Optimization of antibodies that bind to lymphocyte activation gene-3 (LAG-3) and uses thereof

Abstract

A stable therapeutic antibody that specifically binds to LAG-3 is provided. [Solution] The present invention provides isolated monoclonal antibodies that specifically bind to LAG-3 and have optimized functional properties compared to previously described anti-LAG-3 antibodies, such as antibody 25F7. These properties include a reduction in deamidation sites while maintaining high affinity binding to human LAG-3 and physical (i.e., thermal and chemical) stability. Nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells, and methods for expressing the antibodies of the invention, as well as immunoconjugates, bispecific molecules, and pharmaceutical compositions comprising the antibodies, are also provided. The present invention also provides methods for detecting LAG-3 and methods for stimulating immune responses and treating diseases using the anti-LAG-3 antibodies of the invention. Combination therapies in which the antibodies are co-administered with at least one additional immunostimulatory antibody are also provided.

Description

[Technical Field]

[0001] Background of the Invention Therapeutic antibodies are one of the fastest-growing areas in the pharmaceutical industry. To maintain efficacy (i.e., activity) and minimize immunogenicity, antibodies and other protein drugs must be protected from physicochemical degradation during production and storage. Indeed, one of the major challenges in developing antibody therapeutics is the potential immunogenic response upon administration to a subject, which can cause rapid clearance or even life-threatening side effects, including anaphylactic shock. Various factors, such as their physicochemical properties (e.g., purity, stability, or solubility), clinical factors (e.g., dose, administration route, disease heterogeneity, or patient characteristics), and co-treatment with other drugs, affect the immunogenicity of antibodies (Swann et al. (2008) Curr Opinion Immuol 20:493-499). [Background technology]

[0002] Loss of antibody immunogenicity and / or antibody activity is often due to deamidation. Deamidation is a chemical degradation process that occurs spontaneously in proteins (e.g., antibodies). Deamidation removes the amide functional group from amino acid residues, such as asparagine and glutamine, thus damaging their amide-containing side chains. This, in turn, causes structural and biological changes throughout the protein, thus creating heterogeneous forms of the antibody. Deamidation is one of the most common post-translational modifications that occur in recombinantly produced therapeutic antibodies.

[0003] For example, heterogeneity in the heavy chain of monoclonal antibody h1B4 (humanized anti-CD18 antibody) due to deamidation during cell culture was reported by Tsai et al. (Pharm Res 10(11):1580 (1993)). In addition, reduction / loss of biological activity due to deamidation has become a recognized problem. For example, Kroon et al. characterized several deamidation sites in the therapeutic antibody OKT3 and reported that samples from OKT3 production lots (ranging from 14 months to 3 years) had less than 75% activity (Pharm Res 9(11):1386 (1992), p. 1389, column 2). In addition, OKT3 samples showing a large amount of oxidized peptides in their maps had significantly reduced activity in antigen-binding potency assays (p. 1390, column 1). The authors concluded that specific sites of chemical modification that occur during storage of OKT3 were identified by peptide mapping and correlated with changes observed in chemical analysis and biological assays of the antibody (page 1392, column 1). Loss of biological activity has also been reported for a variety of other deamidated therapeutic proteins, including recombinant human DNase (Cacia et al. (1993) J. Chromatogr. 634:229-239) and recombinant soluble CD4 (Teshima et al. (1991) Biochemistry 30:3916-3922).

[0004] Overall, deamidation is a significant and unpredictable problem in the pharmaceutical industry. The efforts associated with monitoring variations caused by deamidation in antibody therapeutics, particularly as well as the FDA concerns associated with this variation, increase costs and delay clinical trials. Furthermore, modifications to address this problem, including shifting conditions (e.g., temperature, pH, and cell type) associated with recombinant production and / or alteration of amino acids susceptible to deamidation (e.g., site-directed mutagenesis), can negatively affect stability and activity, particularly when changes are made within the complementarity-determining regions (CDRs) of antibodies. Thus, there is a need for more stable versions of therapeutic antibodies. Summary of the Invention

[0005] summary The present invention provides isolated monoclonal antibodies (e.g., human monoclonal antibodies) that bind to LAG-3 (e.g., human LAG-3) and have optimized physical stability compared to previously described anti-LAG-3 antibodies. In particular, the present invention relates to modified forms of antibody 25F7 (US 2011 / 0150892 A1) that exhibit significantly improved thermal and chemical stability compared to the unmodified antibody. Specifically, by modifying a critical binding region in the heavy chain CDR2 domain of antibody 25F7, the modified antibody was shown to exhibit significantly higher physical and thermal stability, reduced deamidation, increased thermal reversibility, and reduced aggregation. At the same time, it was unexpectedly observed that the modified antibody maintained the same high binding affinity for human LAG-3 and the functional activity of the unmodified antibody, e.g., the ability to inhibit LAG-3 binding to major histocompatibility (MHC) class II molecules and stimulate antigen-specific T cell responses. The combined substantial increase in stability and maintenance of binding / biological activity of the modified antibodies was surprising, especially considering the criticality of the CDR regions to antibody function.

[0006] The antibodies of the present invention can be used for a variety of applications, including the detection of LAG-3 protein and the stimulation of antigen-specific T cell responses in tumor-bearing or virus-bearing subjects.

[0007] Thus, in one aspect, the present invention relates to an isolated monoclonal antibody (e.g., a human antibody), or antigen-binding portion thereof, having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the antibody further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In another embodiment, the antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3 regions of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (e.g., SEQ ID NOs: 15, 16, and 17, respectively). In another embodiment, the antibody further comprises CDR1, CDR2, and CDR3 regions of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (e.g., SEQ ID NOs: 18, 19, and 20, respectively).

[0008] In preferred embodiments, the antibody exhibits increased physical properties (i.e., thermal and chemical stability) compared to antibody 25F7, while maintaining at least the same binding affinity for human LAG-3 as 25F7. For example, the antibody exhibits reduced sequence variability in the heavy chain CDR2 region due to deamidation compared to antibody 25F7, e.g., about 2.5% or less of the amino acid sequence modified after 12 weeks at 4°C (i.e., under the "real-time" stability test described herein), and / or about 12.0% or less of the amino acid sequence modified after 12 weeks at 40°C (i.e., under accelerated stress conditions described herein), but at least about 1 x 10 -7 K below M D (More preferably, 1x10 -8 K below M D , 5x10 -9 K below M D , or 1x10 -9 K below M D ) binding affinity for human LAG-3. In other embodiments, the antibody exhibits at least about 40% thermoreversibility in PBS at pH 8.0.

[0009] In other embodiments, the antibody is an unmodified antibody (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). In one embodiment, the antibody has a T of 60°C or higher, e.g., 65°C or higher, or 70°C or higher. M1 (temperature of initial unfolding). The melting point of the antibody was determined by differential scanning calorimetry (Chen et al. (2003) Pharm Res 20 :1952-60; Ghirlando et al. (1999) Immunol Lett 68 :47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40 :343-9).

[0010] In other embodiments, the antibody is characterized by resistance to rapid degradation. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67 :3626-32).

[0011] In other embodiments, the antibody exhibits minimal aggregation effects, e.g., 25% or less, e.g., 20% or less, 15% or less, 10% or less, 5% or less, or 4% or less. Aggregation can lead to undesirable immune responses and / or altered or unfavorable pharmacokinetic properties. Aggregation can be measured by several techniques, including size exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

[0012] In other embodiments, the antibody has at least one of the following properties: (a) Binding to monkey LAG-3; (b) no binding to mouse LAG-3; (c) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules; and (d) Stimulating immune responses, especially antigen-specific T cell responses Further shown.

[0013] Preferably, the antibody exhibits at least two of properties (a), (b), (c), and (d). More preferably, the antibody exhibits at least three of properties (a), (b), (c), and (d). Even more preferably, the antibody exhibits all four of properties (a), (b), (c), and (d).

[0014] In other embodiments, the antibody stimulates an antigen-specific T cell response, e.g., interleukin-2 (IL-2) production in an antigen-specific T cell response. In other embodiments, the antibody stimulates an immune response, e.g., an anti-tumor response (e.g., inhibition of tumor growth in vivo in a tumor xenograft model) or an autoimmune response (e.g., the development of diabetes in NOD mice).

[0015] In other embodiments, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21). In other embodiments, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO: 23).

[0016] In other embodiments, the antibody stains pituitary tissue by immunohistochemistry or does not stain pituitary tissue by immunohistochemistry.

[0017] The antibodies of the present invention may optionally contain modifications of the heavy chain constant region hinge region (Angal et al. (1993) Mol. Immunol.) to reduce or eliminate disulfide bridge heterogeneity in the heavy chain. 30 The antibody may be, for example, a full-length antibody of an IgG1, IgG2, or IgG4 isotype, having a serine to proline mutation at amino acid residue 228 (e.g., a position corresponding to position 241 described in SEQ ID NO:105-108). In one aspect, the constant region isotype is IgG4 having a mutation at amino acid residue 228, e.g., S228P. Alternatively, the antibody may be an antibody fragment, e.g., a Fab, Fab', or Fab'2 fragment, or a single-chain antibody.

[0018] In another aspect of the invention, the antibody (or antigen-binding portion thereof) is part of an immunoconjugate that includes a therapeutic agent, e.g., a cytotoxin or a radioisotope, linked to the antibody. In other aspects, the antibody is part of a bispecific molecule that includes a second functional moiety (e.g., a second antibody) that has a different binding specificity than the antibody or antigen-binding portion thereof.

[0019] Compositions comprising the antibodies or antigen-binding portions thereof, immunoconjugates or bispecific molecules of the invention, optionally formulated in a pharmaceutically acceptable carrier, are also provided.

[0020] Nucleic acid molecules encoding the antibodies of the invention or antigen-binding portions thereof (e.g., variable regions and / or CDRs) are also provided, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors. Methods for preparing anti-LAG-3 antibodies using host cells containing such expression vectors are also provided, which may include (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell.

[0021] In another aspect, the present invention provides a method for stimulating an immune response using an anti-LAG-3 antibody of the present invention. In one embodiment, the method comprises stimulating an antigen-specific T cell response by contacting T cells with an antibody of the present invention, such that an antigen-specific T cell response is stimulated. In a preferred embodiment, interleukin-2 production by antigen-specific T cells is stimulated. In another embodiment, the subject is a tumor-bearing subject, and an immune response against the tumor is stimulated. In another embodiment, the subject is a virus-bearing subject, and an immune response against the virus is stimulated.

[0022] In yet another embodiment, the invention provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention, such that tumor growth is inhibited in the subject. In yet another embodiment, the invention provides a method for treating a viral infection in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention, such that viral infection is treated in the subject. In other embodiments, these methods comprise administering a composition, bispecific, or immunoconjugate of the invention.

[0023] In yet another embodiment, the present invention provides a method for stimulating an immune response in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, of the present invention and at least one additional immunostimulatory antibody, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody, to stimulate an immune response in the subject, e.g., to inhibit tumor growth or stimulate an anti-viral response. In one embodiment, the additional immunostimulatory antibody is an anti-PD-1 antibody. In another embodiment, the additional immunostimulatory agent is an anti-PD-L1 antibody. In yet another embodiment, the additional immunostimulatory agent is an anti-CTLA-4 antibody. In yet another embodiment, the antibody, or antigen-binding portion thereof, of the present invention is administered together with a cytokine (e.g., IL-2 and / or IL-21), or a costimulatory antibody (e.g., anti-CD137 and / or anti-GITR antibody). The antibody can be, for example, a human, chimeric, or humanized antibody.

[0024] In other aspects, the invention provides anti-LAG-3 antibodies and compositions of the invention for use in the methods, or for the manufacture of a medicament for use in the methods (eg, for treatment).

[0025] Other features and advantages of the present application will be apparent from the description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited herein are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0026] [Figure 1A] Figure 1A shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the heavy chain variable region of the 25F7 human monoclonal antibody. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6), and CDR3 (SEQ ID NO: 7) regions are shown, with the V, D, and J germline derivations indicated. The CDR regions are shown using the Kabat system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0027] [Figure 1B] Figure 1B shows the nucleotide sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of the kappa light chain variable region of the 25F7 human monoclonal antibody. The CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO:9), and CDR3 (SEQ ID NO:10) regions are indicated, and the V and J germline derivations are indicated. The full-length heavy and light chain amino acid sequences of antibody 25F7 are shown in SEQ ID NOs:32 and 34, respectively.

[0028] [Figure 2A] Figure 2A shows the amino acid sequence of the heavy chain variable region of the LAG3.5 monoclonal antibody (SEQ ID NO: 12). The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO: 16), and CDR3 (SEQ ID NO: 17) regions are shown. The full-length heavy and light chain amino acid sequences of antibody LAG3.5 are shown in SEQ ID NOs: 35 and 37, respectively.

[0029] [Figure 2B]Figure 2B shows the nucleotide sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14) of the kappa light chain variable region of the LAG3.5 monoclonal antibody. The CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 20) regions are indicated.

[0030] [Figure 3] Figure 3 shows the amino acid sequence of the CDR2 heavy chain variable region sequence of LAG-3 variants LAG3.5 (SEQ ID NO: 42), LAG3.6 (SEQ ID NO: 43), LAG3.7 (SEQ ID NO: 44), and LAG3.8 (SEQ ID NO: 45) compared to the amino acid sequence of the CDR2 heavy chain variable region sequence of antibody 25F7 (LAG3.1) (SEQ ID NO: 41) and the corresponding human germline sequence (SEQ ID NO: 27). The CDR2 heavy chain variable region of LAG3.5 differs from the CDR2 heavy chain variable region of 25F7 by an arginine (R) at position 54 (relative to asparagine (N)) and a serine (S) at position 56 (relative to asparagine (N)). The remaining CDRs of LAG3.5 and 25F7 are identical. Figure 3 also includes SEQ ID NO: 40.

[0031] [Figure 4] Figures 4A and 4B are graphs showing the binding activity (EC50 and affinity, respectively) of antibodies LAG3.1 (25F7), LAG3.2, LAG3.5, LAG3.6, LAG3.7, and LAG3.8 to activated human CD4+ T cells. Figure 4B lists SEQ ID NOs: 41, 42, 45, 44, and 43, respectively, in order.

[0032] [Figure 5-1] Figures 5A, B, C, D and E are graphs showing the thermal melting curves (i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively. [Figure 5-2] Figures 5A, B, C, D and E are graphs showing the thermal melting curves (i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively.

[0033] [Figure 6-1] Figures 6A, B, C, D and E are graphs showing the thermoreversibility curves (i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively. [Figure 6-2] Figures 6A, B, C, D and E are graphs showing the thermoreversibility curves (i.e., thermal stability) of antibodies LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7 and LAG3.8, respectively.

[0034] [Figure 7] FIG. 7 is a graph showing the binding activity of antibodies LAG3.1 (25F7) and LAG3.5 to activated human CD4+ T cells and antigen binding (Biacore).

[0035] [Figure 8] Figure 8 shows the results of peptide mapping using mass spectrometry (chemical modification / molecular stability) for antibodies LAG3.1 (25F7) and LAG3.5 reflecting deamidation and isomerization after 5 days of incubation under the accelerated stress conditions described herein. Figure 8 lists SEQ ID NOS: 46-52, respectively, in order.

[0036] [Figure 9] FIG. 9 is a graph comparing the hydrophilicity profiles of antibodies LAG3.1 (25F7) and LAG3.5.

[0037] [Figure 10] Figures 10A, B, C and D are graphs comparing the affinity and physical stability (i.e., thermal and chemical stability) of antibodies LAG3.1 and LAG3.5 at 4°C and 40°C, i.e., under both accelerated stress conditions and in "real-time" stability studies, as described herein.

[0038] [Figure 11]11A and B are graphs comparing the percent modification of the amino acid sequences of antibodies LAG3.1 and LAG3.5 at 4°C and 40°C. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description of the Invention In order that this specification may be more readily understood, certain terms are first defined. Further definitions are provided in the detailed description.

[0040] The terms "25F7," "antibody 25F7," "antibody LAG3.1," and "LAG3.1" refer to the anti-human LAG-3 antibody described in US 2011 / 0150892 A1. The nucleotide sequence (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) encoding the heavy chain variable region of 25F7 (LAG3.1) are shown in Figure 1A (with CDR sequences represented as SEQ ID NOs: 4, 5, and 7, respectively). The nucleotide sequence (SEQ ID NO: 3) and corresponding amino acid sequence (SEQ ID NO: 4) encoding the light chain variable region of 25F7 (LAG3.1) are shown in Figure 1B (with CDR sequences represented as SEQ ID NOs: 8, 9, and 10, respectively).

[0041] 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 some cases, cross-react with LAG-3 protein from species other than human. In other embodiments, an antibody specific for human LAG-3 protein may be completely specific for human LAG-3 protein and may not exhibit species or other types of cross-reactivity, or may cross-react with LAG-3 from certain other species but not all other species (e.g., cross-react with monkey LAG-3 but not mouse LAG-3). The term "human LAG-3" refers to the human sequence LAG-3, e.g., the complete amino acid sequence of human LAG-3 having Genbank accession number NP_002277 (SEQ ID NO: 29). The term "mouse LAG-3" refers to the complete amino acid sequence of mouse sequence LAG-3, e.g., mouse LAG-3 having Genbank accession number NP_032505. LAG-3 is also known in the art, e.g., as CD223. A human LAG-3 sequence may differ from human LAG-3 of Genbank accession number NP_002277, e.g., by having conserved mutations or mutations in non-conserved regions, and the LAG-3 has substantially the same biological function as human LAG-3 of Genbank accession number 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.

[0042] The term "monkey LAG-3" is intended to include LAG-3 proteins expressed by Old World and New World monkeys, and is not limited to cynomolgus monkey LAG-3 and rhesus monkey LAG-3. An exemplary amino acid sequence for monkey LAG-3 is the rhesus monkey LAG-3 amino acid sequence, also deposited in Genbank under accession number XM_001108923. Another exemplary amino acid sequence for monkey LAG-3 is the alternative rhesus monkey sequence of clone pa23-5, described in US 2011 / 0150892 A1. This alternative rhesus monkey sequence exhibits a single amino acid difference at position 419 compared to the Genbank-deposited sequence.

[0043] A particular human LAG-3 sequence is generally at least 90% identical in amino acid sequence to the human LAG-3 sequence of Genbank Accession No. NP_002277 and contains amino acid residues that identify the amino acid sequence as human when compared with the LAG-3 amino acid sequence of another species (e.g., mouse). In some cases, the human LAG-3 may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the LAG-3 sequence of Genbank Accession No. NP_002277. In one embodiment, the human LAG-3 sequence may show up to 10 amino acid differences from the LAG-3 sequence of Genbank Accession No. NP_002277. In one embodiment, the human LAG-3 may show up to 5, or even up to 4, 3, 2, or 1 amino acid differences from the LAG-3 sequence of Genbank Accession No. NP_002277. Percent identity can be determined as described herein.

[0044] The term "immune response" refers to the activities of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by such cells or the liver that cause selective disruption of the destruction or removal from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0045] An "antigen-specific T cell response" refers to a response by a T cell resulting from stimulation of the T cell with an antigen that is specific for the T cell. Non-limiting examples of responses by T cells upon antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production).

[0046] As used herein, the term "antibody" includes whole antibodies or antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region consists of three domains: C H 1. C H 2 and C H Each light chain is composed of a light chain variable region (referred to herein as V L The light chain constant region consists of one domain, C L It consists of V H and V L The regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0047] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a LAG-3 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, V L , V H , C L and C H (ii) a F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H (iv) V H and C H Fv fragment consisting of one domain; (v) V of a single arm of an antibody L and V H (vi) Fv fragment consisting of V H dAb fragments consisting of domains (Ward et al. (1989) Nature 341 (vii) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, heavy chain variable regions comprising a single variable domain and two constant domains. Additionally, the two domains V of the Fv fragment L and V H are encoded by separate genes, they can be synthesized using recombinant methods to L and V H The domains can be linked by synthetic linkers that allow them to be produced as a single protein chain that pairs to form a monovalent molecule (also known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242 :423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85(See, e.g., J. Immunol. 1999:5879-5883.) Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0048] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to LAG-3 protein is substantially free of antibodies that specifically bind to antigens other than LAG-3 protein). However, an isolated antibody that specifically binds to human LAG-3 protein may have cross-reactivity with other antigens, such as LAG-3 protein, from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0049] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0050] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0051] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal fused to an immortalized cell, e.g., a transgenic mouse whose genome includes human heavy chain and light chain transgenes.

[0052] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal to human immunoglobulin genes or hybridomas prepared therefrom (described below); (b) antibodies isolated from host cells, e.g., transfectomas, transformed to express human antibodies; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in one embodiment, such recombinant human antibodies can be subjected to in vitro mutation (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutation), thus allowing the V and V sequences of the recombinant antibodies to be modified. H and V L The amino acid sequence of the region is similar to that of the human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0053] The term "isotype" refers to the antibody class (eg, IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0054] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen."

[0055] The term "human antibody derivatives" refers to any modified form of a human antibody, for example, a conjugate of the antibody and another drug or antibody.

[0056] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0057] The term "chimeric antibody" is intended to denote an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0058] As used herein, an antibody that "specifically binds to human LAG-3" is intended to refer to an antibody that binds to human LAG-3 protein (or LAG-3 proteins from one or more non-human species) but does not substantially bind to non-LAG-3 proteins. Preferably, the antibody has "high affinity," i.e., greater than 1×10 -7 M or less, more preferably 1×10 -8 M or less, more preferably 5 × 10 -9 M or less, more preferably 1×10 -9 K below M D It binds to human LAG-3 protein at

[0059] As used herein, the term "does not substantially bind" to a protein or cell means that the antibody does not bind to the protein or cell or does not bind with high affinity, i.e., binds to the protein or cell at a concentration of 1×10 -6 M or more, more preferably 1 × 10 -5 M or more, more preferably 1 × 10 -4M or more, more preferably 1 × 10 -3 M or more, preferably 1×10 -2 K over M D This means that the bond is formed at

[0060] As used herein, "K assoc " or "K a While the term "K" is intended to refer to the association rate of a particular antibody-antigen interaction, as used herein, dis " or "K d The term "K" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "K d Against K a The ratio of (i.e., K d / K a ) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. D A preferred method for determining is by surface plasmon resonance, preferably a biosensor system, e.g., Biacore (登録商標) By using the system.

[0061] The term "high affinity" for IgG antibodies refers to antibodies with a high affinity of 1 x 10 for the target antigen. -7 M or less, preferably 5×10 -8 M or less, more preferably 1×10 -8 M or less, more preferably 5 × 10 -9 M or less and more preferably 1×10 -9 K below M D However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is generally considered to be 10 -6 M or less, more preferably 10 -7 M or less, more preferably 10 -8 K below M D The antibody having the formula:

[0062] The term "deamidation" refers to a chemical degradation process that occurs spontaneously in proteins (e.g., antibodies). Deamidation removes the amide functional group from amino acid residues, such as asparagine and glutamine, thus damaging their amide-containing side chains. Specifically, the asparagine side chain attacks an adjacent peptide group, forming a symmetric succinimide intermediate. The symmetry of the intermediate results in two hydrolysis products: aspartic acid or isoaspartic acid. A similar reaction can also occur in the aspartic acid side chain, resulting in partial conversion to isoaspartic acid. In the case of glutamine, the rate of deamidation is generally 10-fold less than that of asparagine; however, the mechanism is essentially the same, requiring only a water molecule to proceed.

[0063] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, birds, amphibians, and reptiles, although mammals, e.g., non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

[0064] Various aspects of the invention are described in further detail in the following subsections.

[0065] Anti-LAG-3 antibodies with increased stability and advantageous functional properties The antibodies of the present invention specifically bind to human LAG-3 and have optimized stability compared to previously described anti-LAG-3 antibodies, particularly antibody 25F7 (LAG3.1), including reduced deamidation (e.g., increased chemical stability) and increased thermal refolding (e.g., increased physical stability), while maintaining high-affinity binding to human LAG-3.

[0066] Methods for identifying deamidation sites are known in the art (see, e.g., ion exchange, reverse phase, and hydrophobic interaction chromatography, and peptide mapping of proteolytic digests (LC-MS)). Suitable assays for measuring physical stability include, for example, melting point and / or analysis of refolding of antibody structure after denaturation (e.g., percent reversibility, as described, e.g., in Example 3, Section 3).

[0067] Binding to human LAG-3 can also be assessed using one or more techniques that are well established in the art. For example, antibodies can be tested by flow cytometry assays in which the antibody is reacted with a cell line expressing human LAG-3, such as CHO cells transfected to express LAG-3 on the cell surface (e.g., human LAG-3 or monkey LAG-3 (e.g., rhesus or cynomolgus monkeys) or mouse LAG-3). Other suitable cells for use in flow cytometry assays are anti-CD3-stimulated CD4 T cells expressing native LAG-3. + Additionally or alternatively, binding kinetics (e.g., K D The binding of antibodies containing LAG-3 (e.g., IgG ...

[0068] Preferably, the antibody of the invention is administered at a concentration of 1x10 -7 M or less, more preferably 1x10 -8 M or less, 5x10 -9 M or smaller, or 1x10 -9 K below M D It binds to human LAG-3 protein at

[0069] Generally, the antibody binds to LAG-3 in lymphoid tissues, such as tonsils, spleen or thymus, and can be detected by immunohistochemistry.In one embodiment, the antibody stains pituitary tissue (e.g., maintained in the pituitary gland) when measured by immunohistochemistry.In another embodiment, the antibody does not stain pituitary tissue (i.e., not maintained in the pituitary gland) when measured by immunohistochemistry.

[0070] Additional functional properties include cross-reactivity with LAG-3 from other species. For example, an antibody can bind to monkey LAG-3 (e.g., cynomolgus monkey, rhesus monkey) but not substantially bind to mouse-derived LAG-3. Preferably, the antibody of the present invention binds to human LAG-3 with high affinity.

[0071] Other functional properties include the ability of the antibody to stimulate an immune response, e.g., an antigen-specific T cell response. This can be tested, for example, by assessing the antibody's ability to stimulate interleukin-2 (IL-2) production in an antigen-specific T cell response. In one embodiment, the antibody binds to human LAG-3 and stimulates an antigen-specific T cell response. In another embodiment, the antibody binds to human LAG-3 but does not stimulate an antigen-specific T cell response. Other means for assessing the ability of an antibody to stimulate an immune response include testing the ability to inhibit tumor growth, e.g., in an in vivo tumor xenograft model (see, e.g., Example 6), or to stimulate an autoimmune response, e.g., to promote the development of autoimmune disease in an autoimmune model, e.g., to promote the development of diabetes in a NOD mouse model.

[0072] Preferred antibodies of the invention are human monoclonal antibodies. Additionally, or alternatively, the antibodies can be, for example, chimeric or humanized monoclonal antibodies.

[0073] Monoclonal antibody LAG3.5 A preferred antibody of the present invention is the human monoclonal antibody LAG3.5, which has been structurally and chemically characterized as described below and in the Examples. H The amino acid sequence is shown in SEQ ID NO: 12 (FIG. 2A). L The amino acid sequence is shown in SEQ ID NO: 14 (Figure 2B).

[0074] V of other anti-LAG-3 antibodies that bind to human LAG-3 H and V L The sequence (or CDR sequence) is V of the antibody LAG3.5 H and V L Preferably, V H and V L When chains (or CDRs within such chains) are mixed and matched, a particular V H / V L V from the pair H The sequence is structurally similar to V H Similarly, preferably, a particular V H / V L V from the pair L The sequence is structurally similar to V L Replace with an array.

[0075] Thus, in one embodiment, an antibody or antigen-binding portion thereof of the invention comprises: (a) a heavy chain variable region comprising the amino acid sequence SEQ ID NO:12 (i.e., the V H ); and (b) a light chain variable region comprising the amino acid sequence SEQ ID NO:14 (i.e., the V L ) or another anti-LAG3 antibody V L (i.e., different from LAG3.5); and specifically binds to human LAG-3.

[0076] In other embodiments, an antibody or antigen-binding portion thereof of the invention comprises: (a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region comprising the amino acid sequence SEQ ID NO: 12 (i.e., the CDR sequences of LAG3.5, SEQ ID NOs: 15, 16, and 17, respectively); and (b) the CDR1, CDR2 and CDR3 regions of the light chain variable region comprising the amino acid sequence SEQ ID NO: 14 (i.e., the CDR sequences of LAG3.5, SEQ ID NOs: 18, 19 and 20, respectively) or the CDRs of another anti-LAG3 antibody (i.e., different from LAG3.5); and specifically binds to human LAG-3.

[0077] In yet other embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain variable CDR2 region of LAG3.5 combined with the CDRs of another antibody that binds to human LAG-3, e.g., CDR1 and / or CDR3 from the heavy chain variable region, and / or CDR1, CDR2 and / or CDR3 from the light chain variable region, of a different anti-LAG-3 antibody.

[0078] In addition, it is well known in the art that the CDR3 domain alone, independent of the CDR1 and / or CDR2 domains, can determine the binding specificity of an antibody to a cognate antigen, and that multiple antibodies can be predictably produced with the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et al., British Journal of Cancer 83(2) :252-260 (2000); Beiboer et al., J. Mol. Biol. 296 :833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95 :8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116 :2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA 92 :2529-2533 (1995); Ditzel et al., J. Immunol. 157 :739-749 (1996); Berezov et al., BIAjournal8 :Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117 :452-7 (1995); Bourgeois et al., J. Virol 72 :807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90 :4374-8 (1993); Polymenis and Stoller, J. Immunol. 152 :5218-5329 (1994) and Xu and Davis, Immunity 13 :37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185, each of which is incorporated herein by reference in its entirety.

[0079] Thus, in other embodiments, the antibodies of the present invention comprise CDR2 of the heavy chain variable region of LAG3.5 and at least CDR3 of the heavy and / or light chain variable regions of LAG3.5 (SEQ ID NO: 17 and / or 20), or the heavy and / or light chain variable regions of another LAG-3 antibody, and are capable of specifically binding to human LAG-3. The antibodies preferably (a) compete for binding with LAG3.5; (b) maintain the functional properties of LAG3.5; (c) bind to the same epitope as LAG3.5; and / or (d) have a similar binding affinity to LAG3.5. In yet other embodiments, the antibodies may further comprise CDR2 of the light chain variable region of LAG3.5 (SEQ ID NO: 17 and / or 20), or the light chain variable region of another LAG-3 antibody, and are capable of specifically binding to human LAG-3. In other embodiments, the antibodies of the present invention may further comprise CDR1 of the heavy and / or light chain variable region of LAG3.5 (SEQ ID NO: 17 and / or 20), or CDR1 of the heavy and / or light chain variable region of another LAG-3 antibody, and can specifically bind to human LAG-3.

[0080] conservative modification In other embodiments, the antibodies of the invention comprise heavy and / or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences that differ from those of LAG3.5 by one or more conservative modifications. H Residues 54 and 56 of CDR2 remain as arginine and serine (i.e., are not mutated), respectively. It is understood in the art that certain conservative sequence modifications can be made without eliminating antigen binding. See, e.g., Brummell et al. (1993) Biochem. 32 :1180-8; de Wildt et al. (1997) Prot. Eng. 10 :835-41; Komissarov et al. (1997) J. Biol. Chem. 272 :26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32 :6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10 :341-6 and Beers et al. (2000) Clin. Can. Res. 6 :2835-43. Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising the CDR1, CDR2 and CDR3 sequences and / or a light chain variable region comprising the CDR1, CDR2 and CDR3 sequences: (a) the heavy chain variable region CDR1 sequence comprises SEQ ID NO:15, and / or conservative modifications thereof other than at positions 54 and 56; and / or (b) the heavy chain variable region CDR3 sequence comprises SEQ ID NO: 17, and conservative modifications thereof; and / or (c) the light chain variable region CDR1, and / or CDR2, and / or CDR3 sequences comprise SEQ ID NO:18, and / or SEQ ID NO:19, and / or SEQ ID NO:20, and / or conservative modifications thereof; and (d) The antibody specifically binds to human LAG-3.

[0081] Additionally or alternatively, the antibody may have one or more of the following functional properties described above, e.g., high affinity binding to human LAG-3, binding to monkey LAG-3, non-binding to mouse LAG-3, ability to inhibit binding of LAG-3 to MHC class II molecules and / or ability to stimulate antigen-specific T cell responses.

[0082] In various embodiments, the antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0083] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues within the CDR regions of an antibody of the invention can be substituted with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., the above-mentioned functions) using the functional assays described herein.

[0084] Engineered and modified antibodies The antibodies of the present invention may be prepared by using one or more VL2 fragments of LAG3.5 as starting materials to engineer modified antibodies. H and / or V L The antibody can be prepared using an antibody having one or both variable regions (i.e., V H and / or V L), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant region(s), for example, to alter the effector functions of the antibody.

[0085] In one embodiment, CDR grafting can be used to engineer the variable region of an antibody. Antibodies interact with target antigens primarily through amino acid residues present in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs vary more between individual antibodies than sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing an expression vector containing the CDR sequences of that particular natural antibody grafted onto the framework sequences of a different antibody with different properties (see, for example, Riechmann et al. (1998) Nature 106:101-102). 332 :323-327; Jones et al. (1986) Nature 321 :522-525; Queen et al. (1989) Proc. Natl. Acad. See. USA 86 :10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0086] Accordingly, another aspect of the present invention relates to isolated monoclonal antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 15, 16, 17, respectively, and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 18, 19, 20, respectively (i.e., the CDRs of LAG3.5). These antibodies are similar to the V and V sequences of monoclonal antibody LAG3.5. H and V L It comprises the CDR sequences of but may comprise different framework sequences.

[0087] Such framework sequences can be obtained from public DNA databases or published literature containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat et al. (1991), cited supra; Tomlinson et al. (1992) "The Repertoire of Human Germline V H Sequence Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops” J. Mol. Biol. 227 :776-798; and Cox et al. (1994) “A Directory of Human Germ-line V H Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol. 24:827-836, the contents of each of which are incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available at Genbank Accession Numbers: 1-69 (NG_0010109, NT_024637 & BC070333), 3-33 (NG_0010109 & NT_024637), and 3-7 (NG_0010109 & NT_024637). As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available at Genbank Accession Numbers: 1-69 (NG_0010109, NT_024637 & BC070333), 5-51 (NG_0010109 & NT_024637), 4-34 (NG_0010109 & NT_024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0088] The antibody protein sequence is compared to compiled protein sequence databases using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al. (1997), supra).

[0089] Preferred framework sequences for use in the antibodies of the invention are those that are structurally similar to the framework sequences used by the selected antibodies of the invention, e.g., the V and V sequences used by the preferred monoclonal antibodies of the invention. H 4-34 framework sequence and / or V K It is similar to the L6 framework sequence. H and the CDR1, CDR2 and CDR3 sequences of V KThe CDR1, CDR2, and CDR3 sequences of the above can be grafted onto framework regions having the same sequences as those found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions containing one or more mutations compared to the germline sequences. For example, in some cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370).

[0090] Another type of variable region modification is V H and / or V L Mutations are made in amino acid residues in the CDR1, CDR2, and / or CDR3 regions of an antibody of interest, thereby improving one or more binding characteristics (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effect of antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays described herein and in the Examples. Preferably, conservative modifications (described above) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically up to 1, 2, 3, 4, or 5 residues within the CDR regions are altered.

[0091] Thus, in another aspect, the present invention provides a VV comprising an amino acid sequence of SEQ ID NO:15, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:15. H (b) a V comprising an amino acid sequence of SEQ ID NO: 16, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 16; H (preferably, positions 54 and 56 are the same as SEQ ID NO: 16); (c) a V comprising an amino acid sequence of SEQ ID NO: 17, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 17. H(d) a heavy chain variable region comprising the CDR3 region of SEQ ID NO: 18, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 18; L (e) a V comprising an amino acid sequence of SEQ ID NO: 19, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO: 19; L and (f) a V comprising an amino acid sequence of SEQ ID NO:20, or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:20. L The present invention provides an isolated anti-LAG-3 monoclonal antibody, or an antigen-binding portion thereof, comprising the CDR3 region of

[0092] The engineered antibodies of the invention may be modified, e.g., to improve the properties of the antibody. H and / or V L These framework modifications include those made to framework residues within the framework region. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More particularly, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequence from which the antibody is derived.

[0093] Another type of framework modification involves mutating one or more residues within the framework regions, and even within one or more CDR regions, to remove T-cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0094] In addition to, or as an alternative to, modifications made within the framework or CDR regions, antibodies of the invention can be engineered to contain modifications within the Fc region, generally to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0095] In a preferred embodiment, the antibody is an IgG4 isotype antibody containing a serine to proline mutation at a position corresponding to position 228 in the hinge region of the heavy chain constant region (S228P; EU index). This mutation has been reported to disrupt the heterogeneity of disulfide bridges in the heavy chain in the hinge region (Angal et al., supra; position 241 is based on the Kabat numbering system).

[0096] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased.This approach is further described in U.S. Patent No. 5,677,425.The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light chain and the heavy chain, or to enhance or reduce the stability of the antibody.

[0097] In another embodiment, the Fc-hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has impaired Staphylococcus aureus protein A (SpA) binding compared to the native Fc-hinge domain SpA binding. This approach is described in more detail in U.S. Patent No. 6,165,745.

[0098] In other embodiments, antibodies are modified to extend their biological half-life. Various approaches are possible. For example, as described in U.S. Patent No. 6,277,375, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, to extend biological half-life, antibodies can be modified within the CH1 or CL regions to include salvage receptor binding epitopes taken from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022.

[0099] In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue so that the antibody has altered affinity for an effector ligand while maintaining the antigen binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0100] In other embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551.

[0101] In other embodiments, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described in further detail in PCT Publication WO 94 / 29351.

[0102] In yet other embodiments, the Fc region may comprise any of the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 352, 354, 355, 356, 358, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289 The modifications are made by modifying one or more amino acids at positions 94, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is described in further detail in PCT Publication WO 00 / 42072. Furthermore, the binding sites for FcγR1, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped and mutants with improved binding have been described (Shields et al. (2001) J. Biol. Chem. 276 :6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutations have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0103] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to enhance the affinity of the antibody for its antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can enhance the affinity of the antibody for its antigen. See, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861.

[0104] Additionally or alternatively, antibodies can be produced with altered glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), and antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose in the carbohydrate. Ms704, Ms705, and Ms709 FUT8 - / - The cell line was created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (US Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line in which the FUT8 gene encoding fucosyltransferase has been functionally disrupted, such that antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α-1,6 linkage-associated enzymes. EP 1,176,195 also describes a cell line, e.g., rat myeloma cell line YB2 / 0 (ATCC CRL 1662), that has low enzymatic activity for the addition of fucose to N-acetylglucosamine, which binds to the Fc region of antibodies or has no enzymatic activity. PCT Publication WO 03 / 035835 describes a mutant CHO cell line, Lecl3 cells, that reduces the ability of fucose to bind to Asn(297)-linked carbohydrates and also causes hypofucosylation of antibodies expressed in the host cells (Shields et al. (2002) J. Biol. Chem. 277 (See also Umana et al. (1999) Nat. Biotech. 26733-26740.) Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, e.g., Lemna. Methods for producing antibodies in plant systems are described in U.S. patent application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. PCT Publication WO 99 / 54342 describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increase in bisecting GlcNac structures, which results in enhanced ADCC activity of the antibody (Umana et al. (1999) Nat. Biotech. 26733-26740). Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, e.g., Lemna. Methods for producing antibodies in plant systems are described in U.S. patent application corresponding to Alston & Bird LLP Attorney Docket No. 040989 / 314911, filed August 11, 2006. PCT Publication WO 99 / 54342 describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increase in bisecting GlcNac structures, which results in enhanced ADCC activity of the antibody (Umana 17 :176-180). Alternatively, a fucosidase enzyme can be used to cleave the fucose residues of the antibody; for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem.14 :5516-23).

[0105] A further modification of the antibodies herein contemplated by the present invention is pegylation. Antibodies can be pegylated, for example, to extend the antibody's biological (e.g., serum) half-life. To pegylate an antibody, the antibody or fragment thereof is generally reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG moieties being attached to the antibody or antibody fragment. Preferably, pegylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass all forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In one embodiment, the antibody to be pegylated is an unglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention. See, e.g., EP 0154316 and EP 0401384.

[0106] Antibody physical properties The antibodies of the present invention can be characterized by various physical properties in order to detect and / or distinguish between different classes.

[0107] For example, an antibody can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites can result in increased immunogenicity of the antibody or alteration of the antibody's pK resulting in altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41 :673-702; Gala and Morrison (2004) J Immunol 172 :5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12 :43R-56R; Parekh et al. (1985) Nature 316 :452-7; Mimura et al. (2000) Mol Immunol 37 :697-706). Glycosylation is known to occur at motifs containing NXS / T sequences. In some cases, it is preferable to have an anti-LAG-3 antibody that does not contain variable region glycosylation. This can be achieved either by selecting an antibody that does not contain glycosylation motifs in the variable region or by mutating residues within the glycosylated region.

[0108] In preferred embodiments, the antibody does not contain an asparagine isomerism site. Deamidation of asparagine can occur at NG or DG sequences, resulting in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and reduces stability (the isoaspartic acid effect).

[0109] Each antibody has a unique isoelectric point (pI), generally within the pH range of 6 to 9.5. The pI for IgG1 antibodies is generally within the pH range of 7-9.5, and the pI for IgG4 antibodies is generally within the pH range of 6-8. It is suspected that antibodies with pIs outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-LAG-3 antibody with a pI value within the normal range. This can be achieved either by selecting an antibody with a pI within the normal range or by mutating charged surface residues.

[0110] Nucleic acid molecules encoding antibodies of the invention In another aspect, the present invention provides nucleic acid molecules encoding the heavy and / or light chain variable regions or CDRs of the antibodies of the present invention. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkali / SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis, and other techniques known in the art. See Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0111] Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

[0112] A preferred nucleic acid molecule of the invention is the V H and V L sequences (SEQ ID NOs: 12 and 14, respectively) or those encoding the CDRs. H and V LOnce DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V H The DNA fragment encoding is operably linked to another DNA fragment encoding another protein, for example, an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are ligated so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0113] V H The isolated DNA encoding the region is V H A full-length heavy chain gene can be obtained by operably linking the DNA encoding the heavy chain constant region (CH1, CH2, and CH3) to other DNA molecules encoding the heavy chain constant regions. The sequences of human heavy chain constant region genes are known in the art (e.g., Kabat et al. (1991), supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is more preferably an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0114] V L The isolated DNA encoding the region is V LThe DNA encoding the light chain constant region CL can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking it to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (e.g., Kabat et al., supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0115] To create the scFv gene, H and V L The DNA fragments encoding V are linked by a flexible linker. L and V H V with region H and V L The sequence is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 28), so that the sequence can be expressed as a contiguous single-chain protein (see, e.g., Bird et al. (1988) Science 242 :423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883; McCafferty et al. (1990) Nature 348 :552-554, see).

[0116] Production of monoclonal antibodies of the present invention The monoclonal antibodies (mAbs) of the present invention are prepared according to the method of Kohler and Milstein (1975) Nature 256Monoclonal antibodies can be produced using standard somatic cell hybridization (hybridoma) techniques, such as those described in U.S. Pat. No. 4,816,567; U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,762; and U.S. Pat. No. 6,180,370, the contents of which are expressly incorporated herein by reference in their entirety.

[0117] In a preferred embodiment, the antibodies of the present invention are human monoclonal antibodies. Such human monoclonal antibodies against human LAG-3 can be produced using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic or transchromosomal mice are each referred to herein as HuMAb mice. (登録商標) and KM mice (登録商標) These include mice called "human Ig mice," which are collectively referred to herein as "human Ig mice."

[0118] HuMAb mice (登録商標) (Medarex (登録商標) , Inc.) contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonals (Lonberg et al. (1994), supra; Lonberg (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13 : 65-93 and Harding and Lonberg (1995) Ann. NY Acad. Sci. 764 :536-546). HuMAb mice (登録商標) The preparation and use of the mouse and the genomic modifications carried by such mice are further described in Taylor et al. (1992) Nucleic Acids Research 20 :6287-6295; Chen et al. (1993) International Immunology 5 : 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90 :3720-3724; Choi et al. (1993) Nature Genetics 4 :117-123; Chen et al. (1993) EMBO J. 12 : 821-830; Tuaillon et al. (1994) J. Immunol. 152 :2912-2920; Taylor et al. (1994) International Immunology 6 : 579-591; and Fishwild et al. (1996) Nature Biotechnology 14 : 845-851, the contents of all of which are expressly incorporated herein by reference in their entireties. Further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807; PCT Publication Nos. WO 92 / 03918; WO 93 / 12227; WO 94 / 25585; WO 97 / 13852; WO 98 / 24884; WO 99 / 45962 and WO 01 / 14424, the contents of which are expressly incorporated herein by reference in their entireties.

[0119] In other embodiments, human antibodies of the invention can be prepared using mice harboring human immunoglobulin sequences on transgenes and transchromosomes, e.g., mice harboring a human heavy chain transgene and a human light chain transchromosome. (登録商標) This mouse, referred to herein as the "KM / FCGR2D mouse," is described in detail in PCT Publication WO 02 / 43478. A modified form of this mouse, which further comprises a homozygous disruption of the endogenous FcγRIIB receptor gene, is also described in PCT Publication WO 02 / 43478 and is referred to as the "KM / FCGR2D mouse." (登録商標) Additionally, mice carrying either the HCo7 or HCo12 heavy chain transgene, or both, can be used.

[0120] Additional transgenic animal embodiments include Xenomouse (Abgenix, Inc., U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963). Further embodiments include "TC mice" (Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97 :722-727) and cattle carrying human heavy and light chain transchromosomes (Kuroiwa et al. (2002) Nature Biotechnology 20 :889-894; PCT Publication WO 02 / 092812), the contents of these patents and publications are expressly incorporated herein in their entireties by reference.

[0121] In one embodiment, human monoclonal antibodies of the invention are prepared using phage display methods for screening libraries of human immunoglobulin genes. See, e.g., U.S. Patent Nos. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081, the contents of which are incorporated herein by reference in their entireties.

[0122] Human monoclonal antibodies of the invention can also be prepared using SCID mice reconstituted with human immune cells so that a human antibody response can be produced upon immunization (see, e.g., U.S. Patent Nos. 5,476,996 and 5,698,767, the contents of which are incorporated herein by reference in their entireties).

[0123] In other embodiments, human anti-LAG-3 antibodies are prepared using phage display, where the phage contains nucleic acids encoding antibodies produced in transgenic animals pre-immunized with LAG-3. In preferred embodiments, the transgenic animals are HuMab, KM, or Kirin mice. See, e.g., U.S. Patent No. 6,794,132, the contents of which are incorporated herein by reference in their entirety.

[0124] Immunization of human Ig mice In one embodiment of the present invention, human Ig mice are immunized with a purified or enriched preparation of LAG-3 antigen, recombinant LAG-3 protein, or cells expressing LAG-3 protein. See, e.g., Lonberg et al. (1994), supra; Fishwild et al. (1996), supra; PCT Publication Nos. WO 98 / 24884 or WO 01 / 14424, the contents of which are incorporated herein by reference in their entireties. In a preferred embodiment, 6-16 week old mice are immunized with 5-50 μg of LAG-3 protein. Alternatively, a portion of LAG-3 fused to a non-LAG-3 polypeptide is used.

[0125] In one embodiment, transgenic mice are immunized intraperitoneally (IP) or intravenously (IV) with LAG-3 antigen in complete Freund's adjuvant, followed by IP or IV immunization with antigen in incomplete Freund's adjuvant. In another embodiment, whole cells are used in the absence of adjuvant other than Freund's. Plasma is screened by ELISA, and cells from mice with sufficient titers of anti-LAG-3 human immunoglobulin can be used for fusion.

[0126] Preparation of hybridomas producing human monoclonal antibodies of the present invention To prepare hybridomas producing the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells can be isolated from immunized mice and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. Hybridoma production is known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0127] Preparation of transfectomas producing the monoclonal antibodies of the present invention The antibodies of the present invention can also be prepared in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods known in the art (see, e.g., Morrison, S. (1985) Science 229 :1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0128] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Preferred regulatory sequences for mammalian host cell expression include promoters and / or enhancers derived from viral elements that direct high-level protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and papilloma. Alternatively, non-viral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements include sequences from different sources, including the SV40 early promoter and sequences from the long terminal repeat of human T-cell leukemia virus type 1, e.g., the SRα promoter system (Takebe et al. (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are chosen to be compatible with the expression host cell used.

[0129] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. H The segment is C in the vector H operatively connected to the V segment; L The segment is C in the vector L The variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding heavy and light chain constant regions of the desired isotype so that they are operably linked to the segments. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0130] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been inserted (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, selectable marker genes typically confer resistance to drugs, such as G418, hygromycin, or methotrexate, on host cells into which the vector has been inserted. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0131] For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, more preferably mammalian host cells, is highly preferred, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies.

[0132] Preferred mammalian host cells for expressing the recombinant antibodies of the invention are Chinese hamster ovary cells (CHO cells) (see, e.g., RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159 :601-621, used with a DHFR selectable marker as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77 : dhfr listed in 4216-4220 - These include CHO cells), NSO myeloma cells, COS cells, and SP2 cells. Particularly for use with NSO myeloma cells, a further preferred expression system is the GS gene expression system described in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient time to allow expression of the antibody in the host cells, or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0133] immune complex The antibodies of the present invention can be conjugated with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 39), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO02 / 096910; WO07 / 038658; WO07 / 051081; WO07 / 059404; WO08 / 083312; and WO08 / 103693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295, all of which are incorporated herein by reference.

[0134] bispecific molecules In another aspect, the present invention features bispecific molecules comprising one or more antibodies of the present invention linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), producing a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities. In a preferred embodiment, the bispecific molecule comprises a first binding specificity for LAG-3 and a second binding specificity for a trigger molecule that recruits cytotoxic effector cells capable of killing target cells expressing LAG-3. Examples of suitable trigger molecules are CD64, CD89, CD16, and CD3. See, e.g., Kufer et al., TRENDS in Biotechnology, 22 (5), 238-244 (2004).

[0135] In one embodiment, the bispecific molecule has a third specificity in addition to the anti-Fc binding specificity and the anti-LAG-3 binding specificity. The third specificity can be for an anti-enhancement factor (EF), e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. For example, an anti-enhancement factor can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cells and increase the immune response against the target cell.

[0136] Bispecific molecules can come in many different formats and sizes. At one end of the size spectrum, bispecific molecules maintain the traditional antibody format, except that instead of having two binding arms with the same specificity, they have two binding arms with different specificities. At the other end are bispecific molecules consisting of two single-chain antibody fragments (scFvs) linked by a peptide chain, the so-called Bs(scFv)2 construct. Intermediate-sized bispecific molecules contain two different F(ab) fragments linked by a peptidyl linker. These and other bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Kufer et al., supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), as described herein.

[0137] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition comprising one or more formulated antibodies of the present invention together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention can also be administered in combination with, for example, other immunostimulants, anti-cancer agents, anti-viral agents, or vaccines, so that the anti-LAG-3 antibody enhances the immune response to the vaccine.

[0138] Pharmaceutical compositions can contain any excipient. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003) (this description is incorporated herein by reference).

[0139] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion. Alternatively, the antibodies of the present invention may be administered by non-parenteral routes, such as topical, epidermal, or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0140] The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound but does not impart undesired toxic effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like, as well as non-toxic organic acids, such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0141] The pharmaceutical compositions can be in the form of a sterile aqueous solution or dispersion. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0142] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the particular mode of administration, and is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount ranges from about 0.01% to about 99% of the active ingredient, combined with a pharmaceutically acceptable carrier, preferably from about 0.1% to about 70%, more preferably from about 1% to about 30%.

[0143] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, it may be administered as a single bolus, as several divided doses over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form, particularly for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dose for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the necessary pharmaceutical carrier. Alternatively, when less frequent administration is required, the antibody can be administered as a sustained-release formulation.

[0144] For antibody administration, dosages range from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, of the host's body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg, or within the range of 1-10 mg / kg. Typical treatment regimens involve administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once per three months, or once per three to six months. A preferred administration regimen for the anti-LAG-3 antibody of the present invention involves intravenous administration of 1 mg / kg or 3 mg / kg body weight, with the antibody given using one of the following dosing schedules: (i) six doses every four weeks, then every three months; (ii) every three weeks; or (iii) one dose of 3 mg / kg body weight, then 1 mg / kg body weight every three weeks. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods about 25-300 μg / ml.

[0145] A "therapeutically effective amount" of an anti-LAG-3 antibody of the invention is preferably one that results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of disease symptoms, or prevention of a defect or disability resulting from the affliction of the disease. For example, with respect to treating a subject with a tumor, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, more preferably at least about 60%, and even more preferably at least about 80%, compared to an untreated subject. A therapeutically effective amount of a therapeutic compound can reduce tumor size or ameliorate symptoms in a subject, typically a human or other mammal.

[0146] Pharmaceutical compositions can be controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0147] Therapeutic compositions can be administered via medical devices, such as: (1) needleless hypodermic injection devices (e.g., US Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (US Pat. No. 4,487,603); (3) transdermal devices (US Pat. No. 4,486,194); (4) infusion devices (US Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (US Pat. Nos. 4,439,196 and 4,475,196); which are incorporated herein by reference.

[0148] In one embodiment, the human monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic compounds of the present invention cross the blood-brain barrier, they can be formulated in liposomes, which may further contain targeting molecules to enhance selective delivery to specific cells or organs. See, e.g., US Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29 :685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153 :1038; Bloeman et al. (1995) FEBS Lett. 357 :140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39 :180; Briscoe et al. (1995) Am. J. Physiol. 1233 :134; Schreier et al. (1994) J. Biol. Chem. 269 :9090; Keinanen and Laukkanen (1994) FEBS Lett. 346 :123; and Killion and Fidler (1994) Immunomethods 4 :273, see.

[0149] Uses and Methods of the Invention The antibodies (compositions, bispecifics, and immunoconjugates) of the present invention have numerous in vitro and in vivo utilities, for example, for detecting LAG-3 or enhancing immune responses by blocking LAG-3. In a preferred embodiment, the antibodies are human. Such antibodies can be administered to cells in culture in vitro or ex vivo, or to human subjects in vivo, for example, to enhance immunity in various conditions. Thus, in one aspect, the present invention provides a method for modifying an immune response in a subject, comprising administering an antibody of the present invention, or an antigen-binding portion thereof, to the subject, such that the immune response in the subject is modified. Preferably, the response is enhanced, stimulated, or upregulated.

[0150] Preferred subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients with disorders that can be treated by increasing the immune response (e.g., T cell-mediated immune response). In certain embodiments, the method is particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-LAG-3 antibody is administered together with the antigen of interest, or the antigen is already present in the subject to be treated (e.g., a subject with a tumor or a virus). When an antibody against LAG-3 is administered together with another agent, the two can be administered either sequentially or simultaneously.

[0151] The present invention further provides a method for detecting the presence or amount of human LAG-3 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody, or antigen-binding portion thereof, that specifically binds to human LAG-3 under conditions that allow the formation of a complex between the antibody or portion thereof and human LAG-3. The formation of the complex is then detected (a difference in complex formation between the sample and the control sample indicates the presence of human LAG-3 antigen in the sample). Furthermore, the anti-LAG-3 antibodies of the present invention can be used to purify human LAG-3 via immunoaffinity purification.

[0152] Considering the ability of the anti-LAG-3 antibodies of the present invention to inhibit the binding of LAG-3 to MHC class II molecules and stimulate antigen-specific T cell responses, the present invention also provides methods of using the antibodies of the present invention in vitro or in vivo to stimulate, enhance, or upregulate antigen-specific T cell responses. For example, the present invention provides a method of stimulating an antigen-specific T cell response, comprising contacting T cells with the antibodies of the present invention so that the antigen-specific T cell response is stimulated. Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 production by antigen-specific T cells is stimulated.

[0153] The present invention also provides a method for stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering an antibody of the present invention to the subject, such that an immune response (e.g., an antigen-specific T cell response) in the subject is stimulated. In a preferred embodiment, the subject has a tumor, and an immune response against the tumor is stimulated. In another preferred embodiment, the subject has a virus, and an immune response against the virus is stimulated.

[0154] In another aspect, the invention provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody of the invention, such that tumor growth is inhibited in the subject. In yet another aspect, the invention provides a method for treating a viral infection in a subject, comprising administering to the subject an antibody of the invention, such that the viral infection is treated in the subject.

[0155] These and other methods of the present invention are described in further detail below.

[0156] cancer Blocking LAG-3 with antibodies can enhance the immune response against cancer cells in patients. In one aspect, the present invention relates to the in vivo treatment of a subject using an anti-LAG-3 antibody to inhibit the growth of cancerous tumors. The anti-LAG-3 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-LAG-3 antibody can be used in conjunction with other immunogens, standard cancer treatments, or other antibodies, as described below.

[0157] Thus, in one embodiment, the present invention provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of an anti-LAG-3 antibody, or an antigen-binding portion thereof. Preferably, the antibody is a human anti-LAG-3 antibody (such as any of the human anti-human LAG-3 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized anti-LAG-3 antibody.

[0158] Preferred cancers whose growth can be inhibited using the antibodies of the present invention include cancers that generally respond to immunotherapy. Non-limiting examples of cancers that are preferred for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-resistant prostate cancer), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). In addition, the present invention includes refractory or recurrent malignant tumors whose growth can be inhibited using the antibodies of the present invention.

[0159] Examples of other cancers that can be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, acute myeloid leukemia, chronic myeloid leukemia, and thyroid cancer. The present invention also includes cancers that are induced by PD-L1, such as chronic or acute leukemias including myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers including asbestos-induced cancers, and combinations of the foregoing cancers. The present invention is also useful for treating metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17 :133-144).

[0160] Optionally, antibodies against LAG-3 can be produced by immunogens, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004) J. Immunol. 173 :4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (described further below).

[0161] In humans, some tumors, such as melanoma, have been shown to be immunogenic, and increasing the threshold for T cell activation by blocking LAG-3 can activate tumor responses in the host.

[0162] LAG-3 blockade may be more effective when combined with vaccine protocols. Many experimental strategies for tumor vaccination have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; also see Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be even more effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci USA 90 : 3539-43).

[0163] Studies of gene expression and large-scale gene expression patterns in various tumors have defined so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and the cells from which they arise, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. LAG-3 blockade can be used in conjunction with collections of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are usually viewed by the immune system as self-antigens and are therefore tolerant to them. Tumor antigens can include the protein telomerase, which is required for the synthesis of chromosomal telomeres and is expressed in over 85% of human cancers and in very limited somatic tissues (Kim et al. (1994) Science 266 (These somatic tissues can be protected from immune attack by various means.) Tumor antigens can also be "neoantigens," or idiotypes derived from B-cell tumors, expressed in cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome).

[0164] Other tumor vaccines may contain proteins derived from viruses implicated in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with LAG-3 blockade is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are highly efficient in delivery to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269 :1585-1588; Tamura et al. (1997) Science 278 :117-120).

[0165] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to mount antigen-specific responses. DCs can be produced ex vivo and loaded with a variety of protein and peptide antigens and tumor cell extracts (Nestle et al. (1998) Nature Medicine 4 : 328-332). DCs can also be transduced by genetic means to express these tumor antigens as well. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6 As a vaccination method, DC immunization can be effectively combined with LAG-3 blockade to activate even more potent anti-tumor responses.

[0166] LAG-3 blockade can also be combined with standard cancer treatments. LAG-3 blockade can be effectively combined with chemotherapy regimens. In these cases, it may be possible to reduce the dose of chemotherapy administered (Mokyr et al. (1998) Cancer Research 58 : 5301-5304). An example of such a combination is an anti-LAG-3 antibody combined with dacarbazine for the treatment of melanoma. Another example of such a combination is an anti-LAG-3 antibody combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale supporting the combined use of LAG-3 blockade and chemotherapy is that cell death, a result of the cytotoxic effects of many chemotherapy compounds, should result in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with LAG-3 blockade through cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with LAG-3 blockade. Inhibition of angiogenesis can induce tumor cell death and present tumor antigens to the host's antigen presentation pathway.

[0167] Antibodies that block LAG-3 can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. These responses of T cell populations can be increased by using LAG-3 blockade. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0168] Tumors evade host immune surveillance by a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163 : 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13 : 198-200) and Fas ligand (Hahne et al. (1996) Science 274 Antibodies to each of these entities can be used in combination with anti-LAG-3 to neutralize the effects of immunosuppressants and support a host-mediated tumor immune response.

[0169] Other antibodies that activate host immune responsiveness can be used in combination with anti-LAG-3. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with LAG-3 antibodies (Ito et al. (2000) Immunobiology 201(5) 527-40). For example, CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164 : 2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3 : 682-685 (1997) and ICOS (Hutloff et al. (1999) Nature 397 Activating antibodies against T cell costimulatory molecules, such as IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG8, IgG9, IgG1, IgG1, IgG4, IgG1, IgG4, IgG5, IgG6, IgG7, IgG8, IgG8, IgG1, IgG1, IgG4 ...

[0170] Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Although graft-versus-host disease is a consequence of this treatment, therapeutic benefits can be obtained from the graft-versus-tumor response. LAG-3 blockade can be used to increase the effectiveness of donor-transplanted tumor-specific T cells.

[0171] To stimulate antigen-specific T cells against tumors, there are also several experimental treatment protocols that involve the activation and expansion of antigen-specific T cells ex vivo and the adoptive transfer of these cells into recipients (Greenberg & Riddell (1999) Science 285 These methods can also be used to activate T cell responses to infectious agents, such as CMV. Ex vivo activation in the presence of anti-LAG-3 antibodies can increase the frequency and activity of matched transferred T cells.

[0172] infectious disease Another method of the present invention is used to treat patients who have been exposed to a particular toxin or pathogen. Accordingly, another aspect of the present invention provides a method of treating an infectious disease in a subject, comprising administering an anti-LAG-3 antibody, or an antigen-binding portion thereof, to the subject, such that the subject is treated for the infectious disease. Preferably, the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized antibody.

[0173] Similar to the tumor application described above, antibody-mediated LAG-3 blockade can be used alone or as an adjuvant, in combination with vaccines, to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which there is currently no effective vaccine or for which conventional vaccines are not sufficiently effective. These include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. LAG-3 blockade is particularly useful against established infections caused by infectious agents, such as HIV, whose antigens change over the course of infection. These novel epitopes are recognized as foreign upon administration of anti-human LAG-3, thus stimulating a strong T cell response that is not suppressed by negative signaling via LAG-3.

[0174] Some examples of pathogenic viruses that cause infections treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, polyvirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0175] Some examples of pathogenic bacteria that cause infections treatable by the methods of the present invention include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, anthrax, plague, leptospira, and lyme burgdorferi.

[0176] Some examples of pathogenic fungi that cause infections treatable by the methods of the present invention include Candida (e.g., Candida albicans, Krusei, Glabrata, Tropicalis), Cryptococcus neoformans, Aspergillus (e.g., Fumigatus, Niger), Mucor (e.g., Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0177] Some examples of pathogenic parasites that cause infections treatable by the methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lamblia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia murine, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0178] In all of the above methods, LAG-3 blockade can be combined with other forms of immunotherapy, such as cytokine treatment (e.g., interferon, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy that provides enhanced presentation of tumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2 :1121-1123, see).

[0179] Autoimmune reactions Anti-LAG-3 antibodies can induce and amplify autoimmune responses. Indeed, induction of anti-tumor responses using tumor cell and peptide vaccines indicates that many anti-tumor responses are associated with anti-self reactivity (van Elsas et al. (2001) J. Exp. Med. 194 :481-489; Overwijk et al. (1999) Proc. Natl. Acad. Sci. USA 96 : 2982-2987; Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, it is possible to consider using anti-LAG-3 blockade together with various self-proteins to devise vaccine protocols to efficiently generate immune responses against these self-proteins for disease treatment. For example, Alzheimer's disease is associated with the inappropriate accumulation of Aβ peptides in amyloid deposits in the brain; antibody responses against amyloid can reveal these amyloid deposits (Schenk et al., (1999) Nature 400 : 173-177).

[0180] Other self-proteins, such as IgE for the treatment of allergies and asthma and TNFα for rheumatoid arthritis, can also be used as targets. Finally, antibody responses to various hormones can be induced by the use of anti-LAG-3 antibodies. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors required for the growth of certain tumors can also be considered as possible vaccine targets.

[0181] Similar methods described above for the use of anti-LAG-3 antibodies can be used to induce therapeutic autoimmune responses to treat patients with other self-antigens, such as amyloid deposits including Aβ in Alzheimer's disease, and inappropriate accumulation of cytokines such as TNFα and IgE.

[0182] vaccine Anti-LAG-3 antibodies can be used to stimulate antigen-specific immune responses by co-administering the anti-LAG-3 antibody with an antigen of interest (e.g., a vaccine). Thus, in another aspect, the present invention provides a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) the antigen; and (ii) an anti-LAG-3 antibody, or an antigen-binding portion thereof, such that the immune response to the antigen in the subject is enhanced. Preferably, the antibody is a human anti-human LAG-3 antibody (such as any of the human anti-LAG-3 antibodies described herein). Additionally or alternatively, the antibody can be a chimeric or humanized antibody. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) or antigens derived from viruses, bacteria, or other pathogens.

[0183] Suitable routes of administration of the antibody compositions of the invention (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) in vivo and in vitro are known in the art and can be selected by one of ordinary skill in the art. For example, antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dose of molecule used depends on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0184] As described above, the human anti-LAG-3 antibodies of the present invention can be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressants. The antibody can be combined with the drug (as an immunoconjugate) or administered separately from the drug. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the drug, and can also be co-administered with other known treatments, such as anti-cancer therapies, such as radiation therapy. Such therapeutic agents include, among others, anti-tumor agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea (which are themselves only effective at levels that are toxic or subtoxic to patients). Cisplatin is administered intravenously at a dose of 100 mg / ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg / ml once every 21 days. Co-administration of the human anti-LAG-3 antibody or antigen-binding fragment thereof of the present invention with a chemotherapeutic agent provides two anti-cancer agents that function through different mechanisms to exert cytotoxic effects on human tumor cells. Such co-administration can overcome problems associated with the development of drug resistance or changes in the antigenicity of tumor cells, rendering them unresponsive to the antibody.

[0185] Also within the scope of the present invention are kits comprising an antibody composition of the invention (e.g., a human antibody, bispecific or multispecific molecule, or immunoconjugate) and instructions for use. The kit can further comprise at least one additional reagent or one or more additional human antibodies of the invention (e.g., a human antibody with complementary activity that binds to a different epitope on the LAG-3 antigen than the first human antibody). The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any writing or recorded medium provided on or with the kit, or otherwise accompanying the kit.

[0186] Combination therapy In another aspect, the present invention provides a method of combination therapy in which an anti-LAG-3 antibody (or antigen-binding portion thereof) of the present invention is co-administered with one or more additional antibodies that are effective to stimulate an immune response, thereby further enhancing, stimulating, or upregulating the immune response in a subject. In one embodiment, the present invention provides a method of stimulating an immune response in a subject, comprising administering to the subject an anti-LAG-3 antibody and one or more additional immunostimulatory antibodies, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody, to stimulate an immune response in the subject, e.g., to inhibit tumor growth or stimulate an anti-viral response. In another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-1 antibody. In yet another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-PD-L1 antibody. In yet another embodiment, the subject is administered an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, the anti-LAG-3 antibody is a human antibody, e.g., an antibody described herein. Alternatively, the anti-LAG-3 antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a murine anti-LAG-3 mAb). In other embodiments, at least one additional immunostimulatory antibody (e.g., an anti-PD-1, anti-PD-L1, and / or anti-CTLA-4 antibody) is a human antibody. Alternatively, at least one additional immunostimulatory antibody can be, for example, a chimeric or humanized antibody (e.g., prepared from a murine anti-PD-1, anti-PD-L1, and / or anti-CTLA-4 antibody).

[0187] In another embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) comprising administering an LAG-3 antibody and a CTLA-4 antibody to a subject. In a further embodiment, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. In another embodiment, the present invention provides a method for modifying adverse events associated with the treatment of a hyperproliferative disease with an immune stimulant comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-CTLA-4 antibody to a subject. In one embodiment, the subject is a human. In another embodiment, the anti-CTLA-4 antibody is human sequence monoclonal antibody 10D1 (described in PCT Publication WO 01 / 14424), and the anti-LAG-3 antibody is a human sequence monoclonal antibody, e.g., LAG3.5, described herein. Other anti-CTLA-4 antibodies encompassed by the methods of the present invention are described, for example, in WO98 / 42752; WO00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95 (17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22 (145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58 In one embodiment, the anti-CTLA-4 antibody is administered at a dose of 5×10 -8 K below M D binds to human CTLA-4 at 1 × 10 -8 K below M D binds to human CTLA-4 at 5 × 10 -9 K below M D binds to human CTLA-4 at 1 × 10 -8 M to 1 x 10 -10 K below M D It binds to human CTLA-4 at

[0188] In another embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) comprising administering an LAG-3 antibody and a PD-1 antibody to a subject. In a further embodiment, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-PD-1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. In another embodiment, the present invention provides a method for modifying adverse events associated with the treatment of a hyperproliferative disease with an immune stimulant comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-1 antibody to a subject. In one embodiment, the subject is a human. In one embodiment, the anti-PD-1 antibody is a human sequence monoclonal antibody, and the anti-LAG-3 antibody is a human sequence monoclonal antibody, e.g., LAG3.5, described herein. Examples of human sequence anti-PD-1 antibodies include 17D8, 2D3, 4H1, 5C4, and 4A11, which are described in PCT Publication WO 06 / 121168. Other anti-PD-1 antibodies include, for example, lambrolizumab (WO2008 / 156712), and AMP514 (WO2010 / 027423, WO2010 / 027827, WO2010 / 027828, WO2010 / 098788). In one embodiment, the anti-PD-1 antibody is administered at a dose of 5×10 -8 K below M D binds to human PD-1 at 1 × 10 -8 K below M D binds to human PD-1 at 5 × 10 -9 K below M D binds to human PD-1 at 1 × 10 -8 M to 1 x 10 -10 K below M D It binds to human PD-1 at

[0189] In another embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) comprising administering an LAG-3 antibody and a PD-L1 antibody to a subject. In a further embodiment, the anti-LAG-3 antibody is administered at a subtherapeutic dose, the anti-PD-L1 antibody is administered at a subtherapeutic dose, or both are administered at subtherapeutic doses. In another embodiment, the present invention provides a method for modifying adverse events associated with the treatment of a hyperproliferative disease with an immune stimulant comprising administering an anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-L1 antibody to a subject. In one embodiment, the subject is a human. In another embodiment, the anti-PD-L1 antibody is a human sequence monoclonal antibody and the anti-LAG-3 antibody is a human sequence monoclonal antibody, such as LAG3.5, as described herein. Examples of human sequence anti-PD-L1 antibodies include 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, which are described in PCT Publication WO 07 / 005874. Other anti-PD-L1 antibodies include, for example, MPDL3280A (RG7446) (WO 2010 / 077634), MEDI4736 (WO 2011 / 066389), and MDX1105 (WO 2007 / 005874). In one embodiment, the anti-PD-L1 antibody is administered at a concentration of 5×10 -8 K below M D binds to human PD-L1 at 1 × 10 -8 K below M D binds to human PD-L1 at 5 × 10 -9 K below M D binds to human PD-L1 at 1 × 10 -8 M to 1 x 10 -10 K below M D It binds to human PD-L1 at

[0190] Blocking LAG-3 and one or more second target antigens, such as CTLA-4 and / or PD-1 and / or PD-L1, with antibody can enhance the immune response to cancer cells in patients.Cancer whose growth can be inhibited using the antibody herein includes cancers that generally respond to immunotherapy.Typical examples of cancers for treatment in combination therapy herein include the cancers specifically mentioned above in the discussion of monotherapy with anti-LAG-3 antibody.

[0191] In one embodiment, the therapeutic antibody combinations described herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions with each antibody in a pharmaceutically acceptable carrier. In other embodiments, the therapeutic antibody combinations can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-LAG-3 antibody can be administered sequentially, e.g., an anti-CTLA-4 antibody can be administered first and an anti-LAG-3 antibody can be administered second, or an anti-LAG-3 antibody can be administered first and an anti-CTLA-4 antibody can be administered second. Additionally or alternatively, an anti-PD-1 antibody and an anti-LAG-3 antibody can be administered sequentially, e.g., an anti-PD-1 antibody can be administered first and an anti-LAG-3 antibody can be administered second, or an anti-LAG-3 antibody can be administered first and an anti-PD-1 antibody can be administered second. Additionally or alternatively, the anti-PD-L1 antibody and the anti-LAG-3 antibody may be administered sequentially, for example, the anti-PD-L1 antibody is administered first and the anti-LAG-3 antibody is administered second, or the anti-LAG-3 antibody is administered first and the anti-PD-L1 antibody is administered second.

[0192] Furthermore, when two or more doses of a combination therapy are administered sequentially, the order of sequential administration may be reversed or maintained at each time point of administration, sequential administration may be combined with simultaneous administration, and combinations thereof may be used. For example, a first administration of a combined anti-CTLA-4 antibody and anti-LAG-3 antibody may be simultaneous, a second administration may be sequential with a first anti-CTLA-4 and a second anti-LAG-3, and a third administration may be sequential with a first anti-LAG-3 and a second anti-CTLA-4, etc. Additionally or alternatively, a first administration of a combined anti-PD-1 antibody and anti-LAG-3 antibody may be simultaneous, a second administration may be sequential with a first anti-PD-1 and a second anti-LAG-3, and a third administration may be sequential with a first anti-LAG-3 and a second anti-PD-1, etc. Additionally or alternatively, the first administration of the combined anti-PD-L1 antibody and anti-LAG-3 antibody may be simultaneous, the second administration may be sequential with the first anti-PD-L1 and the second anti-LAG-3, and the third administration may be sequential with the first anti-LAG-3 and the second anti-PD-L1, etc. Another exemplary administration scheme may include the first administration being sequential with the first anti-LAG-3 and the second anti-CTLA-4 (and / or anti-PD-1 and / or anti-PD-L1), and subsequent administrations being simultaneous.

[0193] Optionally, the combination of anti-LAG-3 and one or more additional antibodies (e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies) can be further administered to an immunogen, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1 and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (described further below). Combination LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade can further be combined with a vaccine protocol, such as any of the vaccine protocols described in detail above for monotherapy with anti-LAG-3 antibodies.

[0194] The combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade may also be further combined with standard cancer treatment. For example, the combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade may be effectively combined with chemotherapy regimens. In such cases, it may be possible to reduce the dose of other chemotherapeutic agents administered in the combination herein (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies further combined with dacarbazine for the treatment of melanoma. Another example is a combination of anti-LAG-3 and anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies further combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale supporting the combined use of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade with chemotherapy is that cell death, which is the result of the cytotoxic effect of many chemotherapeutic compounds, should result in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade via cell death are radiation, surgery, and hormone deprivation. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade. Inhibition of angiogenesis can induce tumor cell death and present tumor antigens to the host's antigen presentation pathway.

[0195] Combinations of antibodies that block LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. These responses of T cell populations can be augmented by the use of combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade.

[0196] In another example, the combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 antibody and / or anti-PD-L1 antibody is administered in combination with an anti-tumor antibody, e.g., Rituxan. (登録商標)(rituximab), Herceptin (登録商標) (trastuzumab), Bexxar (登録商標) (tositumomab), Zevalin (登録商標) (ibritumomab), Campath (登録商標) (alemtuzumab), Lymphocide (登録商標) (epratuzumab), Avastin (登録商標) (bevacizumab) and Tarceva (登録商標) (erlotinib), etc. By way of example, and without wishing to be bound by theory, treatment with an anti-cancer antibody or an anti-cancer antibody conjugated to a toxin can cause cancer cell (e.g., tumor cell) death, which enhances the immune response mediated by CTLA-4, PD-1, PD-L1, or LAG-3. In a typical embodiment, treatment of a hyperproliferative disease (e.g., cancer tumor) can include an anti-cancer antibody combined with anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies, simultaneously, sequentially, or in combination thereof, which can enhance the anti-tumor immune response by the host.

[0197] Tumors evade host immune surveillance by a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that are immunosuppressive. These include, among others, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163 : 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13 : 198-200) and Fas ligand (Hahne et al. (1996) Science 274 In other embodiments, antibodies to each of these entities can be used in combination with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies to neutralize the effects of immunosuppressants and support a host tumor immune response.

[0198] Other antibodies that can be used to activate host immune responsiveness can also be used in combination with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies (Ridge et al., supra) can be used with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 (Ito et al., supra). Other antibodies that activate against T cell costimulatory molecules (Weinberg et al., supra, Melero et al., supra, Hutloff et al., supra) can also be used to increase the level of T cell activation.

[0199] As mentioned above, bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. Combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade can be used to increase the efficacy of donor-transplanted tumor-specific T cells.

[0200] To stimulate antigen-specific T cells against tumors, several experimental treatment protocols involve ex vivo activation of antigen-specific T cells and compatible transplantation of these cells into recipients (Greenberg & Riddell, supra). These methods can also be used to activate T cell responses against infectious agents, such as CMV. Ex vivo activation in the presence of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies can be expected to increase the frequency and activity of compatible transplanted T cells.

[0201] In one embodiment, the present invention provides a method for modifying adverse events associated with the treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering to a subject an anti-LAG-3 antibody and a subtherapeutic dose of an anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibody. For example, the method of the present invention provides a method for reducing the incidence of immunostimulatory therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. Because all patients receiving immunostimulatory therapeutic antibodies are at risk of developing colitis or diarrhea induced by such antibodies, all patient populations are suitable for treatment according to the method of the present invention. While steroids have been administered to treat inflammatory bowel disease (IBD) and prevent exacerbations of IBD, they have not been used to prevent (reduce the incidence of) IBD in patients who have not been diagnosed with IBD. Significant side effects associated with steroids, particularly non-absorbable steroids, preclude their prophylactic use.

[0202] In a further embodiment, the combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodies, anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies) can be further combined with the use of any non-absorbed steroid. As used herein, a "non-absorbed steroid" is a glucocorticoid that exhibits extensive first-pass metabolism after hepatic metabolism, such that the bioavailability of the steroid is low, i.e., less than about 20%. In one embodiment of the present invention, the non-absorbed steroid is budesonide. Budesonide is a locally acting glucocorticosteroid that is extensively metabolized, primarily by the liver, after oral administration. ENTOCORT EC (登録商標) ENTOCORT EC (Astra-Zeneca) is a pH- and time-dependent oral formulation of budesonide developed and optimized for drug delivery to the ileum and large intestine. (登録商標)is approved in the United States for the mild treatment of Crohn's disease involving the ileum and / or ascending colon. ENTOCORT EC for the treatment of Crohn's disease (登録商標) The usual oral dose of ENTOCORT EC is 6 to 9 mg / day. (登録商標) is released into the intestine before being absorbed and retained in the intestinal mucosa. Once it has passed through the intestinal mucosa target tissue, ENTOCORT EC (登録商標) is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Therefore, bioavailability is low (approximately 10%). Budesonide's low bioavailability results in an improved therapeutic ratio compared to other glucocorticoids that have less extensive first-pass metabolism. Budesonide produces fewer side effects, including less hypothalamic-pituitary suppression, than systemically acting corticosteroids. However, ENTOCORT EC (登録商標) Chronic administration of PDR 58 can result in systemic glucocorticoid effects, such as hyperadrenocorticism and adrenal suppression. th ed. 2004; 608-610.

[0203] In a further embodiment, the combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade (i.e., immunostimulatory therapeutic antibodies anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies) combined with a non-absorbable steroid can be further combined with salicylate. Salicylate can be a 5-ASA agent, such as sulfasalazine (AZULFIDINE). (登録商標) , Pharmacia & UpJohn); Olsalazine (DIPENTUM (登録商標) , Pharmacia & UpJohn); balsalazide (COLAZAL (登録商標) , Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL (登録商標) , Procter & Gamble Pharmaceuticals;PENTASA (登録商標) , Shire US; CANASA(登録商標) , Axcan Scandipharm, Inc.; ROWASA (登録商標) , Solvay).

[0204] In the methods of the present invention, salicylate administered in combination with anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies and a non-absorbed steroid can include any overlapping or sequential administration of salicylate and a non-absorbed steroid for the purpose of reducing the onset of colitis induced by an immunostimulatory antibody. Thus, for example, the methods of the present invention for reducing the onset of colitis induced by an immunostimulatory antibody include administering salicylate and a non-absorbed steroid simultaneously or sequentially (e.g., administering salicylate 6 hours after the non-absorbed steroid), or any combination thereof. Furthermore, in the present invention, salicylate and a non-absorbed steroid can be administered by the same route (e.g., both orally) or by different routes (e.g., salicylate is administered orally and the non-absorbed steroid is administered rectally) (which may be different from the route used to administer the anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies).

[0205] The present specification is further illustrated by the following examples, which should not be construed as further limiting. All figures and all references in this application, the contents of Genbank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference. In particular, the descriptions of PCT publications WO09 / 045957, WO09 / 073533, WO09 / 073546 and WO09 / 054863 are expressly incorporated herein by reference. [Example]

[0206] Example 1 : Design of mutants of LAG3.1 (antibody 25F7) A variant of the previously described anti-LAG-3 antibody, 25F7, referred to herein as LAG3.1, was generated by first analyzing the amino acid sequence of the antibody for potential sites of degradation. H Site-directed mutagenesis of the expression region was performed using the QuikChange II XL® Site-Directed Mutagenesis Kit (Agilent Technologies). H The regions were subcloned into a UCOE® (EMD Millipore) vector containing the human IgG4-S228P constant region. The various heavy chain vectors were co-transfected with a vector expressing the LAG3.1 kappa chain into CHO-S cells, and stable pools were selected for expression.

[0207] Five potential deamidation motifs were identified within the variable region heavy chain CDR2. These sites were located at positions 52, 54, 56, 58, and 60 of the heavy chain variable region of LAG3.1 (SEQ ID NO: 2) (see Figure 1A). In particular, deamidation of the "NG" sequence within VH CDR2 (SEQ ID NO: 6) as well as further isomerization of the sequence were observed under all conditions. Deamidation of the starting material was approximately 10%. Furthermore, it was found that this "NG" sequence did not correspond to the germline sequence (see Figure 3). However, the consensus germline sequence is a potential glycosylation site and therefore was not included in the antibody mutants.

[0208] As shown in Figure 3, four mutants (referred to herein as LAG3.5, LAG3.6, LAG3.7, and LAG3.8) were designed to address two of the potential deamidation motifs (positions 54 and 56). These mutants were subjected to the conditions summarized in Table 1 below and analyzed for the following properties: (a) chemical and thermal stability (physical stability); (b) size exclusion chromatography (aggregation); (c) isoelectric focusing gel (IEF) (charge heterogeneity); (d) activity by Biacore analysis (binding and functional activity); and (e) peptide mapping by mass spectrometry (chemical modification / molecular stability).

[0209] [Table 1]

[0210] Example 2 Characterization of LAG-3 mutants 1. Activated Human CD4 + T cell binding To test the ability of the antibody variants to bind native human LAG-3 on the surface of activated human T cells, normal healthy donor peripheral blood mononuclear cells were stimulated in 15 cm tissue culture plates at a density of 2 x 10e6 cells / mL with a combination of anti-CD3 (eBioscience, Cat #16-0037-85) and anti-CD28 (BD Bioscience, Cat #555725) antibodies present in solution at 5 μg / mL and 3 μg / mL, respectively. Three days after stimulation, cells were harvested, washed 1X with 1x PFAE buffer (1x PBS + 2% FBS, 0.02% sodium azide, 2 mM Na EDTA), and resuspended in 1x PFAE buffer for staining.

[0211] For the binding reaction, the LAG3.1 variants were serially diluted in cold 1xPFAE buffer, and then 50 μl of the diluted antibody solution was mixed with 50 μl of Fitc-labeled anti-human CD4 (BD Bioscience, Cat # 555346) diluted 1:16 in 1xPFAE buffer. For the binding reaction, 100 μl of the diluted antibody mixture was mixed with 2x10 5 The mixture was added to the cells and incubated at 4°C for 30 minutes. The cells were then washed twice with 1x PFAE buffer. A 1:200 dilution of PE-labeled goat anti-human Fcγ-specific antibody (Jackson ImmunoResearch, Cat. # 109-116-170) was added, the mixture was incubated at 4°C for 30 minutes, and then washed twice with cold 1x PFAE buffer. After the final wash, 150 μl of cold 1x PFAE was added to each solution, and antibody binding analysis was performed by flow cytometry using a FACSCanto flow cytometer (BD Bioscience).

[0212] Flow cytometry analysis showed that EC20 values ​​for antibody binding to activated human CD4+ T cells were significantly higher than those for the control group. 50 This is summarized in Figure 4A, which is a graph showing antibody binding to soluble human LAG-3 / Fc antigen by BIACORE. Figure 4B is a graph showing antibody binding to soluble human LAG-3 / Fc antigen by BIACORE. As shown, the binding affinity of LAG3.5 and LAG3.8 is slightly lower than that of LAG3.1, but their off-rate constants are slightly higher than that of LAG3.1.

[0213] 2. Physical stability The thermal stability and thermal denaturation of the mutants were tested using a Microcal VP-DSC. Specifically, each mutant was diluted in PBS (Mediatech cat # 21-040-CV lot # 21040139). The final concentration of the sample was 250 μg / mL after dilution in PBS. Samples were scanned at 74°C, cooled to 25°C, and reheated to 74°C. PBS buffer was used as a blank control. Data were fitted to a non-two-state model, and curve fitting was performed using Origin software.

[0214] As summarized in Table 2 and shown in Figure 5, LAG3.5 had a higher melting temperature TM2 and showed better overall stability than LAG3.1.

[0215] [Table 2]

[0216] Antibody refolding after denaturation is an inverse measure of the likelihood of long-term aggregation. Therefore, LAG-3 mutants were also tested and compared for thermal reversibility. Specifically, the antibody was heated to 74 ° C, cooled to room temperature, and reheated to 74 ° C. The ratio of the area under the second curve to the first thermogram provides an estimate of thermal reversibility, which is a direct measure of structural reversibility.

[0217] As summarized in Table 3 and shown in Figure 6, LAG3.5 had substantially higher thermal reversibility than all other mutants. In particular, the percent reversibility of LAG3.5 (47%) was more than twice that of LAG3.1 (20%). Thermal reversibility is strongly correlated with the likelihood of long-term aggregation. Lower reversibility corresponds to a higher likelihood of aggregation. Based on this observation, LAG3.1 may exhibit a substantially higher likelihood of aggregation over time compared to LAG3.5. Similarly, all other mutants may exhibit a substantially higher likelihood of aggregation over time compared to LAG3.5.

[0218] [Table 3]

[0219] 3.Agglutination The variants were also tested for stability as a measure of protein aggregation using standard size-exclusion HPLC (SEC-HPLC) according to the following protocol: antibody test samples were diluted to 1.0 mg / ml in phosphate-buffered saline (PBS), and 10 μL was applied to an HPLC (Waters, model 2795). Separation was achieved on a gel filtration column (TOSOH Bioscience, TSKgel G3000 SWxl, 7.8 mm x 300 mm, product #08541) using a mobile phase of 0.1 M sodium phosphate, 0.15 M sodium chloride, 0.1 M sodium sulfate, pH 7.2. Analytes were detected by monitoring UV absorbance at 280 nm, and antibody peak area percent composition was determined using Empower software. As shown in Table 4, LAG3.5 exhibited substantially reduced aggregation compared to LAG3.1.

[0220] [Table 4]

[0221] Example 3 : Mutant selection Based on the above studies, the antibody variant LAG3.5 was selected for further analysis, given its significantly improved physicochemical stability compared to its unmodified form (LAG3.1), particularly its high ability to undergo structural refolding (thermoreversibility). This analysis involved a two-step approach: (a) accelerated stress, followed by (b) a 12-week real-time stability assessment. Specifically, LAG3.5 was incubated at 1.0 mg / ml in 50 mM ammonium bicarbonate at pH 8.0 for 5 days at 40°C. After 5 days, the extent of modification and its effect on activity and stability were analyzed. The LAG3.5 variant was then subjected to 12-week real-time stability in PBS and then analyzed. The results of these studies are described below.

[0222] 1. Antigen binding As shown in Figure 7 (and Table 5), no change in antigen binding was observed after 5 days. As also shown in Figures 10A and B, LAG3.5 showed no change in antigen binding or physical stability after 12 weeks. Notably, LAG3.5 maintains higher affinity than LAG3.8 over the entire 12 weeks at both 4°C and 40°C.

[0223] [Table 5]

[0224] 2.Chemical modification / molecular stability Mass spectrometric peptide mapping was used to analyze the chemical / molecular stability of LAG3.5 relative to LAG3.1. Specifically, the purified antibody was reduced, alkylated, dialyzed, and digested with trypsin (Promega Cat. V5111) and GluC (Roche Cat. 11047817001). The digests were analyzed by nano-LC MSMS mass spectrometry (Thermo Fisher LTQ Orbitrap).

[0225] As shown in Figure 8, LAG3.1 exhibits a V compared to LAG3.5 when subjected to enhanced stability at higher pHs where asparagine residues are deamidated. H This indicated increased heterogeneity in the isomerized peaks (step 1). No mass change due to isomerization could be detected under the current experimental conditions. Percent changes are shown as a proportion of all changes combined with the parent peak.

[0226] In addition, as shown in Figure 11, LAG3.1 exhibited a V H showed increased heterogeneity in the pores (step 2).

[0227] 3. physical stability Thermoreversibility was measured in PBS and pH 8.0. Under both conditions, LAG3.5 again exhibited approximately twice the level of refolding compared to LAG3.1. Specifically, as shown in Tables 6-8, LAG3.5 exhibited 43% refolding in PBS compared to 18% for LAG3.1. LAG3.5 also exhibited 48% refolding at pH 8.0 compared to 29% refolding for LAG3.1.

[0228] [Table 6]

[0229] [Table 7]

[0230] [Table 8]

[0231] 4. Charge non-uniformity To assess charge heterogeneity, the variants were analyzed using isoelectric focusing (IEF) with standard markers of pI 5.5 and pI 10.0 relative to LAG3.1. Briefly, the antibody solution was loaded onto a 1 mm thick IEF pI 3-7 pre-formed gel (Invitrogen, Cat# EC6648BOX) along with a pI 3-10 marker (SERVA, Cat# 39212). Electrophoresis was performed using IEF 3-7 Cathode Buffer (Invitrogen, Cat# LC5370) and IEF Anode Buffer (Invitrogen, Cat# LC5300) with a current of 100 V constant for 1 hour, 200 V constant for 1 hour, and 500 V constant for 30 minutes. The IEF gel was stained with Coomassie blue to detect protein bands and destained with a methanol-acetic acid solution. The IEF gels were then analyzed by ImageQuant TL software. Based on this analysis (data not shown), LAG3.5 showed significantly less heterogeneity compared to LAG3.1.

[0232] 5. HIC-HPLC To assess solubility, the variants were analyzed using standard hydrophobic interaction chromatography (HIC-HPLC) according to the following protocol: 50 μL of 2 M ammonium sulfate was added to 50 μL of antibody test sample at 1 mg / mL. Next, 80 μL of the test sample was applied to an HPLC (Waters, model 2795) connected in series with an HIC column (TOSOH Bioscience, Ether-5PW TSK-gel, 7.5 mm x 75 mm, product #07573). The sample was eluted with a gradient of 100% Buffer A (2 M ammonium sulfate, 0.1 M sodium phosphate, pH 7.0) to 100% Buffer B (0.1 M sodium phosphate, pH 7.0) over 50 minutes at a flow rate of 1.0 ml / min. The antibody was detected by monitoring UV absorbance at 280 nm, and data were analyzed using Empower software. As shown in Figure 9, the hydrophilicity of LAG3.5 indicated its solubility at high concentrations of ammonium sulfate.

[0233] Example 4 : Reversal of T cell-mediated inhibition of immune responses The activity of LAG3.5 was determined by means of a functional assay utilizing an antigen-specific murine T cell hybridoma (3A9). Hybridoma 3A9 expresses a T cell receptor specific for a peptide derived from hen egg white lysozyme (HEL48-62) and secretes IL-2 when cocultured with peptide-pulsed MHC-matched antigen-presenting cells (LK35.2). Because huLAG-3-Fc can bind to MHC class II-positive murine B cell lines, expression of huLAG-3 in the 3A9 line could exert an inhibitory effect through engagement with class II on the murine presenting system. Comparison of the peptide response profile of the 3A9 parental line with that of human LAG-3-transduced 3A9 cells cocultured with MHC-matched antigen-presenting cells demonstrated that expression of human LAG-3 inhibited peptide responsiveness compared to control 3A9 cells. This inhibition was reversed by LAG-3 blockade using LAG3.5. Thus, blocking of LAG-3-mediated inhibition was demonstrated for LAG3.5.

[0234] Example 5 : T cell activation by LAG3.5 The functional activity of LAG3.5 in primary T cells was assessed using human PBMC cultures stimulated with the superantigen SEB. Total PBMCs were isolated from the blood of 18 human donors and stimulated for 72 hours in one of two assay formats: (i) a fixed amount of antibody (20 μg / mL) and serial dilutions of SEB, or (ii) a fixed amount of SEB (85 ng / mL) and serial dilutions of antibody. Secreted IL-2 was monitored by ELISA as a measure of T cell activity. Anti-PD-1 antibodies and ipilimumab were used as positive controls, and the activity of LAG3.5 in combination with anti-PD-1 or anti-CTLA-4 was also assessed in a subset of donors.

[0235] Enhanced IL-2 secretion was observed across a range of SEB concentrations from 15 of 18 donors treated with LAG3.5 alone compared with isotype control antibody treatment. In most cases, stimulation was less than that observed with treatment with anti-PD-1 or ipilimumab. For LAG3.5, the results of the two assay formats (described above) were consistent with each other. Furthermore, in five of six donors tested, the combination of LAG3.5 with anti-PD-1 or ipilimumab elicited higher levels of stimulation than those observed with isotype control antibody combined with anti-PD-1 or ipilimumab. These data demonstrated that LAG3.5 can function in normal human T cell assays and further activate responses mediated by inhibition of PD-1 and CTLA-4 function.

[0236] Sequence Listing Summary [Table 9]

[0237] [Table 10]

[0238] [Table 11]

[0239] [Table 12]

[0240] [Table 13]

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof that binds to human LAG-3, comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 regions from the heavy chain variable region of SEQ ID NO:

12.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain CDR1, CDR2 and CDR3 regions comprise the amino acid sequences of SEQ ID NOs: 15, 16 and 17, respectively.

3. The antibody or antigen-binding portion thereof of claim 1, wherein the light chain variable region comprises the CDR1, CDR2 and CDR3 regions from the light chain variable region of SEQ ID NO:

14.

4. The antibody or antigen-binding portion thereof of claim 3, wherein the light chain CDR1, CDR2 and CDR3 regions comprise the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively.

5. The antibody or antigen-binding portion thereof of claim 1 or 3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

12.

6. An antibody or antigen-binding portion thereof according to any one of claims 1 to 5, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

14.

7. An isolated monoclonal antibody or antigen-binding portion thereof that binds to human LAG-3, comprising heavy and light chain CDR1, CDR2 and CDR3 regions comprising the amino acid sequences of SEQ ID NOs: 15, 16, 17, and SEQ ID NOs: 18, 19 and 20, respectively.

8. An isolated monoclonal antibody or antigen-binding portion thereof that binds to human LAG-3, comprising heavy and light chain variable regions comprising the amino acid sequences of SEQ ID NOs: 12 and 14, respectively.

9. One or a combination of the following properties: (a) Binds to monkey LAG-3; (b) does not bind to mouse LAG-3; (c) binding to major histocompatibility (MHC) class II molecules of LAG-3; (d) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules; or (e) stimulating the immune response 9. The antibody or antigen-binding portion thereof according to claim 1 , wherein:

10. The antibody or antigen-binding portion thereof according to any one of claims 1 to 9, which stimulates interleukin-2 (IL-2) production in an antigen-specific T cell response.

11. An antibody or antigen-binding portion thereof according to any one of claims 1 to 10, which stimulates an anti-tumor immune response.

12. 12. The antibody or antigen-binding portion thereof according to any one of claims 1 to 11, which binds to an epitope of human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21).

13. The antibody or antigen-binding portion thereof according to any one of claims 1 to 12, which binds to an epitope of human LAG-3 comprising the amino acid sequence HPAAPSSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO: 23).

14. 0.27 x 10 －９ K below M Ｄ The antibody or antigen-binding portion thereof according to any one of claims 1 to 13, which binds to human LAG-3 at the

15. 15. An antibody or antigen-binding portion thereof according to any one of claims 1 to 14, which is a human, humanized or chimeric antibody.

16. 16. An antibody or antigen-binding portion thereof according to any one of claims 1 to 15, which is of the IgG1, IgG2 or IgG4 isotype.

17. 17. An antibody or antigen-binding portion thereof according to any one of claims 1 to 16, which is an antibody fragment or a single chain antibody.

18. A bispecific molecule comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 17 and a second antibody or antigen-binding portion thereof.

19. 18. An immunoconjugate comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 17 linked to a therapeutic agent.

20. 20. The immunoconjugate of claim 19, wherein the therapeutic agent is a cytotoxin or a radioisotope.

21. A composition comprising an antibody or antigen-binding portion thereof described in any one of claims 1 to 17, a bispecific molecule described in claim 18, or an immunoconjugate described in claim 19 or 20, and a pharmaceutically acceptable carrier.

22. 22. The composition of claim 21, further comprising an anti-cancer agent.

23. 23. The composition of claim 22, wherein the agent is an antibody or a chemotherapeutic agent.

24. An isolated nucleic acid encoding the heavy and / or light chain variable region of the antibody or antigen-binding portion thereof of any one of claims 1-8.

25. 25. An expression vector comprising the nucleic acid of claim 24.

26. A host cell comprising the expression vector of claim 25.

27. 27. A method for producing an anti-LAG-3 antibody, comprising expressing the antibody in the host cell of claim 26 and isolating the antibody from the host cell.

28. 21. A method for stimulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof described in any of claims 1 to 17, a bispecific molecule of claim 18, or an immunoconjugate of claim 19 or 20, such that an immune response in the subject is stimulated.

29. 29. The method of claim 28, wherein the subject has a tumor and an immune response against the tumor is stimulated.

30. 29. The method of claim 28, wherein the subject has a virus and an immune response against the virus is stimulated.

31. 29. The method of claim 28, wherein the immune response is an antigen-specific T cell response, such that an antigen-specific T cell response is stimulated.

32. 32. The method of claim 31, wherein interleukin-2 production by antigen-specific T cells is stimulated.

33. 21. A method for inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof described in any of claims 1-17, a bispecific molecule of claim 18, or an immunoconjugate of claim 19 or 20, such that tumor growth is inhibited in the subject.

34. 21. A method for treating a viral infection in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof described in any of claims 1-17, a bispecific molecule of claim 18, or an immunoconjugate of claim 19 or 20, such that the viral infection is treated in the subject.

35. 31. The method of claim 30, further comprising administration of at least one additional immunostimulatory antibody.

36. 36. The method of claim 35, wherein the at least one additional immunostimulatory antibody is an anti-PD-1 antibody.

37. 37. The method of claim 36, wherein the at least one additional immunostimulatory antibody is an anti-PD-L1 antibody.

38. 37. The method of claim 36, wherein the at least one additional immunostimulatory antibody is an anti-CTLA-4 antibody.

39. Use of an antibody or antigen-binding portion thereof according to any of claims 1 to 17, a bispecific molecule according to claim 18, or an immunoconjugate according to claim 19 or 20, for stimulating an immune response in a subject, optionally an antigen-specific T cell response, or for inhibiting the growth of tumor cells, or for treating a viral infection.

40. Use of an antibody or antigen-binding portion thereof according to any of claims 1 to 17, a bispecific molecule according to claim 18, or an immunoconjugate according to claim 19 or 20 for the manufacture of a medicament for stimulating an immune response, optionally an antigen-specific T cell response, in a subject, or for inhibiting the growth of tumor cells, or for treating a viral infection.

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