis The pharmacokinetic effects on TIL, MIL and other important tumor markers in subjects who have undergone biopsy are summarized using summary statistics and plots.In addition, the correlation between TIL or MIL changes and tumor marker expression in peripheral blood marker measurements can be explored graphically using suitable modeling methods based on data availability.The association of biomarker measurements from peripheral blood or tumor biopsy with clinical outcomes can also be explored graphically and, if necessary, further evaluated by methods such as, but not limited to, logistic regression, and characterized by suitable statistics.

[0196] 6. Immunogenicity analysis A list is provided for all available immunogenicity data. The number and percentage of subjects meeting specific endpoint definitions are summarized. To examine possible relationships between immunogenicity and safety, tables summarizing the frequency and type of AEs of special interest can be explored by immunogenicity status. In addition, possible relationships between immunogenicity and efficacy, pharmacokinetic markers and / or PK can also be explored.

[0197] Overview of Arrays [Table 4] [Table 5] [Table 6] [Table 7]

Claims

1. 1. A method of treating a hematological malignancy in a human patient, comprising administering to the patient: 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. administering an effective amount of A method comprising at least one administration cycle, the cycle being 8 weeks, and for each of the at least one cycle, the anti-LAG-3 antibody is administered as follows: (a) 4 doses of 20 mg anti-LAG-3 antibody; (b) 4 doses of 80 mg anti-LAG-3 antibody; (c) four doses of 240 mg anti-LAG-3 antibody; or (d) 4 doses of 800 mg anti-LAG-3 antibody.

2. The method of claim 1, wherein the anti-LAG-3 antibody is formulated for intravenous administration.

3. 3. The method of claim 1 or 2, wherein the treatment consists of up to 12 cycles.

4. The method of any of claims 1-3, wherein the anti-LAG-3 antibody is administered on days 1, 15, 29 and 43 of each cycle.

5. The method of any of claims 1-4, wherein the treatment results in at least one therapeutic effect selected from reducing the number of malignant cells, inhibiting malignant cell invasion, inhibiting malignant cell metastasis, preventing the development of malignant cells, and reducing one or more symptoms associated with a malignant tumor.

6. The method of any one of claims 1 to 5, wherein the malignant tumor is selected from leukemia, lymphoma, and myeloma.

7. The method according to any one of claims 1 to 6, wherein the malignant tumor is selected from relapsed or refractory chronic lymphocytic leukemia and lymphoma.

8. 8. The method of claim 7, wherein the malignant tumor is selected from chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma (NHL).

9. The anti-LAG-3 antibody (a) a heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:7; (b) a heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:8; (c) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:9; (d) a light chain variable region CDR1 having the sequence set forth in SEQ ID NO: 10; (e) a light chain variable region CDR2 having the sequence set forth in SEQ ID NO: 11; and (f) a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 12 The method of any one of claims 1 to 8, comprising:

10. The method of any of claims 1-9, wherein the anti-LAG-3 antibody comprises heavy and light chain variable regions having the sequences set forth in SEQ ID NO:3 and SEQ ID NO:5, respectively.

11. The method of any of claims 1-10, wherein the anti-LAG-3 antibody comprises a heavy chain and a light chain having the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively.

12. 1. A kit for treating relapsed or refractory chronic lymphocytic leukemia or lymphoma in a human patient, comprising: (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 an anti-LAG-3 antibody in the method of any one of claims 1-11. Including the kit.

13. 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, for administration in at least one cycle, wherein for each cycle, the anti-LAG-3 antibody is administered as follows: (a) 4 doses of 20 mg anti-LAG-3 antibody; (b) 4 doses of 80 mg anti-LAG-3 antibody; (c) four doses of 240 mg anti-LAG-3 antibody; or (d) 4 doses of 800 mg anti-LAG-3 antibody.

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