Anti-LAG-3 antibodies

Abstract

FIELD: biotechnology.SUBSTANCE: isolated agonistic antibody to the lymphocyte activation gene-3 (LAG-3) or its antigen-binding fragment, as well as to a pharmaceutical composition containing it. Also disclosed are a nucleic acid encoding the above antibody or fragment thereof, as well as a cell and vector containing it.EFFECT: treating a T-cell-mediated immune disorder selected from an inflammatory disease and an autoimmune disorder.39 cl, 31 dwg, 36 tbl, 23 ex

Description

[0001] This invention relates to antibodies or antigen-binding fragments thereof that bind to the lymphocyte activation gene-3 (LAG-3), in particular, antibodies or antigen-binding fragments thereof that are LAG-3 agonists, and the use of antibodies or fragments as medicaments, in particular for the treatment of conditions associated with the proliferation and / or activation of CD4 + and / or CD8 + T cells, particularly inflammatory and autoimmune disorders.

[0002] Lymphocyte activation gene 3 (LAG-3) is a homolog of the type I membrane protein CD4, containing four extracellular domains of the Ig superfamily. Similar to CD4, LAG-3 oligomerizes on T cell surfaces and binds to MHC class II molecules on antigen-presenting cells (APCs), but with significantly higher affinity than CD4. LAG-3 is expressed on activated CD4-positive and CD8-positive T lymphocytes, where it associates with the cell-surface CD3-TCR complex and negatively regulates signal transduction. Consequently, it negatively regulates T cell proliferation, function, and homeostasis. When the MHC class II-peptide complex is recognized by a specific TCR, intracellular signals are transduced into the T cell via the TCR and into the APC via MHC class II molecules. The negative regulatory role of LAG-3 signaling in T cells is exerted in human primary CD4 and CD8 T cell responses ( et at., Immunology. 2005 Jun; 115(2): 170-178).

[0003] Also, LAG-3 encodes an alternative splicing variant that is converted to a soluble form of LAG-3 (sLAG-3). As a soluble molecule, LAG-3 activates antigen-presenting cells (APCs) through MHC class II signaling, leading to increased antigen-specific T cell responses in vivo (Triebel, Trends Immunol., 2003, 24: 619-622).

[0004] The amino acid sequence of human and mouse LAG-3 protein is shown in Figure 1 in Huard et al (Proc. Natl. Acad. Sci. USA, 11: 5744-5749, 1997). The sequence of human LAG-3 protein is reproduced in Figure 1 below (SEQ ID NO: 27). The amino acid sequences of the four extracellular domains of the Ig superfamily (D1, D2, D3, and D4) of human LAG-3 are located at amino acid residues: 1-149 (D1) (SEQ ID NO: 28); 150-239 (D2) (SEQ ID NO: 29); 240-330 (D3) (SEQ ID NO: 39); and 331-412 (D4) (SEQ ID NO: 51).

[0005] Baixeras et al. (J. Exp. Med., 1992, Vol. 176: 327-337) reported the production of 17B4, a murine monoclonal antibody (IgG1 isotype) to human LAG-3 protein. This antibody recognizes the outer 30-amino acid loop of the first N-terminal D1 domain of human LAG-3. 17 B4 inhibits LAG-3 / MHC class interactions and increases T cell proliferation as an antagonist of LAG-3 signaling (Huard et al, Eur J Immunol. 1996; 26:1180-6). Monoclonal antibody (mAb) 17B4 lacks agonist activity as determined by its inability to induce an increase in intracellular free calcium levels in T cells in the absence of a secondary cross-linking reagent (Hannier et al, J Immunol. 1998; 161:4058-65.).

[0006] Agents capable of modulating the activation and / or effector functions of CD4-positive and CD4-positive T cells are highly desirable. In particular, autoreactive T cells and autoantibodies are known to be involved in many autoimmune disorders. Therefore, there is a need for agents that can inhibit or eliminate autoreactive lymphocytes without compromising the immune system's ability to defend against pathogens.

[0007] Poirier et al (Clinical and Experimental Immunology, 2011, 164: 265-274) described the evaluation of a cytotoxic chimeric antibody against LAG-3 (chimeric A9H12). In vivo, the antibody depleted LAG-3 +-activated activated T cells in lymph nodes and demonstrated efficacy in reducing skin inflammation in a baboon model of tuberculin-induced delayed-type hypersensitivity (DTH). Antibodies that specifically deplete activated T cells represent a promising therapeutic strategy for the prevention and / or treatment of autoimmune disorders.

[0008] As an alternative strategy, the applicant has established that LAG-3 agonists will negatively regulate T cell proliferation and / or function without T cell exhaustion, and that such agonists can also be used to treat inflammatory or autoimmune disorders.

[0009] The applicant was able to obtain monoclonal anti-LAG-3 agonist antibodies. These antibodies inhibit antigen-induced proliferation of CD4-positive and CD8-positive T cells. These antibodies and their antigen-binding fragments can be used to treat immune disorders, particularly T-cell-mediated immune disorders, including inflammatory and autoimmune disorders.

[0010] According to the invention, an agonistic anti-LAG-3 antibody or an antigen-binding fragment thereof is provided. In particular, the antibody is a monoclonal agonistic anti-LAG-3 antibody or an antigen-binding fragment thereof.

[0011] The term "LAG-3" as used herein refers to the lymphocyte activation gene-3. The term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs. For example, antibodies specific for the human LAG-3 protein may, in some cases, cross-react with the LAG-3 protein of non-human species. In other embodiments, antibodies specific for the human LAG-3 protein may be completely specific for the human LAG-3 protein and may not exhibit species-specific or other types of cross-reactivity, or may cross-react with the LAG-3 of some 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 a human LAG-3 sequence, such as the complete amino acid sequence of human LAG-3 having Genbank accession number NP 002277 (SEQ ID NO: 38), or the amino acid sequence of the human LAG-3 protein shown in Figure 1 (SEQ ID NO: 27). The term "mouse LAG-3" refers to a mouse LAG-3 sequence, such as the complete amino acid sequence of mouse LAG-3 having Genbank accession number NP 032505. In the art, LAG-3 is also known, for example, as CD223. The sequence of human LAG-3 may differ from the sequence of human LAG-3 having Genbank accession number NP 002277 by the presence of, for example, conservative mutations or mutations in non-conserved regions, and LAG-3 has substantially the same biological function as human LAG-3 having Genbank accession number NP 002277.For example, the biological function of human LAG-3 is the presence of an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibody of the present invention, or the biological function of human LAG-3 is binding to MHC class II molecules.

[0012] The term "simian LAG-3" encompasses LAG-3 proteins expressed by Old World and New World monkeys, including, but not limited to, cynomolgus macaque LAG-3 and rhesus macaque LAG-3. A representative amino acid sequence of simian LAG-3 is the amino acid sequence of rhesus macaque LAG-3, which is also deposited in GenBank under accession number XM 001108923. Another representative amino acid sequence of simian LAG-3 is the alternative sequence of rhesus macaque clone pa23-5, described in US 2011 / 0150892 A1. This alternative amino acid sequence of rhesus macaque has a single difference at position 419 from the sequence deposited in GenBank.

[0013] A particular human LAG-3 sequence is typically at least 90% identical to the amino acid sequence of human LAG-3 having Genbank accession number NP 002277 and contains amino acid residues that identify the amino acid sequence as human when compared to the amino acid sequences of LAG-3 of other species (e.g., mice). In some cases, the amino acid sequence of human LAG-3 may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence of LAG-3 having Genbank accession number NP 002277. In some embodiments, the human LAG-3 sequence will exhibit no more than 10 amino acid differences from the LAG-3 sequence having Genbank accession number NP 002277.In some embodiments, human LAG-3 may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the LAG-3 sequence having Genbank accession number NP 002277. Percent identity may be determined as described herein.

[0014] According to some embodiments, the agonistic anti-LAG-3 antibody of the invention or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + and / or CD8 + T cells or antigen-induced CD4 activation + and / or CD8 + T cells.

[0015] The agonistic anti-LAG-3 antibody of the invention or an antigen-binding fragment thereof may be an isolated agonistic anti-LAG-3 antibody or an antigen-binding fragment thereof.

[0016] The term "agonistic" is used herein interchangeably with the term "agonist".

[0017] Also, according to the invention, there is provided an isolated antibody or antigen-binding fragment thereof that binds to LAG-3 and inhibits antigen-induced CD4 proliferation. + and / or CD8 + T cells or antigen-induced CD4 activation + and / or CD8 + T cells.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells and / or antigen-induced CD8 proliferation + T cells. In some embodiments, the antibody or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells and antigen-induced CD8 proliferation + T cells. In specific embodiments, the antibody or antigen-binding fragment thereof inhibits antigen-induced CD8 proliferation + T cells are greater than antigen-induced CD4 proliferation +T cells.

[0019] Figure 21 shows the differences between depleting anti-LAG-3 antibodies, antagonist anti-LAG-3 antibodies, and antibodies of the invention (i.e., agonist anti-LAG-3 antibodies and antibodies that bind to LAG-3 and inhibit antigen-induced CD4 proliferation + and / or CD8 + T cells or antigen-induced CD4 activation + and / or CD8 + T cells).

[0020] An anti-LAG-3 depleting antibody depletes activated T cells by binding to LAG-3 expressed on the cell surface. Depletion can be achieved through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of the depleting antibody binds to Fc receptors (FcγRs) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to lysis of target cells. In CDC, the Fc region of the depleting antibody binds to the Clq component of complement, and the target cell is killed by triggering the complement cascade on the cell surface. Thus, anti-LAG-3 depleting antibodies inhibit T cell-mediated immune responses. It should be noted that the effects of depleting anti-LAG-3 antibodies are long-lasting and irreversible, as they cause destruction of activated T cells.Such antibodies are useful, for example, for the treatment of inflammatory and autoimmune disorders, and for preventing transplant rejection.

[0021] Antagonist anti-LAG-3 antibody binds to LAG-3 on the surface of activated T cells and prevents LAG-3 from interacting with MHC class II molecules on the surface of antigen-presenting cells (APCs). This blocks the downregulation of signal transduction that occurs when APCs bind to LAG-3 on the surface of activated T cells. Accordingly, antagonist anti-LAG-3 antibodies prevent the downregulation of T cell proliferation, function, and homeostasis normally mediated by LAG-3. Such antibodies are useful, for example, for the treatment of cancer and infectious diseases.

[0022] The antibodies of the invention bind to LAG-3 on the surface of activated T cells and negatively regulate signal transduction through LAG-3 agonism, causing negative regulation of T cell proliferation and / or activation. Thus, the antibodies of the invention inhibit T cell-mediated immune responses, in particular by inhibiting antigen-induced CD4 proliferation. + and / or CD8 + T cells and / or antigen-induced CD4 activation + and / or CD8 + T cells. The effects of such antibodies are reversible and may be shorter-lasting than the effects of anti-LAG-3 depleting antibodies, as they do not cause destruction of activated T cells. It should be noted that the period of effectiveness of the antibody according to the present invention will depend on the plasma half-life of the antibody.

[0023] Inhibition of antigen-induced CD4 proliferation + and / or CD8 +T-cell counts can be determined using any suitable method known to those skilled in the art. An example of a suitable method is measuring CD4 proliferation. + and / or CD8 + T cells induced by antigen peptides in the presence of antibody or fragment, compared with the corresponding proliferation in the presence of a negative control antibody of the same CD4 isotype + and CD8 + T cells can be present, for example, in a sample of peripheral blood mononuclear cells (PBMCs) obtained from a healthy donor. Cell proliferation can be induced by any suitable antigenic peptides, such as a pool of peptides encompassing the CMV pp35 sequence. Cell proliferation can be measured by labeling the cells, for example, with a fluorescent dye such as carboxyfluorescein succinimidyl ester (CFSE). An example of a method for determining the inhibition of antigen-induced CD4 proliferation + and / or CD8 +T cells are described in more detail in Example 10 below.

[0024] Percentage inhibition of antigen-induced CD4 proliferation + and / or CD8 + T cells can be determined as a percentage inhibition of the proliferation index (PI), calculated as the sum of the percentage of CD4 + and / or CD8 + T cells under each division peak (assessed by FACS), multiplied by the number of divisions, as described in more detail in Example 10 below.

[0025] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to antigen-induced CD4 proliferation + T cells in the absence of antibody or fragment.

[0026] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits antigen-induced proliferation of CD8 + T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to antigen-induced CD8 proliferation + T cells in the absence of antibody or fragment.

[0027] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells and antigen-induced CD8 proliferation + T cells, each, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to antigen-induced CD4 proliferation + T cells and antigen-induced CD8 proliferation + T cells, respectively, in the absence of an antibody or fragment.

[0028] In one embodiment, the antibody of the invention or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells by at least 20% compared to antigen-induced CD4 proliferation + T cells in the absence of antibody or fragment, and inhibits antigen-induced CD8 proliferation + T cells by at least 30% compared to antigen-induced CD8 proliferation + T cells in the absence of antibody or fragment.

[0029] Inhibition of antigen-induced CD4 proliferation + and / or CD8 + T cells by the antibody of the invention or a fragment thereof can be compared with antigen-induced CD4 proliferation + and / or CD8 + T cells in the presence of a negative control antibody of the same isotype or its fragment.

[0030] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits antigen-induced CD8 proliferation + At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more T cells than the antibody or fragment inhibits antigen-induced CD4 proliferation + T cells.

[0031] In accordance with some embodiments, inhibiting antigen-induced CD8 proliferation + T cell activation is LAG-3-dependent and IL-2-independent.

[0032] In some embodiments, the antibody of the invention or antigen-binding fragment thereof inhibits antigen-induced CD4 activation + and / or CD8 + T cells. In some embodiments, the antibody of the invention or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + and / or CD8 +T cells and antigen-induced CD4 activation + and / or CD8 + T cells.

[0033] In particular, the antibody of the invention or an antigen-binding fragment thereof can bind to LAG-3 and inhibit antigen-induced CD4 activation + and / or CD8 + T cells. In some embodiments, an antibody of the invention or an antigen-binding fragment thereof binds to LAG-3 and inhibits antigen-induced CD4 activation. + T cells and antigen-induced CD8 activation + T cells.

[0034] Inhibition of CD4 activation + and / or CD8 + T cell counts can be determined using any suitable method known to those skilled in the art. An example of a suitable method is measuring the effect of an antibody or fragment on the expression of the CD4 activation marker. + and / or CD8 + T cells or secretion of a T cell activation marker. For example, CD8 activation +T cells can be measured by measuring the expression of CD25, as a marker of activation, on CD8 +T cells induced by antigenic peptides in the presence of an antibody or fragment compared to the corresponding CD25 expression in the presence of a negative control antibody of the same isotype. Alternatively, T cell activation can be measured by measuring the secretion of IFN-γ in the cellular supernatant of the T cells induced by antigenic peptides in the presence of an antibody or fragment compared to the corresponding secretion in the presence of a negative control antibody of the same isotype. T cells can be present, for example, in a sample of PBMCs obtained from a healthy donor. Cell activation can be induced by any suitable antigenic peptides, such as a pool of peptides covering the CMV pp35 sequence. An example of a method for determining inhibition of antigen-induced T cell activation by measuring secretion of a T cell activation marker is described in more detail in Example 15 below. An example of a method for determining inhibition of antigen-induced CD8 activation.+ T cells by measuring the expression of the activation marker CD8 + T cells are described in more detail in Example 16 below.

[0035] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits antigen-induced CD4 activation + and / or CD8 + T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to antigen-induced CD4 activation + and / or CD8 + T cells in the absence of antibody or fragment.

[0036] Inhibition of antigen-induced CD4 activation + and / or CD8 + T cells by the antibody of the invention or a fragment thereof can be compared with antigen-induced activation of CD4 + and / or CD8 + T cells in the presence of a negative control antibody of the same isotype or its fragment.

[0037] According to some embodiments, the antibody of the invention or antigen-binding fragment thereof inhibits the binding of IMP321 to MHC class II-positive cells.

[0038] IMP321 (also referred to as "LAG-3Ig" below) is a recombinant soluble human LAG-3Ig fusion protein. The fusion protein is produced as a 200 kDa dimer in Chinese hamster ovary (CHO) cells transfected with a plasmid encoding the extracellular domain of human LAG-3 fused to the Fc region of human IgG1. The sequence of IMP321 is provided in SEQ ID NO: 17 in US Patent Application 2011 / 0008331.

[0039] Binding of IMP321 to MHC class II-positive cells can be determined by measuring the binding of an IMP321-label conjugate (e.g., an IMP321-Alex 488 conjugate) to Raji cells (which are MHC class II-positive B cells), such as described in Example 8 below.

[0040] In some embodiments, an antibody of the invention or antigen-binding fragment thereof inhibits IMP321 binding to MHC class II-positive cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to IMP321 binding to MHC class II-positive cells in the absence of the antibody or fragment.

[0041] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits the binding of IMP321 to MHC class II-positive cells by at least 30% compared to the binding of IMP321 to MHC class II-positive cells in the absence of the antibody or fragment, wherein the concentration ratio of the antibody or fragment to IMP321 is 0.1:1.

[0042] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits the binding of IMP321 to MHC class II-positive cells by at least 80% compared to the binding of IMP321 to MHC class II-positive cells in the absence of the antibody or fragment, wherein the concentration ratio of the antibody or fragment to IMP321 is 0.3:1 or 1:1.

[0043] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits IMP321-induced monocyte activation.

[0044] IMP321 is capable of activating cells of the human monocytic cell line THP-1. Activation of THP-1 cells can be determined by the level of secretion of chemokine ligand 4 (CCL4, also known as macrophage inflammatory protein 1β, MIP-1β) by THP-1 cells. Pre-incubation of the antibody or fragment of the invention with IMP321 before incubation of the mixture with THP-1 cells can be used to determine whether the antibody or fragment inhibits IMP321-induced monocyte activation. The method for determining the inhibition of IMP321-induced monocyte activation is described in more detail in Example 9 below.

[0045] In some embodiments, an antibody of the invention or antigen-binding fragment thereof inhibits IMP321-induced monocyte activation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the amount of IMP321-induced monocyte activation in the absence of the antibody or fragment.

[0046] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits IMP321-induced monocyte activation by at least 70% compared to the amount of IMP321-induced monocyte activation in the absence of the antibody or fragment, wherein the concentration ratio of the antibody or fragment to IMP321 is 1:1.

[0047] Huard et al (Proc. Natl. Acad. Sci. USA, 11: 5744–5749, 1997) described the characterization of the MHC class II binding site on the LAG-3 protein. Many residues important for MHC class II protein binding are grouped based on the large 30-amino acid outer loop structure in the D1 domain of LAG-3. The amino acid sequence of the outer loop structure of the D1 domain of human LAG-3 is GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 40), the underlined sequence in Figure 1.

[0048] The antibody of the invention or an antigen-binding fragment thereof can bind to an epitope of human LAG-3 that overlaps with the MHC class II binding site of LAG-3.

[0049] The antibody of the invention or an antigen-binding fragment thereof can bind to an epitope that overlaps with the outer 30-amino acid loop of the first N-terminal domain of D1 of human LAG-3.

[0050] In other embodiments, an antibody of the invention or an antigen-binding fragment thereof does not bind to the sequence of the outer 30 amino acid loop (SEQ ID NO: 40) of the first N-terminal domain of D1 of human LAG-3.

[0051] The antibody of the invention can inhibit the binding of LAG-3 to MHC class II molecules in vivo. In particular, the antibody of the invention can antagonize the MHC class II activating signal in antigen-presenting cells (APCs). Thus, the antibody of the invention can inhibit LAG-3-induced APC activation, such as dendritic cell activation, such as LAG-3-induced monocyte or macrophage activation.

[0052] In some embodiments, an antibody of the invention or antigen-binding fragment thereof inhibits LAG-3 binding to MHC class II-positive cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to LAG-3 binding to MHC class II-positive cells in the absence of the antibody or fragment.

[0053] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof inhibits LAG-3-induced APC activation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the amount of LAG-3-induced APC activation in the absence of the antibody or fragment.

[0054] The monoclonal antibody of the invention or its antigen-binding fragment may comprise one, two or three complementarity determining regions (CDRs) of the variable region of the heavy chain (VH) of the antibody comprising the amino acid sequence of SEQ ID NO: 7, and / or one, two or three CDRs of the variable region of the light chain (VL) of the antibody comprising the amino acid sequence of SEQ ID NO: 8.

[0055] Also, according to the invention, an anti-LAG-3 antibody or an antigen-binding fragment thereof is provided, which comprises one, two or three complementarity determining regions (CDRs) of the variable region of the heavy chain (VH) of the antibody, comprising the amino acid sequence of SEQ ID NO: 7, and / or one, two or three CDR regions of the variable region of the light chain (VL) of the antibody, comprising the amino acid sequence of SEQ ID NO: 8.

[0056] The CDR regions of the VH region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the CDR regions of the VL region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0057] The antibody according to the invention or its antigen-binding fragment may comprise a VH region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 1, 2 and 3, and / or a VL region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 4, 5 and 6.

[0058] CDR regions having the amino acid sequences of SEQ ID NOs: 1, 2 and 3 may be presented in any order in the VH region, and CDR regions having the amino acid sequences of SEQ ID NOs: 4, 5 and 6 may be presented in any order in the VL region. However, in a preferred embodiment, the antibody or fragment thereof comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and / or CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0059] The CDR regions of the VH region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 21, 22, and 23, and the CDR regions of the VL region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 24, 25, and 26.

[0060] The antibody according to the invention or its antigen-binding fragment may comprise the VH region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 21, 22 and 23, and / or the VL region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 24, 25 and 26.

[0061] CDR regions having the amino acid sequences of SEQ ID NOs: 21, 22 and 23 may be presented in any order in the VH region, and CDR regions having the amino acid sequences of SEQ ID NOs: 24, 25 and 26 may be presented in any order in the VL region. However, in a preferred embodiment, the antibody or fragment thereof comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 21, CDR-H2 having the amino acid sequence of SEQ ID NO: 22, and CDR-H3 having the amino acid sequence of SEQ ID NO: 23, and / or CDR-L1 having the amino acid sequence of SEQ ID NO: 24, CDR-L2 having the amino acid sequence of SEQ ID NO: 25, and CDR-L3 having the amino acid sequence of SEQ ID NO: 26.

[0062] In some embodiments, the CDR regions of the VH region of the antibody are selected from the CDR regions having the amino acid sequences of SEQ ID NO: 1, 2, 3, 21, 22 and 23, and the CDR regions of the VL region of the antibody are selected from the CDR regions having the amino acid sequences of SEQ ID NO: 4, 5, 6, 24, 25 and 26.

[0063] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises a VH region of an antibody comprising CDR1 VH, CDR2 V H and CDR3 VH, where CDR1 V H has an amino acid sequence selected from SEQ ID NO: 1 and 21, and / or CDR2 V H has an amino acid sequence selected from SEQ ID NO: 2 and 22, and / or CDR3 V H has an amino acid sequence selected from SEQ ID NO: 3 and 23.

[0064] In some embodiments:

[0065] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 1 and 21, and CDR2 V Hhas an amino acid sequence selected from SEQ ID NO: 2 and 22:

[0066] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 1 and 21, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 3 and 23;

[0067] CDR2 V H has an amino acid sequence selected from SEQ ID NO: 2 and 22, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 3 and 23; or

[0068] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 1 and 21, CDR2 V H has an amino acid sequence selected from SEQ ID NO: 2 and 22, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 3 and 23.

[0069] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises a VL region of an antibody comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has an amino acid sequence selected from SEQ ID NOs: 4 and 24, and / or the VL CDR2 has an amino acid sequence selected from SEQ ID NOs: 5 and 25, and / or the VL CDR3 has an amino acid sequence selected from SEQ ID NOs: 6 and 26.

[0070] In some embodiments:

[0071] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 4 and 24, and CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 5 and 25;

[0072] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 4 and 24, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 6 and 26;

[0073] CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 5 and 25, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 6 and 26; or

[0074] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 4 and 24, CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 5 and 25, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 6 and 26.

[0075] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises a VH region of an antibody comprising: a VH CDR1 having an amino acid sequence selected from SEQ ID NOs: 1 and 21; a VH CDR2 having an amino acid sequence selected from SEQ ID NOs: 2 and 22; and a VH CDR3 having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and a VL region of an antibody comprising: a VL CDR1 having an amino acid sequence selected from SEQ ID NOs: 4 and 24; a VL CDR2 having an amino acid sequence selected from SEQ ID NOs: 5 and 25; and a VL CDR3 having an amino acid sequence selected from SEQ ID NOs: 6 and 26.

[0076] The antibody of the invention or an antigen-binding fragment thereof may comprise a VH region of an antibody comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 7, and / or a VL region of an antibody comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 8.

[0077] In a preferred embodiment, the antibody of the invention or an antigen-binding fragment thereof comprises an antibody VH region comprising the amino acid sequence of SEQ ID NO: 7 and / or an antibody VL region comprising the amino acid sequence of SEQ ID NO: 8.

[0078] The antibody of the invention or an antigen-binding fragment thereof may comprise a VH region of an antibody and / or a VL region of an antibody comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, or that is identical to, the amino acid sequence of a V H and / or VL regions of the murine monoclonal anti-LAG-3 antibody 13E2 described herein in Examples 1, 2 and 3 (amino acid sequence of V H 13E2: SEQ ID NO: 7; amino acid sequence VL 13E2: SEQ ID NO: 8).

[0079] Also, in accordance with the invention, an antibody or an antigen-binding fragment thereof is provided that competes for binding to LAG-3 with an antibody that comprises a VH region of an antibody comprising the amino acid sequence of SEQ ID NO: 7, and a VL region of an antibody comprising the amino acid sequence of SEQ ID NO: 8.

[0080] Furthermore, the present invention provides an antibody or antigen-binding fragment thereof that competes for binding to LAG-3 with the mouse monoclonal anti-LAG-3 antibody 13E2.

[0081] The monoclonal antibody of the invention or its antigen-binding fragment may comprise one, two or three complementarity determining regions (CDRs) of the variable region of the heavy chain (VH) of the antibody comprising the amino acid sequence of SEQ ID NO: 17, and / or one, two or three CDRs of the variable region of the light chain (VL) of the antibody comprising the amino acid sequence of SEQ ID NO: 18.

[0082] Also, according to the invention, an anti-LAG-3 antibody or an antigen-binding fragment thereof is provided, which comprises one, two or three complementarity determining regions (CDRs) of the variable region of the heavy chain (VH) of the antibody, comprising the amino acid sequence of SEQ ID NO: 17, and / or one, two or three CDR regions of the variable region of the light chain (VL) of the antibody, comprising the amino acid sequence of SEQ ID NO: 18.

[0083] The CDR regions of the VH region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, and the CDR regions of the VL region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 14, 15 and 16.

[0084] The antibody according to the invention or its antigen-binding fragment may comprise a VH region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 11, 12 and 13, and / or a VL region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 14, 15 and 16.

[0085] CDR regions having the amino acid sequences of SEQ ID NOs: 11, 12 and 13 may be presented in any order in the VH region, and CDR regions having the amino acid sequences of SEQ ID NOs: 14, 15 and 16 may be presented in any order in the VL region. However, in a preferred embodiment, the antibody or fragment thereof comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 11, CDR-H2 having the amino acid sequence of SEQ ID NO: 12, and CDR-H3 having the amino acid sequence of SEQ ID NO: 13, and / or CDR-L1 having the amino acid sequence of SEQ ID NO: 14, CDR-L2 having the amino acid sequence of SEQ ID NO: 15, and CDR-L3 having the amino acid sequence of SEQ ID NO: 16.

[0086] The CDR regions of the VH region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 31, 32, and 33, and the CDR regions of the VL region of the antibody may be CDR regions having the amino acid sequences of SEQ ID NOs: 34, 35, and 36.

[0087] The antibody according to the invention or its antigen-binding fragment may comprise the VH region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 31, 32 and 33, and / or the VL region of an antibody with CDR regions having the amino acid sequences of SEQ ID NO: 34, 35 and 36.

[0088] CDR regions having the amino acid sequences of SEQ ID NOs: 31, 32 and 33 may be presented in any order in the VH region, and CDR regions having the amino acid sequences of SEQ ID NOs: 34, 35 and 36 may be presented in any order in the VL region. However, in a preferred embodiment, the antibody or antigen-binding fragment thereof comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 31, CDR-H2 having the amino acid sequence of SEQ ID NO: 32, and CDR-H3 having the amino acid sequence of SEQ ID NO: 33, and / or CDR-L1 having the amino acid sequence of SEQ ID NO: 34, CDR-L2 having the amino acid sequence of SEQ ID NO: 35, and CDR-L3 having the amino acid sequence of SEQ ID NO: 36.

[0089] In some embodiments, the CDR regions of the VH region of the antibody are selected from the CDR regions having the amino acid sequences of SEQ ID NO: 11, 12, 13, 31, 32 and 33, and the CDR regions of the VL region of the antibody are selected from the CDR regions having the amino acid sequences of SEQ ID NO: 14, 15, 16, 34, 35 and 36.

[0090] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises a VH region of an antibody comprising CDR1 VH, CDR2 V H and CDR3 VH, where CDR1 V H has an amino acid sequence selected from SEQ ID NO: 11 and 31, and / or CDR2 V H has an amino acid sequence selected from SEQ ID NO: 12 and 32, and / or CDR3 V H has an amino acid sequence selected from SEQ ID NO: 13 and 33.

[0091] In some embodiments:

[0092] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 11 and 31, and CDR2 V Hhas an amino acid sequence selected from SEQ ID NO: 12 and 32;

[0093] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 11 and 31, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 13 and 33;

[0094] CDR2 V H has an amino acid sequence selected from SEQ ID NO: 12 and 32, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 13 and 33; or

[0095] CDR1 V H has an amino acid sequence selected from SEQ ID NO: 11 and 31, CDR2 V H has an amino acid sequence selected from SEQ ID NO: 12 and 32, and CDR3 V H has an amino acid sequence selected from SEQ ID NO: 13 and 33.

[0096] In some embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises a VL region of an antibody comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 has an amino acid sequence selected from SEQ ID NOs: 14 and 34, and / or the VL CDR2 has an amino acid sequence selected from SEQ ID NOs: 15 and 35, and / or the VL CDR3 has an amino acid sequence selected from SEQ ID NOs: 16 and 36.

[0097] In some embodiments:

[0098] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 14 and 34, and CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 15 and 35;

[0099] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 14 and 34, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 16 and 36;

[0100] CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 15 and 35, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 16 and 36; or

[0101] CDR1 VL has an amino acid sequence selected from SEQ ID NOs: 14 and 34, CDR2 VL has an amino acid sequence selected from SEQ ID NOs: 15 and 35, and CDR3 VL has an amino acid sequence selected from SEQ ID NOs: 16 and 36.

[0102] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises: a VH region of an antibody comprising: a VH CDR1 having an amino acid sequence selected from SEQ ID NOs: 11 and 31, a VH CDR2 having an amino acid sequence selected from SEQ ID NOs: 12 and 32, and a VH CDR3 having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and a VL region of an antibody comprising: a VL CDR1 having an amino acid sequence selected from SEQ ID NOs: 14 and 34, a VL CDR2 having an amino acid sequence selected from SEQ ID NOs: 15 and 35, and a VL CDR3 having an amino acid sequence selected from SEQ ID NOs: 16 and 36.

[0103] The antibody of the invention or an antigen-binding fragment thereof may comprise a VH region of an antibody comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17, and / or a VL region of an antibody comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18.

[0104] In a preferred embodiment, the antibody of the invention or an antigen-binding fragment thereof comprises an antibody VH region comprising the amino acid sequence of SEQ ID NO: 17 and / or an antibody VL region comprising the amino acid sequence of SEQ ID NO: 18.

[0105] The antibody of the invention or an antigen-binding fragment thereof may comprise an antibody VH region and an antibody VL region comprising an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, or that is identical to, the amino acid sequence of the VH and VL regions of the murine monoclonal anti-LAG-3 antibody 34F4 described herein in Examples 4, 5 and 6 (the amino acid sequence of the V H 34F4: SEQ ID NO: 17; amino acid sequence VL 34F4: SEQ ID NO: 18).

[0106] Also, according to the invention, there is provided an antibody or an antigen-binding fragment thereof that competes for binding to LAG-3 with an antibody that comprises a VH region of an antibody comprising the amino acid sequence of SEQ ID NO: 17, and a VL region of an antibody comprising the amino acid sequence of SEQ ID NO: 18.

[0107] Furthermore, according to the invention, there is provided an antibody or an antigen-binding fragment thereof that competes for binding to LAG-3 with the murine monoclonal anti-LAG-3 antibody 34F4.

[0108] In certain embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences that differ from the sequences of the 13E2 or 34F4 antibody by one or more conservative modifications, such as five conservative modifications. It is understood in the art that certain modifications can be made to the conserved sequences that do not impair antigen binding. See, for example, 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.

[0109] The term "conservative sequence modifications" as used herein refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody of the invention by conventional methods 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 by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibody can be tested for retained function (i.e., the functions described above) using the functional assays described herein.

[0110] The antibodies of the invention can be produced using an antibody having one or more V sequences H and / or VL 13E2 or 34F4 as starting material for constructing a modified antibody. The antibody can be constructed by modifying one or more residues within one or both variable regions (i.e., V H and / or VL), for example, within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues within the constant region(s), for example, to alter the effector function(s) of the antibody.

[0111] In some embodiments, CDR grafting can be used to engineer the variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located within the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDRs of individual antibodies are more diverse than sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from another antibody with different properties (see, e.g., Riechmann et al. (1998) Nature 332:323-327; Jones et al.(1986) Nature 321: 522–525; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029–10033; U.S. Patents 5,225,539; 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0112] Also, according to the invention, there is provided an antibody of the invention or an antigen-binding fragment thereof, for example, a monoclonal antibody or an antigen-binding fragment thereof, comprising CDR1, CDR2 and CDR3 of the variable region of the heavy chain of an antibody having the amino acid sequence of SEQ ID NO: 7, and / or comprising CDR1, CDR2 and CDR3 of the variable region of the light chain of an antibody having the amino acid sequence of SEQ ID NO: 8 (i.e., the CDR regions of 13E2). Although such antibodies comprise the CDR sequences of the VH and VL regions of the monoclonal antibody 13E2, they may comprise different framework sequences.

[0113] Similarly, according to the invention, there is provided an antibody of the invention or an antigen-binding fragment thereof, for example, a monoclonal antibody or an antigen-binding fragment thereof, comprising CDR1, CDR2 and CDR3 of the variable region of the heavy chain of an antibody comprising the amino acid sequence of SEQ ID NO: 17, and / or comprising CDR1, CDR2 and CDR3 of the variable region of the light chain of an antibody comprising the amino acid sequence of SEQ ID NO: 18 (i.e., the CDR regions 34F4). Although such antibodies comprise CDR V sequences Hand VL of the 34F4 monoclonal antibody, they may contain different framework sequences. Such framework sequences can be obtained from public DNA databases or published reference materials that include germline antibody gene sequences. For example, germline DNA sequences for the human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" (available online at www.mrc-cpe.cam.ac.uk / vbase), and in Kabat et al. (1991), see above; Tomlinson et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops" J. Mol. Biol. 221:116-19%; and Cox et al. (1994) "A Directory of Human Germ-line V HSegments Reveals a Strong Bias in Their Usage" Eur. J. Immunol. 24:827-836; the contents of which are expressly 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 in accompanying Genbank accession numbers: 1-69 (NG_0010109, NT_024637, and BC070333), 3-33 (NG_0010109 and NT_024637), and 3-7 (NG_0010109 and NT_024637). As another example, the following heavy chain germline sequences found in the HCol2 HuMAb mouse are available in accompanying Genbank accession numbers: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

[0114] The protein sequences of the antibodies were compared with the compiled protein sequence database using one of the sequence similarity search methods called Gapped BLAST (Altschul et al. (1997), supra), which is well known to those skilled in the art.

[0115] Preferred framework sequences for use in the antibodies of the invention are sequences structurally similar to the framework sequences of the 13E2 or 34F4 antibodies.

[0116] Sequences showing significant alignment with the nucleic acid sequence encoding domain V H monoclonal antibody 13E2, include the following germline genes: IGHV8-8*01, IGHV8-11*01, IGHV8-12*01, IGHD2-12*01, IGHD1-1*01, IGHJ1*01, IGHJ1*02, IGHJ1*03.

[0117] Sequences showing significant alignment with the nucleic acid sequence encoding the VL domain of monoclonal antibody 13E2 include the following germline genes: IGKV6-17*01, IGKV6-25*01, IGKV6-23*01, IGKJ2*01, IGKJ2*03, IGKJ2*02.

[0118] Sequences showing significant alignment with the nucleic acid sequence encoding domain V H monoclonal antibody 34F4, include the following germline genes: IGHV8-8*01, IGHV8-12*01, IGHV8-11*01, IGHD1-1*01, IGHD1-2*01, IGHD2-3*01, IGHJ2*01, IGHJ2*02, IGHJ2*03.

[0119] Sequences showing significant alignment with the nucleic acid sequence encoding the VL domain of monoclonal antibody 34F4 include the following germline genes: IGKV6-17*01, IGKV6-25*01, IGKV6-23*01, IGKJ1*01, IGKJ1*02, IGKJ2*01.

[0120] Preferred heavy chain framework sequences for use in the antibodies of the invention are sequences that are structurally similar to the framework sequences encoded by the germline V gene of IGHV8-8*01, IGHV8-11*01 or IGHV8-12*01, especially IGHV8-8*01. Preferred light chain framework sequences for use in the antibodies of the invention are sequences that are structurally similar to the framework sequences encoded by the germline V gene of IGKV6-17*01, IGKV6-25*01 or IGKV6-23*01, especially IGKV6-17*01.

[0121] CDR1, CDR2, and CDR3 V sequences H and the sequences CDR1, CDR2 and CDR3 V Lmay be grafted onto framework regions that have a sequence identical to that found in the germline immunoglobulin gene from which the sequence is derived, or such CDR sequences may be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, it has been found that in some cases it is advantageous to mutate residues in framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762; and 6,180,370).

[0122] Another type of variable region modification is the mutation of amino acid residues within the CDR1, CDR2 and / or CDR3 regions of V Hand / or VL to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding or another functional property of interest can be assessed in the in vitro or in vivo assays described herein and provided in the examples. Conservative modifications (described above) are preferably introduced. Such mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically no more than one, two, three, four, or five residues within a CDR region are changed. In some embodiments, no more than one, two, three, four, or five residues in total are changed for all six CDR regions.

[0123] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises:

[0124] A VH region of an antibody comprising: a VH CDR1 having an amino acid sequence selected from SEQ ID NOs: 1 and 21; a VH CDR2 having an amino acid sequence selected from SEQ ID NOs: 2 and 22; and a VH CDR3 having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and a VL region of an antibody comprising: a VL CDR1 having an amino acid sequence selected from SEQ ID NOs: 4 and 24; a VL CDR2 having an amino acid sequence selected from SEQ ID NOs: 5 and 25; and a VL CDR3 having an amino acid sequence selected from SEQ ID NOs: 6 and 26; or

[0125] a variant thereof in which no more than one, two, three, four or five amino acid residues are altered by substitution, addition or deletion of amino acids within the CDR sequences.

[0126] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof comprises:

[0127] A VH region of an antibody comprising: a VH CDR1 having an amino acid sequence selected from SEQ ID NOs: 11 and 31, a VH CDR2 having an amino acid sequence selected from SEQ ID NOs: 12 and 32, and a VH CDR3 having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and a VL region of an antibody comprising: a VL CDR1 having an amino acid sequence selected from SEQ ID NOs: 14 and 34, a VL CDR2 having an amino acid sequence selected from SEQ ID NOs: 15 and 35, and a VL CDR3 having an amino acid sequence selected from SEQ ID NOs: 16 and 36; or

[0128] a variant thereof in which no more than one, two, three, four or five amino acid residues are altered by substitution, addition or deletion of amino acids within the CDR sequences.

[0129] In another embodiment, the invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region having SEQ ID NO: 1, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 1; (b) a VH CDR2 region having SEQ ID NO: 2, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 2; and (c) a VH CDR3 region having SEQ ID NO: 3, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 3;and / or a light chain variable region comprising: (a) a VL CDR1 region having SEQ ID NO: 4, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 4; (b) a VL CDR2 region comprising SEQ ID NO: 5, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 5; and (c) a VL CDR3 region comprising SEQ ID NO: 6, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 6.

[0130] In another embodiment, the invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region having SEQ ID NO: 11, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 11; (b) a VH CDR2 region having SEQ ID NO: 12, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 12; and (c) a VH CDR3 region having SEQ ID NO: 13, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 13;and / or a light chain variable region comprising: (a) a VL CDR1 region of SEQ ID NO: 14, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 14; (b) a VL CDR2 region of SEQ ID NO: 15, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 15; and (c) a VL CDR3 region of SEQ ID NO: 16, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 16.

[0131] In another embodiment, the invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region having SEQ ID NO: 21, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 21; (b) a VH CDR2 region having SEQ ID NO: 22, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 22; and (c) a VH CDR3 region having SEQ ID NO: 23, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 23;and / or a light chain variable region comprising: (a) a VL CDR1 region of SEQ ID NO: 24, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 24; (b) a VL CDR2 region of SEQ ID NO: 25, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 25; and (c) a VL CDR3 region of SEQ ID NO: 26, or or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 26.

[0132] In another embodiment, the invention provides an anti-LAG-3 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (a) a VH CDR1 region having SEQ ID NO: 31, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 31; (b) a VH CDR2 region having SEQ ID NO: 32, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 32; and (c) a VH CDR3 region having SEQ ID NO: 33, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 33;and / or a light chain variable region comprising: (a) a VL CDR1 region of SEQ ID NO: 34, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 34; (b) a VL CDR2 region of SEQ ID NO: 35, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 35; and (c) a VL CDR3 region of SEQ ID NO: 36, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 36.

[0133] The engineered antibodies of the invention include antibodies in which modifications have been made to framework residues in V Hand / or VL, for example, to improve the properties of a given antibody. Such framework modifications are typically performed to reduce the immunogenicity of the antibody. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has 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's framework sequences with the germline sequences from which the antibody is derived.

[0134] Another type of framework modification involves mutating one or more residues in the framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication 20030153043.

[0135] As a complement to or alternative to modifications made in the framework regions or CDR regions, the antibodies of the invention can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-independent cell-mediated cytotoxicity. In addition, the antibody of the invention can also be chemically modified (e.g., one or more chemical groups can be attached to the antibody), or can be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region is the EU Kabat index numbering.

[0136] In one embodiment, the hinge region of the CF11 domain is modified such 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 5,677,425. The number of cysteine ​​residues in the hinge region of the CF11 domain is altered, for example, to facilitate the assembly of light or heavy chains, or to increase or decrease the stability of the antibody.

[0137] In another embodiment, the Fc hinge region of an antibody is mutated to increase or decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the contact region of the CF12-CF13 domains of the Fc hinge fragment such that the antibody exhibits impaired binding to staphylococcal protein A (SpA) compared to the binding of the native Fc hinge domain to SpA. This approach is described in more detail in U.S. Patent 6,165,745.

[0138] In another embodiment, the antibody is modified to increase its biological half-life. The IgG class is the most stable and has a serum half-life of 20 days, while IgM and IgA only last 5-8 days (Brekke & Sandlie, 2003, Nature Reviews Drug Discovery 2, 52-62). Various approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375. Alternatively, to increase the biological half-life, the antibody may be altered within the CF11 or CL region such that it contains a salvage receptor binding epitope formed from two loops of the CP12 domain of the Fc region of IgG, as described in U.S. Patents 5,869,046 and 6,121,022.

[0139] The antibody of the invention or antigen-binding fragment thereof should be devoid of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), such that the antibody or fragment can be used to negatively regulate the proliferation and / or function of T cells without depleting T cells as a result of ADCC or CDC.

[0140] In ADCC, the Fc region of an antibody binds to Fc receptors (FcγRs) on the surface of immune effector cells such as natural killer cells and macrophages, leading to lysis of target cells. In CDC, the Fc region binds to the complement component C1q, and target cells are killed by initiating the complement cascade on the cell surface. The ADCC and CDC activity of an antibody depends on its isotype. Both IgM and IgG can mediate complement fixation, whereas only IgG can promote antibody-dependent cellular cytotoxicity (ADCC). IgG isoforms exhibit different levels of CDC and ADCC:

[0141] IgG: CDC (hIgG3>hIgG1>hIgG2>hIgG4; mIgG2a>mIgG1);

[0142] ADCC (hIgG1≥hIgG3>hIgG2≥IgG4; mIgG2a>mIgG1).

[0143] Thus, in some embodiments, the antibody of the invention or antigen-binding fragment thereof comprises the Fc fragment of mouse IgG1 or human IgG4 to ensure that the antibody or fragment does not have ADCC and CDC activity.

[0144] The ADCC and CDC activity of the antibody of the invention or its antigen-binding fragment can be determined using any suitable method known to those skilled in the art. Examples of suitable CDC and ADCC activity assays are described in WO 2008 / 132601 and below.

[0145] Anti-LAG-3 antibody exhibiting CDC activity will consistently destroy LAG-3 + cells in the presence of complement compared to its isotype control.

[0146] For CDC testing, target cells used to evaluate anti-LAG-3 antibodies can be a transfected LAG-3 cell line or primary T cells activated to induce LAG-3 expression. For example, in one possible assay for CDC testing, the target cells are LAG-3+ CHO cells compared to wt CHO cells. Both cell types (i.e., LAG-3-expressing cells and equivalent cells that do not express LAG-3) were incubated for 1 hour at 37°C with either the anti-LAG-3 antibody being tested or its isotype-matched negative control antibody and rabbit serum containing active complement. Cell viability was then assessed using the fluorescent dye 7-amino-actinomycin D (7-AAD), which labels cells that have lost their membrane integrity, a phenomenon that occurs rapidly after death.The percentage of 7-AAD-positive CHO cells (i.e., dead target cells) was determined by flow cytometry. An antibody exhibiting CDC activity will kill only LAG-3+ cells (e.g., LAG-3+ CHO cells) in the presence of complement. The anti-LAG-3 antibody can be titrated to determine the antibody's effectiveness in activating CDC at low antibody concentrations.

[0147] The CDC assay can also be performed on PBMCs stimulated with the SEB superantigen. The cytotoxicity of the test antibody was analyzed on both activated (i.e., CD25+ / LAG-3+ cells) and non-activated (i.e., CD25- / LAG-3- cells) CD4+ helper T cells and CD8+ cytotoxic T cells. Only activated CD4+ and CD8+ T cells were specifically killed by the CDC-expressing antibody.

[0148] In ADCC testing, PBMCs were stimulated for one day with IL-2 to serve as effector cells, and LAG-3-expressing cells (e.g., LAG-3+ CHO cells) were labeled with the vital dye CFSE to serve as target cells. In the presence of a test anti-LAG-3 antibody, if the effector cells (PBMCs) are able to kill a significant percentage of LAG-3-expressing cells (e.g., LAG-3+ CHO cells) compared to an isotype-matched negative control antibody, then the test antibody exhibits ADCC. This effect should increase with the number of effector cells. The test antibody can be titrated to determine the effectiveness of the antibody in inducing ADCC at low antibody concentrations.

[0149] A test anti-LAG-3 antibody can be considered to lack CDC or ADCC when it kills less than twice as many LAG-3+ cells as an isotype-matched negative control antibody in any of the assays described above.

[0150] Target cell death is exploited in classical ADCC bioassays, which utilize donor peripheral blood mononuclear cells (PBMCs) or a subset of natural killer (NK) cells as effector cells. However, these cells can vary in response and can lead to high background readings. The ADCC Reporter Bioassay, available from Promega, utilizes an alternative readout at an earlier point in the ADCC activation pathway: activation of gene transcription via the NFAT (nuclear factor of activated T cells) pathway in the effector cell. Additionally, the ADCC Reporter Bioassay utilizes engineered Jurkat cells stably expressing the FcγRIIIa receptor, the high-affinity V158 variant, and the NFAT response element driving firefly luciferase expression as effector cells.The biological activity of antibodies in ADCC was quantified using luciferase produced by NFAT pathway activation; luciferase activity in the effector cell was quantified using luminescence readings. Using this assay, the signal is high, and the background level in the assay is low. A satisfactory response in the assay is achieved only in the presence of target cells with the correct surface antigen, the correct specific antibody, and effector cells expressing FcγRIIIa. If any of the above is absent, there is no response.

[0151] When assessing the ADCC activity of an antibody using the ADCC Reporter Bioassay, the test antibody can be considered to not exhibit ADCC when the measured increase in bioluminescence is two-fold less than that of an isotype-matched negative control antibody.

[0152] In other embodiments, the Fc region comprises a mutant human IgG4 Fc sequence with an S228P mutation to abolish Fab fragment exchange (as shown in Figure 20(A) for the chimeric antibody Chim13E2IgG4 comprising the heavy chain sequence 13E2IgG4mut).

[0153] In other embodiments, the Fc region comprises the C portion of the wild-type human Ig (IgK) kappa chain (13E2IgK) (as shown in Figure 20(B) for the chimeric antibody Chim13E2IgG4 comprising the light chain sequence of 13E2IgK).

[0154] The numbering of residues in the Fc region used for the human IgG4 Fc mutant described above is the standard Eu index numbering as in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)).

[0155] In another embodiment, the glycosylation of the antibody is modified. For example, a deglycosylated antibody (i.e., an antibody lacking glycosylation) can be produced. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such modifications of the carbohydrate composition can be accomplished, for example, by altering one or more glycosylation sites in the amino acid sequence of the antibody. For example, one or more amino acid residues can be substituted that results in the elimination of one or more glycosylation sites of the variable region framework, thereby eliminating glycosylation at that site. Such deglycosylation can increase the affinity of the antibody for an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0156] Another modification of the antibodies of the present application that is contemplated by the present invention is PEGylation. An antibody can be PEGylated, for example, to increase its biological (e.g., serum) half-life. To accomplish PEGylation of an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), for example, a reactive ester or aldehyde derivative of PEG, under conditions whereby one or more PEG groups are attached to the antibody or fragment thereof. Preferably, PEGylation is accomplished by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer).The term "polyethylene glycol" as used herein is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(C1-C10)alkoxy or aryloxy polyethylene glycols or polyethyleneglycol maleimide. In some embodiments, the antibody to be PEGylated is a deglycosylated antibody. Methods for PEGylation of proteins are known in the art and can be used to PEGylate the antibodies of the invention. See, for example, European Patents EP 0 154 316 and EP 0 401 384.

[0157] The antibody according to the invention may be a monoclonal antibody or an antigen-binding fragment thereof.

[0158] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, antibody fragments that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAbs), single-domain antibodies (dAbs), single-domain heavy-chain antibodies, single-domain light-chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. The term also includes Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, and monoclonal antibodies. An antibody may be monovalent or bivalent.An antibody can be an Ig monomer, which is a "Y-shaped" molecule composed of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds. Antibodies can be labeled with a detectable label, such as a radioisotope, an enzyme that produces a detectable product, a fluorescent protein, etc. Antibodies can also be conjugated to other molecules, such as members of specific binding pairs, such as biotin (a member of the biotin-avidin specific binding pair), etc. Antibodies can also be bound to a solid support, including, but not limited to, polystyrene plates or particles, etc.

[0159] “Antibody fragments” comprise portions of an intact antibody, such as the binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057–1062 (1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, the name of which reflects its ability to readily crystallize. Pepsin treatment results in the formation of an F(ab')2 fragment that contains two antigen-binding sites and is still capable of causing antigen cross-linking.

[0160] "Fv" represents the smallest antibody fragment that contains the antigen recognition and binding site. This region consists of a variable domain dimer of one heavy and one light chain, tightly linked by non-covalent bonds. It is in this configuration that the three CDR regions of each variable domain interact, forming the antigen-binding site on the surface of the V dimer. H -V L Together, these six CDR regions provide the antibody with antigen-binding specificity. However, even a single variable domain (or half of an Fv, containing only three CDR regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the full binding region.

[0161] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of several residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of antibodies. In this document, Fab'-SH refers to Fab' fragments in which the cysteine ​​residue(s) in the constant domains contain a free thiol group. F(ab')2 fragments of antibodies were originally produced as a pair of Fab' fragments containing hinge cysteines between them. Other chemical conjugates of antibody fragments are also known.

[0162] The "light chains" of antibodies from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further subdivided into types, such as IgG2a and IgG2b.

[0163] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V domains H and V L antibodies, wherein these domains are in the same polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V domains. H and V L, which enables sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269–315 (1994).

[0164] The term "diabodies" refers to small antibody fragments with two antigen-binding sites that contain the heavy chain variable domain (V H ), linked to the light chain variable domain (V L ) in the same polypeptide chain (V H -V L ). When using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444–6448.

[0165] As used herein, a "monoclonal antibody" is an antibody produced by a group of identical cells, all of which were derived from a single cell by repeated cellular replication. That is, a clone of cells produces only one type of antibody. Although a monoclonal antibody can be produced using hybridoma technology, other production methods known to those skilled in the art can also be used (e.g., antibodies obtained from phage display antibody libraries).

[0166] As used herein, the term "CDR" or "complementarity determining region" refers to non-contiguous antigen-binding sites located in the variable regions of heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609–6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901–917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732–745 (1996). An additional system is the International ImMunoGeneTics (IMGT) numbering system (Lefranc et al., Nucleic Acids Research 27:209–212 (1999)). Definitions include overlapping or subgroups of amino acid residues when compared to each other.However, the application of any of these definitions to the CDRs of antibodies or grafted antibodies or variants thereof shall be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDR regions defined in each of the above references are presented below for comparison in Table 1. The CDR regions listed in Table 4 in Example 2 and in Table 9 in Example 5 were defined according to Lefranc 1999 and Kabat 1991.

[0167]

[0168] The terms "CDR-L1," "CDR-L2," and "CDR-L3" as used herein refer to the first, second, and third CDR regions in the variable region of the light chain, respectively. The terms "CDR-H1," "CDR-H2," and "CDR-H3" as used herein refer to the first, second, and third CDR regions in the variable region of the heavy chain, respectively. The terms "CDR-1," "CDR-2," and "CDR-3" as used herein refer to the first, second, and third CDR regions of each variable domain of the chain, respectively.

[0169] The term "affinity" used herein refers to the equilibrium constant for the reversible binding of two agents (e.g., antibody and antigen) and is expressed as the dissociation constant (K D). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more than the affinity of the antibody to unrelated amino acid sequences.The affinity of an antibody for a target protein can be, for example, about 100 nM (nanomolar concentration) to 0.1 nM, about 100 nM to 1 pM (picomolar concentration), or about 100 nM to 1 fM (femtomolear concentration), or more. As used herein, the term "avidy" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactive" and "preferentially binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0170] The term “binding” refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic interactions and / or hydrogen bonds, including interactions such as salt bridges and water bridges. The antibody of the invention specifically binds to an epitope within the LAG-3 protein, in particular the human LAG-3 protein. “Specific binding” refers to binding with an affinity of at least about 5×10 -7 M or more, for example 10 -7 M, 5×10 -8 M, 10 -8 M or greater. "Non-specific binding" refers to binding with an affinity of less than about 10 -7 M, for example, binding with an affinity of 10 -6 M, 10 -5 M, 10 -4M, etc. As used herein, an antibody that "specifically binds to human LAG-3" refers to an antibody that binds to human LAG-3 protein (and possibly LAG-3 protein from one or more other non-human species), but does not substantially bind to proteins other than the LAG-3 protein. Preferably, the antibody binds to human LAG-3 protein with "high affinity," namely, with a Kd of 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 M or less.

[0171] The expression “substantially does not bind” to a protein or cells as used herein means does not bind or does not bind with high affinity to a protein or cells, that is, binds to a protein or cells with K D 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, even more preferably 1×10 -2 M or more.

[0172] The term “K” used here assoc " or "K a " refers to the association rate constant of a particular antibody-antigen interaction, whereas the term "K" used herein dis " or "K d " refers to the dissociation rate constant of a particular antibody-antigen interaction. The term "K" used herein D » refers to the dissociation constant, which is obtained from the ratio K d to K a (that is, K d / K a ) and expressed as molar concentration (M).

[0173] The term “high affinity” in relation to an IgG antibody refers to an antibody that has a K D 1×10 -7 M or less, more preferably 5×10 -8 M or less, even more preferably lxlO"8 M or less, even more preferably 5×10 -9M or less and even more preferably 1×10 -9 M or less for the target antigen. However, "high-affinity" binding may vary for other antibody isotypes. For example, "high-affinity" binding for the IgM isotype refers to an antibody having a K D 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less.

[0174] In some embodiments, an antibody of the invention or antigen-binding fragment thereof binds to human LAG-3 protein with higher affinity (i.e., lower dissociation constant) than the antagonist anti-LAG-3 monoclonal antibody 17B4 (Baixeras, et al., J. Exp. Med., 1992, Vol. 176: 327-337). Example 7 below describes the results of a Biacore assay for binding of antibody 17B4 to human LAG-3Ig protein. The results showed that the dissociation constant of 17B4 for human LAG-3Ig was 3.69 nM. Thus, in some embodiments, an antibody of the present invention binds to human LAG-3 protein (or human LAG-3Ig protein) with a dissociation constant (K D ) less than 3.69 nM, for example, as determined by Biacore analysis.

[0175] In some embodiments, an antibody of the present invention binds to human LAG-3 protein (or a derivative thereof, such as human LAG-3Ig protein) with a dissociation constant (K D ) no more than 3.5 nM, no more than 2.5 nM, no more than 2 nM, no more than 1 nM, no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM. In some embodiments, an anti-LAG-3 antibody of the present invention binds to human LAG-3 protein with a K D no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, no more than 20 pM, no more than 10 pM, no more than 9 pM, no more than 8 pM, no more than 7 pM, no more than 6 pM, no more than 5 pM, no more than 4 pM, no more than 3 pM, no more than 2 pM or no more than 1 pM.

[0176] Example 20 below describes the results of a Biacore assay for the binding of the chimeric IgG4 13E2 antibody (described below) and humanized IgG4 13E2 (described below) to human LAG-3Ig protein. The results showed that the dissociation constant of the chimeric IgG4 13E2 antibody to human LAG-3Ig was 21.9 pM, while that of the humanized IgG4 13E2 to human LAG-3Ig was 22.8 pM.

[0177] In specific embodiments, an antibody of the present invention binds to a human LAG-3 protein (or a human LAG-3Ig protein) with a dissociation constant (Kd) of no more than 100 pM, no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, or no more than 25 pM, for example, as determined by a Biacore assay.

[0178] In some embodiments, the affinity of an antibody of the invention or an antigen-binding fragment thereof may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than the affinity of 17B4 for the human LAG-3 protein. In some embodiments, an antibody of the invention or an antigen-binding fragment thereof may be at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, or 3.5-fold higher than the affinity of 17B4 for the human LAG-3 protein.

[0179] The affinity of an antibody for the human LAG-3 protein can be determined by one skilled in the art, for example, using surface plasmon resonance, for example, using a biosensor system such as the Biacore system (Murphy et al, Using Biacore to measure the binding kinetics of an antibody-antigen interaction; Curr Protoc Protein Sci. 2006 Sep; Chapter 19:Unit 19.14). For example, the Biacore assay can be used to determine the dissociation constant between the antibody of the invention and the human LAG-3 protein.

[0180] Binding to human LAG-3 can be assessed using one or more other techniques also well known in the art. For example, the antibody can be tested using a flow cytometric assay in which the antibody reacts with a cell line expressing human LAG-3, such as CHO cells that have been transfected to express human LAG-3 on their cell surface. Other suitable cells for use in flow cytometric assays include SEB-stimulated PBMCs or anti-CD3-stimulated CD4 + Activated T cells expressing native LAG-3. Other suitable binding assays include ELISAs, such as those using recombinant LAG-3 protein.

[0181] The antibody of the invention is an isolated antibody. An "isolated" antibody is one that has been identified and separated, and / or removed, from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In some embodiments, the antibody is purified (1) to greater than 90%, greater than 95%, or greater than 98% by weight of the antibody as determined by the Lowry method, such as greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibody includes antibody in situ in recombinant cells, since at least one component of the antibody's natural environment is absent. In some cases, isolated antibody will be obtained through at least one purification step.

[0182] In preferred embodiments, the antibody of the invention is a humanized antibody or an antigen-binding fragment thereof, in particular a humanized monoclonal antibody or an antigen-binding fragment thereof.

[0183] The humanized antibody of the invention or an antigen-binding fragment thereof may comprise a humanized light chain framework region and / or a humanized heavy chain framework region.

[0184] The term "humanized antibody" as used herein refers to an immunoglobulin comprising portions of immunoglobulins of different origins, wherein at least one portion comprises amino acid sequences of human origin. For example, a humanized antibody may comprise portions derived from an immunoglobulin of non-human origin with the required specificity, such as a mouse, and from sequences of an immunoglobulin of human origin (e.g., an immunoglobulin), linked together chemically by conventional means (e.g., synthetically) or produced as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of a chimeric antibody can be expressed to produce a continuous polypeptide chain).Another example of a humanized immunoglobulin is an immunoglobulin comprising one or more immunoglobulin chains comprising a CDR region derived from an antibody of non-human origin and a framework region derived from a light and / or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework alterations). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., Cabilly et al., U.S. Patent 4,816,567; Cabilly et al., European Patent 0125,023 B1; Boss et al., U.S. Patent 4,816,397; Boss et al., European Patent 0120,694 B1; Neuberger, M.S. et al., WO 86 / 01533; Neuberger, M.S. et al., European Patent 0194276 B1; Winter, U.S. Patent 5,225,539; Winter, European Patent 0239400 B1; Padlan, E.A. et al., European Patent Application 0519596 A1. See also Ladner et al., U.S. Patent 4,946,778; Huston, U.S. Patent 5,476,786; and Bird, R.E. et al., Science, 242: 423–426 (1988) concerning single-chain antibodies.

[0185] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. The amino acid sequence of the variable region may be identical to the amino acid sequence of the variable region of the species from which it is obtained (e.g., a mouse sequence), or may be at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to this variable region sequence.For example, the amino acid sequence of the variable region of a chimeric antibody of the invention may contain one or more amino acid deletions, substitutions, or additions (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, substitutions, or additions) compared to the amino acid sequence of the variable region of the species from which it is derived.

[0186] Similarly, the amino acid sequence of the constant region may be identical to the amino acid sequence of the constant region of the species from which it is derived (e.g., a human sequence), or may be at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the constant region. For example, the amino acid sequence of the constant region of a chimeric antibody of the invention may contain one or more amino acid deletions, substitutions, or additions (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, substitutions, or additions) compared to the amino acid sequence of the constant region of the species from which it is derived.

[0187] For example, as described above, the amino acid sequence of the Fc hinge region of the chimeric antibody can be mutated to decrease the biological half-life of the antibody, or the amino acid sequence of the Fc region can be mutated to increase the biological half-life of the chimeric antibody.

[0188] For example, in some embodiments of the chimeric antibodies of the invention, the heavy chain variable region sequence comprises or is derived from a murine antibody, and the heavy chain constant region sequence comprises or is derived from an IgG4 Fc sequence. In other embodiments, the light chain variable region sequence comprises or is derived from a murine antibody, and the light chain constant region sequence comprises or is derived from a human IgK C chain kappa (IgK) sequence.

[0189] In other embodiments, the Fc region comprises a mutant human IgG4 Fc sequence with an S228P mutation to abolish Fab fragment exchange (as shown in Figure 20(A) for the chimeric antibody Chim13E2IgG4 comprising the heavy chain sequence 13E2IgG4mut).

[0190] In other embodiments, the Fc region comprises a portion of the C chain of a wild-type human Ig (IgK) kappa (13E2IgK) (as shown in Figure 20(B) for the chimeric antibody Chim13E2IgG4 comprising the light chain sequence of 13E2IgK).

[0191] The numbering of residues in the Fc region used for the human IgG4 Fc mutant described above is the standard Eu index numbering as in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)).

[0192] Humanized antibodies can be produced using synthetic and / or recombinant nucleic acids to produce genes (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences encoding humanized variable regions can be engineered using PCR mutagenesis techniques to alter DNA sequences encoding a human or humanized chain, such as template DNA, from a previously humanized variable region (see, e.g., Kamman, M., et al., Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, BL et al., Nucleic Acids Res., 19(9): 2471–2476 (1991); and Lewis, AP and JS Crowe, Gene, 101: 297–302 (1991)). Using these or other suitable methods, variants can also be obtained.For example, cloned variable regions can be mutagenized and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see, e.g., Krebber et al., US Pat. No. 5,514,548; Hoogenboom et al., WO 93 / 06213, published Apr. 1, 1993)).

[0193] The term "framework" as used herein in relation to the variable region of an antibody refers to all amino acid residues within the variable region of the antibody outside the CDR regions. The framework of a variable region typically represents a discontinuous amino acid sequence approximately 100-120 amino acids long, but it refers only to those amino acids outside the CDR regions. The term "framework region" as used herein refers to those regions of the framework that are separated by the CDR regions.

[0194] Humanization of the framework region(s) reduces the risk of developing antibodies that elicit a human anti-mouse antibody (HAMA) response in humans. Art-recognized methods for determining the immune response can be used to monitor the HAMA response in a specific patient or during clinical trials. Patients administered humanized antibodies can be assessed for immunogenicity at the start and during therapy. The HAMA response is measured, for example, by detecting antibodies to the humanized therapeutic reagent in serum samples from patients using a method known in the art, including surface plasmon resonance technology (BIACORE) and / or enzyme-linked immunosorbent assay (ELISA). In many cases, the humanized antibody of the invention does not substantially elicit a HAMA response in human subjects.

[0195] Certain amino acid substitutions within the human variable region framework are selected based on their potential impact on CDR conformation and / or antigen binding. The unnatural alignment of mouse CDR regions with the human variable region framework can lead to unnatural conformational constraints that, if not corrected by substituting specific amino acid residues, result in loss of binding affinity.

[0196] The selection of amino acid residues for replacement can be determined, in particular, by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Typically, molecular models are created starting from the resolved structures of immunoglobulin chains or their domains. The chains for modeling are compared by amino acid sequence similarity with the chains or domains of the resolved three-dimensional structure, and the chains or domains exhibiting the greatest sequence similarity are selected as the starting point for constructing the molecular model. Chains or domains possessing at least 50% sequence identity are selected for modeling, for example, chains or domains that possess at least 60%, at least 70%, at least 80%, at least 90% sequence identity, or more.The resolved initial structures are modeled in a way that allows for differences between the actual amino acids in the immunoglobulin chains or domains being modeled and those in the initial structure. The modified structures are then assembled into the composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from one another and that bond lengths and angles are within chemically acceptable limits.

[0197] CDRs and framework regions can be defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). An alternative structural definition was proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol. 186:651 (1989) (collectively referred to as "Chothia"). When the framework residues, as defined by Kabat above, are loop structural residues, as defined by Chothia above, amino acids present in the mouse antibody can be selected for substitution in the humanized antibody. Residues that are “adjacent to a CDR region” include amino acid residues at positions immediately adjacent to one or more CDRs in the primary sequence of a humanized immunoglobulin chain, such as at positions immediately adjacent to a CDR as defined by Kabat or a CDR as defined by Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids are particularly likely to interact with amino acids in the CDR regions and, if selected from the acceptor, distort the donor CDRs and reduce affinity. Furthermore, adjacent amino acids may interact directly with the antigen (Amit et al., Science, 233:747 (1986)) and selecting these amino acids from the donor may be desirable to preserve all contacts with the antigen that confer affinity to the parent antibody. Alternatively, the CDRs and framework regions may be as defined by MacCallum et al. or Lefranc et al. (see Table 1 above).

[0198] In some cases, humanized framework region V H or frame region V L represents a consensus humanized framework region. The consensus humanized framework region may represent the most abundant amino acid residue in the selection of V framework sequences. L or VH human immunoglobulin.

[0199] In some embodiments, an antibody of the invention or a fragment thereof comprises one or more humanized framework regions (FRs). In some embodiments, the subject anti-LAG-3 antibody comprises a light chain variable region comprising one, two, three or four light chain FRs that have been humanized. In some embodiments, the subject antibody comprises a light chain variable region comprising, from N-terminus to C-terminus: a humanized light chain FR1; CDR-L1 as described herein; a humanized light chain FR2; CDR-L2 as described herein; a humanized light chain FR3; CDR-L3 as described herein; and a humanized light chain FR4. In some embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are as follows: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0200] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having the amino acid sequence of SEQ ID NO: 1; a humanized light chain FR2; a CDR-L2 having the amino acid sequence of SEQ ID NO: 2; a humanized light chain FR3; a CDR-L3 having the amino acid sequence of SEQ ID NO: 3; and a humanized light chain FR4.

[0201] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0202] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR; CDR-L1 having the amino acid sequence of SEQ ID NO: 4; a humanized light chain FR2; CDR-L2 having the amino acid sequence of SEQ ID NO: 5; a humanized light chain FR3; CDR-L3 having the amino acid sequence of SEQ ID NO: 6; and a humanized light chain FR4.

[0203] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

[0204] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having the amino acid sequence of SEQ ID NO: 24; a humanized light chain FR2; a CDR-L2 having the amino acid sequence of SEQ ID NO: 25; a humanized light chain FR3; a CDR-L3 having the amino acid sequence of SEQ ID NO: 26; and a humanized light chain FR4.

[0205] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are selected from: SEQ ID NO: 4 and 24 (CDR-Ll); SEQ ID NO: 5 and 25 (CDR-L2); and SEQ ID NO: 6 and 26 (CDR-L3).

[0206] For example, the antibody in question may comprise a light chain variable region comprising, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having an amino acid sequence selected from SEQ ID NOs: 4 and 24; a humanized light chain FR2; a CDR-L2 having an amino acid sequence selected from SEQ ID NOs: 5 and 25; a humanized light chain FR3; a CDR-L3 having an amino acid sequence selected from SEQ ID NOs: 6 and 26; and a humanized light chain FR4.

[0207] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0208] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having the amino acid sequence of SEQ ID NO: 11; a humanized light chain FR2; a CDR-L2 having the amino acid sequence of SEQ ID NO: 12; a humanized light chain FR3; a CDR-L3 having the amino acid sequence of SEQ ID NO: 13; and a humanized light chain FR4.

[0209] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0210] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having the amino acid sequence of SEQ ID NO: 14; a humanized light chain FR2; a CDR-L2 having the amino acid sequence of SEQ ID NO: 15; a humanized light chain FR3; a CDR-L3 having the amino acid sequence of SEQ ID NO: 16; and a humanized light chain FR4.

[0211] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36.

[0212] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having the amino acid sequence of SEQ ID NO: 34; a humanized light chain FR2; a CDR-L2 having the amino acid sequence of SEQ ID NO: 35; a humanized light chain FR3; a CDR-L3 having the amino acid sequence of SEQ ID NO: 36; and a humanized light chain FR4.

[0213] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are selected from: SEQ ID NO: 14 and 34 (CDR-Ll); SEQ ID NO: 15 and 35 (CDR-L2); and SEQ ID NO: 16 and 36 (CDR-L3).

[0214] For example, the antibody in question may comprise a light chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized light chain FR1; a CDR-L1 having an amino acid sequence selected from SEQ ID NOs: 14 and 34; a humanized light chain FR2; a CDR-L2 having an amino acid sequence selected from SEQ ID NOs: 15 and 35; a humanized light chain FR3; a CDR-L3 having an amino acid sequence selected from SEQ ID NOs: 16 and 36; and a humanized light chain FR4.

[0215] In some embodiments, the subject anti-LAG-3 antibody comprises a heavy chain variable region comprising one, two, three or four heavy chain FR regions that have been humanized. In some embodiments, the subject antibody comprises a heavy chain variable region comprising, from N-terminus to C-terminus: a humanized heavy chain FR1; CDR-H1 as described herein; a humanized heavy chain FR2; CDR-H2 as described herein; a humanized heavy chain FR3; CDR-H3 as described herein; and a humanized heavy chain FR4.

[0216] In some embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0217] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 4; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 5; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and a humanized heavy chain FR4.

[0218] In other embodiments, the corresponding amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 are as follows: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0219] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 1; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 2; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 3; and a humanized heavy chain FR4.

[0220] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are as follows: SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0221] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 21; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 22; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 23; and a humanized heavy chain FR4.

[0222] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are selected from the following: SEQ ID NOs: 1 and 21 (CDR-H1); SEQ ID NOs: 2 and 22 (CDR-H2); and SEQ ID NOs: 3 and 23 (CDR-H3).

[0223] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having an amino acid sequence selected from SEQ ID NOs: 1 and 21; a humanized heavy chain FR2; a CDR-H2 having an amino acid sequence selected from SEQ ID NOs: 2 and 22; a humanized heavy chain FR3; a CDR-H3 having an amino acid sequence selected from SEQ ID NO: 23; and a humanized heavy chain FR4.

[0224] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are selected from the following: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0225] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 14; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 15; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 16; and a humanized heavy chain FR4.

[0226] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are as follows: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13

[0227] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 11; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 12; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 13; and a humanized heavy chain FR4.

[0228] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are as follows: SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33

[0229] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having the amino acid sequence of SEQ ID NO: 31; a humanized heavy chain FR2; a CDR-H2 having the amino acid sequence of SEQ ID NO: 32; a humanized heavy chain FR3; a CDR-H3 having the amino acid sequence of SEQ ID NO: 33; and a humanized heavy chain FR4.

[0230] In other embodiments, the corresponding amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are selected from: SEQ ID NO: 11 and 31 (CDR-H1); SEQ ID NO: 12 and 32 (CDR-H2); and SEQ ID NO: 13 and 33 (CDR-H3).

[0231] For example, the antibody in question may comprise a heavy chain variable region that comprises, in the direction from the N-terminus to the C-terminus: a humanized heavy chain FR1; a CDR-H1 having an amino acid sequence selected from SEQ ID NOs: 11 and 31; a humanized heavy chain FR2; a CDR-H2 having an amino acid sequence selected from SEQ ID NOs: 12 and 32; a humanized heavy chain FR3; a CDR-H3 having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and a humanized heavy chain FR4.

[0232] Examples of suitable humanized framework sequences include:

[0233] option 1 VH (VH1)

[0234] VH1FR1: QVTLKESGPALVKPTQTLTLTCTFS (SEQ ID NO: 52);

[0235] VH1FR2: WIRQPPGKALEWLA (SEQ ID NO: 53);

[0236] VH1FR3: RLTISKDTSKSQVILNMTNMDPVDTATYYC (SEQ ID NO: 54); And

[0237] VH1FR4: WGQGTTVTVSS (SEQ ID NO: 55);

[0238] option 2 VH (VH2)

[0239] VH2FR1: QITLKESGPALVKPTQTLTLTCSFS (SEQ ID NO: 56);

[0240] VH2FR2: WIRQPPGKALEWLA (SEQ ID NO: 57);

[0241] VH2FR3: RLTISKDTSKNQVVLTMANMDPVDTATYYC (SEQ ID NO: 58);

[0242] VH2FR4: WGQGTTVTVSS (SEQ ID NO: 59);

[0243] variant 3 V H (VH3)

[0244] VH3FR1: QITLKETGPTLVKPTQTLTLTCTFS (SEQ ID NO: 60);

[0245] VH3FR2: WIRQPPGKALEWVT (SEQ ID NO: 61);

[0246] VH3FR3: RVTIRKDTSKNQVALTMTNMDPLDTGTYYC (SEQ ID NO: 62);

[0247] VH3FR4: WGQGTLVTVSS (SEQ ID NO: 63);

[0248] variant 4 V H (VH4)

[0249] VH4FR1: QITLKESGPTLVKPTQTLTLTCTFS (SEQ ID NO: 64);

[0250] VH4FR2: WIRQPPGKTLEWLT (SEQ ID NO: 65);

[0251] VH4FR3: RLSITKDTSKNQVVLTMTNMDPLDTGTYYC (SEQ ID NO: 66);

[0252] VH4FR4: WGQGTLVTVSS (SEQ ID NO: 67);

[0253] вариант 1 VL (VL1)

[0254] VL1FR1: DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 68);

[0255] VL1i FR2: WYQQKPGQPPKLLIY (SEQ ID NO: 69);

[0256] VL i FR3: GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 70); и

[0257] VL1FR4: FGQGTKLEIK (SEQ ID NO: 71);

[0258] вариант 2 VL (VL2)

[0259] VL2FR1: DIQMTQSPSSLS ASVGDRVTITC (SEQ ID NO: 72);

[0260] VL2FR2: WYQQKPGQAPKLLIF (SEQ ID NO: 73);

[0261] VL2FR3: GVPSRFSGSGSGTDFTLTLSSLQPEDFATYYC (SEQ ID NO: 74); и

[0262] VL2FR4: FGQGTKVEIK (SEQ ID NO: 75);

[0263] вариант 3 VL (VL3)

[0264] VL3FR1: DIVMTQTPS S LS AS VGDRVTITC (SEQ ID NO: 76);

[0265] VL3FR2: WYQQRPGQAPKLLIY (SEQ ID NO: 77);

[0266] VL3FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 78); и

[0267] VL3FR4: FGQGTRLDIK (SEQ ID NO: 79);

[0268] вариант 4 VL (VL4)

[0269] VL4FR1: EIVLTQSPDSLAVSLGERATINC (SEQ ID NO: 80);

[0270] VL4FR2: WYQQKAGQSPKLLIY (SEQ ID NO: 81);

[0271] VL4FR3: GVPDRFSGSGSGTDFTLTIDSLQAEDVAVYYC (SEQ ID NO: 82); And

[0272] VL4FR4: FGGGTKVEIK (SEQ ID NO: 83).

[0273] Figure 22 shows the sequences of the variable regions of humanized variants 1-4 V H and variants 1–4 VL, aligned with the corresponding sequence of the parental mouse antibody 13E2. The CDR sequences are highlighted in gray. Changes in the humanized framework sequences of the variants compared to the parental mouse sequence are shown underlined and in bold. The altered residues in the humanized sequence for each variant are also shown in Tables 26 (heavy chain sequences) and 27 (light chain sequences) in Example 18.

[0274] In some embodiments, a humanized antibody of the invention (or antigen-binding fragment thereof) comprises a humanized heavy chain that comprises any of the amino acid substitutions shown for VH1, VH2, VH3, or VH4 in Table 26, and / or a humanized light chain that comprises any of the amino acid substitutions shown for VL1, VL2, VL3, or VL4 in Table 27.

[0275] The antibody of the invention or an antigen-binding fragment thereof (in particular, the humanized antibody of the invention or an antigen-binding fragment thereof) may comprise any of the above-mentioned humanized framework sequences (SEQ ID NOs: 52-83), or any combination of the above-mentioned humanized framework sequences (SEQ ID NOs: 52-83).

[0276] In some embodiments, the humanized heavy chain framework region may comprise an amino acid sequence shown in any of: SEQ ID NO: 52, 53, 54, or 55; any of SEQ ID NO: 56, 57, 58, or 59; any of SEQ ID NO: 60, 61, 62, or 63; or any of SEQ ID NO: 64, 65, 66, or 67.

[0277] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof (in particular, a humanized antibody of the invention or an antigen-binding fragment thereof) may comprise:

[0278] frame area 1 V H 1 (FR1 VH) sequence SEQ ID NO: 52; FR2 V H sequence SEQ ID NO: 53; FR3 V H sequences SEQ ID NO: 54; and FR4 V H sequence SEQ ID NO: 55;

[0279] frame area 1 V H (FR1 VH) sequences SEQ ID NO: 56; FR2 V H sequence SEQ ID NO: 57; FR3 V Hsequences SEQ ID NO: 58; and FR4 V H sequence SEQ ID NO: 59;

[0280] frame area 1 V H (FR1 VH) sequences SEQ ID NO: 60; FR2 V H sequence SEQ ID NO: 61; FR3 V H sequences SEQ ID NO: 62; and FR4 V H sequence SEQ ID NO: 63; or

[0281] frame area 1 V H (FR1 VH) sequences SEQ ID NO: 64; FR2 V H sequence SEQ ID NO: 65; FR3 V H sequences SEQ ID NO: 66; and FR4 V H sequence SEQ ID NO: 67.

[0282] Alternatively or additionally, in some embodiments, the humanized light chain framework region may comprise an amino acid sequence shown in any of: SEQ ID NO: 68, 69, 70, or 71; any of SEQ ID NO: 72, 73, 74, or 75; any of SEQ ID NO: 76, 77, 78, or 79; or any of SEQ ID NO: 80, 81, 82, or 83.

[0283] In specific embodiments, an antibody of the invention or an antigen-binding fragment thereof (in particular, a humanized antibody of the invention or an antigen-binding fragment thereof) may comprise:

[0284] VL framework region 1 (FR1 VL) of the sequence SEQ ID NO: 68; VL FR2 of the sequence SEQ ID NO: 69; VL FR3 of the sequence SEQ ID NO: 70; and VL FR4 of the sequence SEQ ID NO: 71;

[0285] VL framework region 1 (FR1 VL) of the sequence SEQ ID NO: 72; VL FR2 of the sequence SEQ ID NO: 73; VL FR3 of the sequence SEQ ID NO: 74; and VL FR4 of the sequence SEQ ID NO: 75;

[0286] VL framework region 1 (FR1 VL) of the sequence SEQ ID NO: 76; VL FR2 of the sequence SEQ ID NO: 77; VL FR3 of the sequence SEQ ID NO: 78; and VL FR4 of the sequence SEQ ID NO: 79; or

[0287] VL framework region 1 (FR1 VL) of the sequence SEQ ID NO: 80; VL FR2 of the sequence SEQ ID NO: 81; VL FR3 of the sequence SEQ ID NO: 82; and VL FR4 of the sequence SEQ ID NO: 83.

[0288] In particular embodiments, an antibody of the invention or an antigen-binding fragment thereof (in particular, a humanized antibody of the invention or an antigen-binding fragment thereof) may comprise any of the following combinations of humanized framework sequences:

[0289] FR1-FR4 VH1; and FR1-FR4 VL1;

[0290] FR1-FR4 VH1; and FR1-FR4 VL2;

[0291] FR1-FR4 VH1; and FR1-FR4 VL3;

[0292] FR1-FR4 VH1; and FR1-FR4 VL4;

[0293] FR1-FR4 VH2; and FR1-FR4 VL1;

[0294] FR1-FR4 VH2; and FR1-FR4 VL2;

[0295] FR1-FR4 VH2; and FR1-FR4 VL3;

[0296] FR1-FR4 VH2; and FR1-FR4 VL4;

[0297] FR1-FR4 VH3; and FR1-FR4 VL1;

[0298] FR1-FR4 VH3; and FR1-FR4 VL2;

[0299] FR1-FR4 VH3; and FR1-FR4 VL3;

[0300] FR1-FR4 VH3; and FR1-FR4 VL4;

[0301] FR1-FR4 VH4; and FR1-FR4 VL1;

[0302] FR1-FR4 VH4; and FR1-FR4 VL2;

[0303] FR1-FR4 VH4; and FR1-FR4 VL3;

[0304] FR1-FR4 VH4; and FR1-FR4 VL4.

[0305] In a specific embodiment, the antibody of the invention or antigen-binding fragment thereof (in particular, the humanized antibody of the invention or antigen-binding fragment thereof) comprises the following humanized framework sequences:

[0306] FR1 VH4: QITLKESGPTLVKPTQTLTLTCTFS (SEQ ID NO: 64);

[0307] FR2 VH4: WIRQPPGKTLEWLT (SEQ ID NO: 65);

[0308] FR3 VH4: RLSITKDTSKNQVVLTMTNMDPLDTGTYYC (SEQ ID NO: 66); And

[0309] FR4 VH4: WGQGTLVTVSS (SEQ ID NO: 67); And

[0310] FR1 VL3: DIVMTQTPSSLS ASVGDRVTITC (SEQ ID NO: 76);

[0311] FR2 VL3: WYQQRPGQAPKLLIY (SEQ ID NO: 77);

[0312] FR3 VL3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 78); And

[0313] FR4 VL3: FGQGTRLDIK (SEQ ID NO: 79).

[0314] In specific embodiments, an isolated antibody of the invention or antigen-binding fragment thereof comprises a VH region of an antibody comprising:

[0315] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 1 and 21; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 2 and 22; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0316] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having the amino acid sequence of SEQ ID NO: 1; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having the amino acid sequence of SEQ ID NO: 2; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having the amino acid sequence of SEQ ID NO: 3; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0317] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having the amino acid sequence of SEQ ID NO: 21; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having the amino acid sequence of SEQ ID NO: 22; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having the amino acid sequence of SEQ ID NO: 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0318] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 1 and 21; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 2 and 22; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0319] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having the amino acid sequence of SEQ ID NO: 1; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having the amino acid sequence of SEQ ID NO: 2; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having the amino acid sequence of SEQ ID NO: 3; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0320] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having the amino acid sequence of SEQ ID NO: 21; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having the amino acid sequence of SEQ ID NO: 22; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having the amino acid sequence of SEQ ID NO: 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0321] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 1 and 21; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 2 and 22; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0322] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having the amino acid sequence of SEQ ID NO: 1; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having the amino acid sequence of SEQ ID NO: 2; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having the amino acid sequence of SEQ ID NO: 3; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0323] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having the amino acid sequence of SEQ ID NO: 21; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having the amino acid sequence of SEQ ID NO: 22; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having the amino acid sequence of SEQ ID NO: 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0324] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 1 and 21; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 2 and 22; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 3 and 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 67;

[0325] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having the amino acid sequence of SEQ ID NO: 1; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having the amino acid sequence of SEQ ID NO: 2; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having the amino acid sequence of SEQ ID NO: 3; and FR4 VH having the amino acid sequence of SEQ ID NO: 67; or

[0326] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having the amino acid sequence of SEQ ID NO: 21; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having the amino acid sequence of SEQ ID NO: 22; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having the amino acid sequence of SEQ ID NO: 23; and FR4 VH having the amino acid sequence of SEQ ID NO: 67.

[0327] In a specific embodiment, an isolated antibody of the invention or an antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 84.

[0328] Alternatively or additionally, in specific embodiments, an isolated antibody of the invention or antigen-binding fragment thereof comprises a VL region of an antibody comprising:

[0329] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 4 and 24; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 5 and 25; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 6 and 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0330] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having the amino acid sequence of SEQ ID NO: 4; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having the amino acid sequence of SEQ ID NO: 5; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having the amino acid sequence of SEQ ID NO: 6; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0331] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having the amino acid sequence of SEQ ID NO: 24; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having the amino acid sequence of SEQ ID NO: 25; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having the amino acid sequence of SEQ ID NO: 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0332] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 4 and 24; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 5 and 25; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 6 and 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0333] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having the amino acid sequence of SEQ ID NO: 4; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having the amino acid sequence of SEQ ID NO: 5; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having the amino acid sequence of SEQ ID NO: 6; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0334] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having the amino acid sequence of SEQ ID NO: 24; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having the amino acid sequence of SEQ ID NO: 25; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having the amino acid sequence of SEQ ID NO: 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0335] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 4 and 24; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 5 and 25; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 6 and 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0336] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having the amino acid sequence of SEQ ID NO: 4; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having the amino acid sequence of SEQ ID NO: 5; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having the amino acid sequence of SEQ ID NO: 6; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0337] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having the amino acid sequence of SEQ ID NO: 24; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having the amino acid sequence of SEQ ID NO: 25; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having the amino acid sequence of SEQ ID NO: 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0338] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 4 and 24; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 5 and 25; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 6 and 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 83;

[0339] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having the amino acid sequence of SEQ ID NO: 4; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having the amino acid sequence of SEQ ID NO: 5; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having the amino acid sequence of SEQ ID NO: 6; and FR4 VL having the amino acid sequence of SEQ ID NO: 83;

[0340] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having the amino acid sequence of SEQ ID NO: 24; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having the amino acid sequence of SEQ ID NO: 25; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having the amino acid sequence of SEQ ID NO: 26; and FR4 VL having the amino acid sequence of SEQ ID NO: 83.

[0341] In a specific embodiment, an isolated antibody of the invention or an antigen-binding fragment thereof comprises an antibody light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0342] In a further specific embodiment, the isolated antibody of the invention or antigen-binding fragment thereof comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0343] In other embodiments, an isolated antibody of the invention or an antigen-binding fragment thereof comprises a VH region of an antibody comprising:

[0344] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 11 and 31; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 12 and 32; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0345] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having the amino acid sequence of SEQ ID NO: 11; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having the amino acid sequence of SEQ ID NO: 12; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having the amino acid sequence of SEQ ID NO: 13; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0346] FR1 VH having the amino acid sequence of SEQ ID NO: 52; CDR1 VH having the amino acid sequence of SEQ ID NO: 31; FR2 VH having the amino acid sequence of SEQ ID NO: 53; CDR2 VH having the amino acid sequence of SEQ ID NO: 32; FR3 VH having the amino acid sequence of SEQ ID NO: 54; CDR3 VH having the amino acid sequence of SEQ ID NO: 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 55;

[0347] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 11 and 31; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 12 and 32; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0348] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having the amino acid sequence of SEQ ID NO: 11; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having the amino acid sequence of SEQ ID NO: 12; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having the amino acid sequence of SEQ ID NO: 13; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0349] FR1 VH having the amino acid sequence of SEQ ID NO: 56; CDR1 VH having the amino acid sequence of SEQ ID NO: 31; FR2 VH having the amino acid sequence of SEQ ID NO: 57; CDR2 VH having the amino acid sequence of SEQ ID NO: 32; FR3 VH having the amino acid sequence of SEQ ID NO: 58; CDR3 VH having the amino acid sequence of SEQ ID NO: 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 59;

[0350] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 11 and 31; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 12 and 32; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0351] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having the amino acid sequence of SEQ ID NO: 11; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having the amino acid sequence of SEQ ID NO: 12; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having the amino acid sequence of SEQ ID NO: 13; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0352] FR1 VH having the amino acid sequence of SEQ ID NO: 60; CDR1 VH having the amino acid sequence of SEQ ID NO: 31; FR2 VH having the amino acid sequence of SEQ ID NO: 61; CDR2 VH having the amino acid sequence of SEQ ID NO: 32; FR3 VH having the amino acid sequence of SEQ ID NO: 62; CDR3 VH having the amino acid sequence of SEQ ID NO: 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 63;

[0353] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having an amino acid sequence selected from SEQ ID NOs: 11 and 31; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having an amino acid sequence selected from SEQ ID NOs: 12 and 32; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having an amino acid sequence selected from SEQ ID NOs: 13 and 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 67;

[0354] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having the amino acid sequence of SEQ ID NO: 11; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having the amino acid sequence of SEQ ID NO: 12; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having the amino acid sequence of SEQ ID NO: 13; and FR4 VH having the amino acid sequence of SEQ ID NO: 67; and

[0355] FR1 VH having the amino acid sequence of SEQ ID NO: 64; CDR1 VH having the amino acid sequence of SEQ ID NO: 31; FR2 VH having the amino acid sequence of SEQ ID NO: 65; CDR2 VH having the amino acid sequence of SEQ ID NO: 32; FR3 VH having the amino acid sequence of SEQ ID NO: 66; CDR3 VH having the amino acid sequence of SEQ ID NO: 33; and FR4 VH having the amino acid sequence of SEQ ID NO: 67.

[0356] Alternatively or additionally, in specific embodiments, an isolated antibody of the invention or antigen-binding fragment thereof comprises a VL region of an antibody comprising:

[0357] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 14 and 34; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 15 and 35; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 16 and 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0358] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having the amino acid sequence of SEQ ID NO: 14; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having the amino acid sequence of SEQ ID NO: 15; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having the amino acid sequence of SEQ ID NO: 16; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0359] FR1 VL having the amino acid sequence of SEQ ID NO: 68; CDR1 VL having the amino acid sequence of SEQ ID NO: 34; FR2 VL having the amino acid sequence of SEQ ID NO: 69; CDR2 VL having the amino acid sequence of SEQ ID NO: 35; FR3 VL having the amino acid sequence of SEQ ID NO: 70; CDR3 VL having the amino acid sequence of SEQ ID NO: 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 71;

[0360] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 14 and 34; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 15 and 35; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 16 and 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0361] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having the amino acid sequence of SEQ ID NO: 14; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having the amino acid sequence of SEQ ID NO: 15; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having the amino acid sequence of SEQ ID NO: 16; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0362] FR1 VL having the amino acid sequence of SEQ ID NO: 72; CDR1 VL having the amino acid sequence of SEQ ID NO: 34; FR2 VL having the amino acid sequence of SEQ ID NO: 73; CDR2 VL having the amino acid sequence of SEQ ID NO: 35; FR3 VL having the amino acid sequence of SEQ ID NO: 74; CDR3 VL having the amino acid sequence of SEQ ID NO: 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 75;

[0363] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 14 and 34; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 15 and 35; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 16 and 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0364] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having the amino acid sequence of SEQ ID NO: 14; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having the amino acid sequence of SEQ ID NO: 15; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having the amino acid sequence of SEQ ID NO: 16; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0365] FR1 VL having the amino acid sequence of SEQ ID NO: 76; CDR1 VL having the amino acid sequence of SEQ ID NO: 34; FR2 VL having the amino acid sequence of SEQ ID NO: 77; CDR2 VL having the amino acid sequence of SEQ ID NO: 35; FR3 VL having the amino acid sequence of SEQ ID NO: 78; CDR3 VL having the amino acid sequence of SEQ ID NO: 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 79;

[0366] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having an amino acid sequence selected from SEQ ID NOs: 14 and 34; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having an amino acid sequence selected from SEQ ID NOs: 15 and 35; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having an amino acid sequence selected from SEQ ID NOs: 16 and 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 83;

[0367] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having the amino acid sequence of SEQ ID NO: 14; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having the amino acid sequence of SEQ ID NO: 15; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having the amino acid sequence of SEQ ID NO: 16; and FR4 VL having the amino acid sequence of SEQ ID NO: 83; or

[0368] FR1 VL having the amino acid sequence of SEQ ID NO: 80; CDR1 VL having the amino acid sequence of SEQ ID NO: 34; FR2 VL having the amino acid sequence of SEQ ID NO: 81; CDR2 VL having the amino acid sequence of SEQ ID NO: 35; FR3 VL having the amino acid sequence of SEQ ID NO: 82; CDR3 VL having the amino acid sequence of SEQ ID NO: 36; and FR4 VL having the amino acid sequence of SEQ ID NO: 83. In some cases, the antibody in question comprises an immunoglobulin constant region (e.g., an Fc region). In some embodiments, the Fc region, if present, is a human Fc region. If constant regions are present, the antibody may comprise light chain and heavy chain constant regions. Suitable heavy chain constant regions include the CH1, hinge, CH2, CH3, and CH4 regions.

[0369] An example of a suitable heavy chain Fc region is the Fc region of the human IgG4 isotype. The light chain constant regions may be lambda or kappa. A subject antibody (e.g., a subject humanized antibody) may contain sequences from more than one class or isotype. Antibodies may be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which the variable regions of the light and heavy chains are connected by a spacer.

[0370] In some cases, the heavy chain region may be an IgG4 isotype region. In some of these embodiments, the hinge region comprises an S241P substitution. See, e.g., Angal et al. (1993) Mot. Immunol. 30:105. In some of these embodiments, the hinge region comprises an L236E substitution (or L235E, using EU numbering; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. US Dept. Health and Human Services, Bethesda, MD, NIH Publication No. 91-3242). See, e.g., Reddy et al. (2000) J. Immunol. 164:1925; and Klechevsky et al. (2010) Blood 116:1685. In some of these embodiments, the hinge region comprises a S241P substitution and a L236E substitution.

[0371] The amino acid sequence of the constant region may be identical to the amino acid sequence of the constant region of the species from which it is derived (e.g., a human sequence), or may be at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the constant region. For example, the amino acid sequence of the constant region of an antibody of the invention may contain one or more amino acid deletions, substitutions, or additions (e.g., one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, substitutions, or additions) compared to the amino acid sequence of the constant region of the species from which it is derived.

[0372] For example, as described above, the amino acid sequence of the Fc hinge region of the antibody of the invention can be mutated to decrease the biological half-life of the antibody, or the amino acid sequence of the Fc region can be mutated to increase the biological half-life of the antibody.

[0373] It is necessary to ensure that the antibody of the invention or its antigen-binding fragment is devoid of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) so that the antibody or fragment can be used to negatively regulate the proliferation and / or function of T cells without depleting T cells as a result of ADCC or CDC.

[0374] For example, in some embodiments, the Fc region comprises a wild-type human IgG4 Fc region sequence.

[0375] In other embodiments, the Fc region comprises a mutant human IgG4 Fc sequence with an S228P mutation to eliminate Fab fragment exchange (as shown in Figure 20(A) for the chimeric antibody Chim13E2IgG4 comprising the heavy chain sequence 13E2IgG4mut).

[0376] In other embodiments, the Fc region comprises the C portion of a wild-type human Ig (IgK) kappa chain (13E2IgK) (as shown in Figure 20(B) for the chimeric antibody Chim 13E2IgG4 comprising the light chain sequence of 13E2IgK).

[0377] The numbering of residues in the Fc region used for the human IgG4 Fc mutant described above represents the standard EU index numbering as in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)).

[0378] The antibody in question may contain a free thiol (-SH) group at the carboxyl terminus, wherein the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody, including the antibody in question), a framework, a carrier, etc.

[0379] In some embodiments, the subject antibody comprises one or more unnatural amino acids. In some embodiments, the unnatural amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. See, for example, U.S. Patent 7,632,924, which describes typical unnatural amino acids. Incorporation of an unnatural amino acid can provide attachment to a polymer, a second polypeptide, a scaffold, and the like. For example, the subject antibody linked to a water-soluble polymer can be prepared by reacting a water-soluble polymer (e.g., PEG) containing a carbonyl group with an antibody containing an unnatural amino acid containing an aminooxy, hydrazine, hydrazide, or semicarbazide group.As another example, the subject antibody linked to a water-soluble polymer can be prepared by reacting the subject antibody containing an alkyne-containing amino acid with a water-soluble polymer (e.g., PEG) containing an azide group; in some embodiments, the azide or alkyne group is linked to the PEG molecule via an amide bond. "Non-naturally occurring amino acid" means an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally occurring amino acid" include "non-natural amino acid," "non-naturally occurring amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated variations thereof.The term "unnatural amino acid" also includes, but is not limited to, amino acids that arise from modification (e.g., post-translational modification) of naturally occurring amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but are not themselves incorporated into the growing polypeptide chain by the translation complex. Examples of such unnatural amino acids include, but are not limited to, N-acetylglucosaminyl-L-seri, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0380] In some embodiments, the antibody in question is linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers are, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and polymers of natural origin. Suitable polymers include, for example, substituted or unsubstituted linear or branched polyalkylene, polyalkenylene, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homo- or heteropolysaccharides. Suitable polymers include, for example, ethylene-vinyl alcohol copolymers (commonly known collectively as EVOH or under the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride;poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphoester; polyphosphoester urethane; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonate); Polyether-ester copolymers (e.g. poly(ethylene oxide)-poly(lactic acid) copolymers (PEO / PLA); polyalkylene oxides; polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene-ethylene-alpha-olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones, polyvinyl aromatic polymers such as polystyrene; polyvinyl esters such as polyvinyl acetate;Copolymers of vinyl monomers with each other and with olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyesters; epoxy resins; polyurethanes; viscose; viscose triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate-butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; poly(ethylene glycol); and carboxymethyl cellulose.

[0381] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) and their derivatives, for example, substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) and its derivatives. Suitable natural polymers include, for example, albumin, amylose, dextran, glycogen and their derivatives.

[0382] The average molecular weight of suitable polymers can be from 500 Da to 50,000 Da, such as from 5,000 Da to 40,000 Da, or from 25,000 Da to 40,000 Da. For example, in some embodiments in which the antibody in question comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer can have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0383] As noted above, in some embodiments, the subject antibody is covalently linked to a non-peptide synthetic polymer. In some embodiments, the subject antibody is covalently linked to a PEG polymer. In some embodiments, the subject scFv multimer is covalently linked to a PEG polymer. See, e.g., Albrecht et al. (2006) J. Immunol. Methods 310:100. Methods and reagents suitable for PEGylation of a protein are well known in the art and can be found, for example, in U.S. Patent 5,849,860. PEG suitable for conjugation to a protein is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2) n OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and where n is an integer from 1 to 1000. When R is a protecting group, it typically contains from 1 to 8 carbon atoms.

[0384] In some embodiments, the PEG conjugated to the antibody in question is linear. In some embodiments, the PEG conjugated to the antibody in question is branched. Branched PEG derivatives are described in U.S. Patent 5,643,575, "star PEGs" and multi-arm PEGs are described in the Shearwater Polymers, Inc. catalog "Polyethylene Glycol Derivatives 1997-1998." Star PEGs are described in the art, including, for example, U.S. Patent 6,046,305.

[0385] The antibody in question may be glycosylated; for example, the antibody in question may contain a covalently linked carbohydrate or polysaccharide moiety. Antibody glycosylation is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, represent recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0386] Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be accomplished by adding or substituting one or more serine or threonine residues in the original antibody sequence (for O-linked glycosylation sites). Similarly, removal of glycosylation sites can be accomplished by modifying amino acids in the antibody's natural glycosylation sites.

[0387] The antibody in question, in some embodiments, comprises a "radiopaque" label, e.g., a label that can be readily visualized, such as by x-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials are iodine, bromine, or barium salts. Other radiopaque materials are also known, including, but not limited to, organic bismuth derivatives (see, e.g., U.S. Patent 5,939,045), radiopaque multiurethanes (see U.S. Patent 5,346,981), organobismuth composites (see, e.g., U.S. Patent 5,256,334), radiopaque barium multimeric complexes (see, e.g., U.S. Patent 4,866,132), and the like.

[0388] The antibody in question can be covalently linked to a second molecule (e.g., a lipid, a polypeptide other than the antibody in question, a synthetic polymer, a carbohydrate, etc.) using, for example, glutaraldehyde, a homobifunctional crosslinking reagent, or a heterobifunctional crosslinking reagent. Glutaraldehyde crosslinks polypeptides through its amino groups. Homobifunctional crosslinking reagents (e.g., a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl-reactive crosslinking reagent) contain two or more identical reactive moieties and can be used in one-step reactions in which the crosslinking reagent is added to a solution containing a mixture of polypeptides to be crosslinked. Homobifunctional NHS ester and imidoesters crosslink amino-containing polypeptides.At slightly alkaline pH, imidoesters react only with primary amines to form imidoamides and do not affect the overall charge of cross-linked polypeptides. Homobifunctional sulfhydryl-reactive cross-linking reagents include bismaleimidehexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propionamidobutane (DPDPB).

[0389] Heterobifunctional crosslinking reagents contain two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety) and crosslink to one polypeptide via the amine- or sulfhydryl-reactive moiety and then react with the other polypeptide via the unreacted moiety. There are many heterobifunctional haloacetyl crosslinking reagents, as well as pyridyl disulfide crosslinking reagents. Carbodiimides are a classic example of heterobifunctional crosslinking reagents for conjugating carboxyls with amines, resulting in an amide bond.

[0390] The antibody in question can be immobilized on a solid support. Suitable supports are well known in the art and include, in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicone chips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracites, reaction plate wells (e.g., multi-well plates), plastic tubes, etc. The solid support may include any of a variety of materials, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamides, agarose, and magnetite. Suitable methods for immobilizing the antibody in question on a solid support are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, etc.Solid supports may be soluble or insoluble, such as in aqueous solution. In some embodiments, a suitable solid support is typically insoluble in aqueous solution.

[0391] The subject antibody, in some embodiments, comprises a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable labels include, but are not limited to, magnetic particles (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 With or 32P), enzymes (e.g. horseradish peroxidase, alkaline phosphatase, luciferase and others commonly used in enzyme-linked immunosorbent assay (ELISA)) and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

[0392] In some embodiments, the antibody in question comprises a contrast agent or radioisotope, wherein the contrast agent or radioisotope is suitable for use in imaging, such as visualization procedures, performed on humans. Non-limiting examples of labels include a radioisotope such as 1231 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (Indian) and 67 Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron.

[0393] Radioactive isotopes of Gd ( 153Gd) are also available and suitable for imaging procedures in non-human mammals.

[0394] The antibody in question can be labeled using standard methods. For example, the antibody in question can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycuryl. In fluorination, fluorine is added to the antibody in question during synthesis by a fluoride ion displacement reaction. For reviews of protein synthesis with such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122–130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst, 16(2):209–244 (1999). The antibody in question can also be labeled with a contrast agent using standard methods. For example, the antibody in question can be labeled with Gd by conjugating low molecular weight Gd chelates such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA) to the antibody. See Caravan et al., Chem. Rev. 99:2293–2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11–16 (1985).The antibody in question can be labeled with Gd, for example, by conjugating polylysine-Gd chelates to the antibody. See, for example, Curtet et al., Invest. Radiol., 33(10):752–761 (1998). Alternatively, the antibody in question can be labeled with Gd by incubating paramagnetic polymerized liposomes containing the Gd lipid chelator with avidin and biotinylated antibody. See, for example, Sipkins et al., Nature Med., 4:623–626 (1998).

[0395] Suitable fluorescent proteins that can be linked to the antibody of interest include, but are not limited to, green fluorescent protein from Aequoria victoria or a mutant or derivative thereof, e.g., as described in U.S. Patents 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, many such GFPs being commercially available, e.g., from Clontech, Inc.; red fluorescent protein; yellow fluorescent protein; any of the various fluorescent and colored proteins from Anthozoan species, as described, e.g., in Matz et al. (1999) Nature Biotechnol. 17:969-973; etc.

[0396] In some embodiments, the subject antibody is conjugated to a therapeutic agent. Any of the subject antibodies described herein can be used to form an antibody-agent conjugate. The agent can be attached to the N-terminus of the light chain, the C-terminus of the light chain, the N-terminus of the heavy chain, or the C-terminus of the heavy chain. In some embodiments, the agent is attached to the hinge region of the antibody or to one or more other regions on the antibody. For a single-chain antibody, the agent can be attached to the N- or C-terminus of the single-chain antibody. The agent can be conjugated to the antibody directly or via a linker using techniques known to those skilled in the art. The linker can be cleavable or non-cleavable. Examples of such therapeutic agents (e.g., for use in therapy) are known to those skilled in the art.

[0397] In some embodiments, the antibody in question will be linked (e.g., covalently or non-covalently) to a fusion partner, such as a ligand; an epitope tag; a peptide; a protein other than an antibody; and the like. Suitable fusion partners include peptides and polypeptides that confer enhanced stability in vivo (e.g., increased serum half-life); provide ease of purification, such as (His) n, for example, 6His, etc.; provide for secretion of the fusion protein from the cell; provide an epitope tag, for example, GST, hemagglutinin (HA; for example, YPYDVPDYA; SEQ ID NO: 41), FLAG (for example, DYKDDDDK; SEQ ID NO: 42), c-myc (for example, EQKLISEEDL; SEQ ID NO: 43), etc.; provide a detectable signal, for example, an enzyme generating a detectable product (for example, β-galactosidase, luciferase), or a protein that is itself detectable, for example, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.; provide multimerization, for example, a multimerization domain such as the Fc portion of an immunoglobulin; and the like.

[0398] The fusion can also encompass an affinity domain, including peptide sequences that can interact with a binding partner, such as one immobilized on a solid support used for identification or purification. Sequential single amino acids, such as histidine, when fused to a protein can be used for one-step purification of the fusion protein by high-affinity binding to a resin on a column such as nickel sepharose. Typical affinity domains are His5 (HHHHH) (SEQ ID NO: 44), HisX6 (HHHHHH) (SEQ ID NO: 45), c-myc (EQKLISEEDL) (SEQ ID NO: 46), Flag (DYKDDDDK) (SEQ ID NO: 42), StrepTag (WSHPQFEK) (SEQ ID NO: 47), hemagglutinin, e.g., HA-Tag (YPYDVPDYA;SEQ ID NO: 41), glutathione S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 48), Phe-His-His-Thr (SEQ ID NO: 49), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 50), metal binding domains, such as zinc binding domains or calcium binding domains, such as those of calcium binding proteins, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, large subunit of calpain, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, inteins, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose-binding protein.

[0399] In relation to nucleotide and amino acid sequences, the term "identical" or "identity" means the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

[0400] The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions, multiplied by 100%), taking into account the number of gaps and the length of each gap required to insert them for optimal alignment of the two sequences. Sequence comparison and determination of the percentage identity between the two sequences can be performed using the mathematical algorithm described below.

[0401] The percent identity between a query nucleic acid sequence and a subject nucleic acid sequence is the "identity" value, expressed as a percentage, calculated using the BLASTN algorithm when the subject nucleic acid sequence has 100% coverage with the query nucleic acid sequence after performing a pairwise BLASTN alignment. Such pairwise BLASTN alignments between the query nucleic acid sequence and the subject nucleic acid sequence were performed using the default parameters of the BLASTN algorithm available on the National Center for Biotechnology Institute website without filtering out low-complexity regions.It is important that the claimed nucleotide sequence may be described using a nucleic acid sequence identified in one or more claims of the present document.

[0402] The percent identity between a query amino acid sequence and a subject amino acid sequence is the "identity" value, expressed as a percentage, calculated using the BLASTP algorithm when the subject amino acid sequence has 100% of the requested coverage with the query amino acid sequence after performing a pairwise BLASTP alignment. Such pairwise BLASTN alignments between the query amino acid sequence and the subject amino acid sequence were performed using the default parameters of the BLASTN algorithm available on the website of the National Center for Biotechnology Institute without filtering out low-complexity regions. Importantly, the query nucleotide sequence can be described using a nucleic acid sequence identified in one or more claims herein.

[0403] Methods for obtaining the antibody in question

[0404] The antibody in question can be produced by any known method, for example, standard protein synthesis methods; recombinant DNA methods; etc. In some embodiments, the antibody in question was produced by a method selected from the group consisting of recombinant production and chemical synthesis.

[0405] When the antibody in question is a single-chain polypeptide, it can be synthesized using standard methods of chemical peptide synthesis. When the polypeptide is synthesized chemically, the synthesis can occur in the liquid phase or the solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of a given sequence is fixed to an insoluble support, after which the remaining amino acids in the sequence are sequentially added, is an example of a suitable method for chemically synthesizing the antibody in question. Various forms of SPPS, such as Fmoc and Boe, are suitable for the synthesis of the antibody in question. Solid-phase synthesis methods are described in Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc, 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem. Co., Rockford, 111. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3–10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723–8. Briefly, small, insoluble, porous beads are loaded with functional units that build peptide chains. After multiple cycles of addition / deblocking, the free N-terminal amine of a solid-phase-anchored peptide is conjugated to a single N-blocked amino acid unit. This unit is then deblocked, revealing a new N-terminal amine to which the next amino acid can be attached. The peptide remains immobilized on the solid phase and undergoes a filtration process, after which it is cleaved.

[0406] Standard recombinant techniques can be used to produce the antibody in question. For example, nucleic acids encoding the variable regions of the heavy and light chains, optionally linked to constant regions, are introduced into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of the immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., native or heterologous promoters), signal sequences, enhancer elements, repressor elements, and transcription termination sequences.Expression control sequences can be represented by eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). After insertion of the vector into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and for antibody extraction and purification.

[0407] Due to the degeneracy of the genetic code, each amino acid sequence of immunoglobulins can be encoded by different nucleotide sequences. The desired nucleic acid sequences can be obtained by de novo solid-phase DNA synthesis, polymerase chain reaction (PCR), or mutagenesis of a previously obtained variant of a given polynucleotide. Oligonucleotide-mediated mutagenesis is an example of a suitable method for producing substitutions, deletions, and insertions in the DNA of a target polypeptide. See Adelman et al., DNA 2:183 (1983). Briefly, the DNA of the target polypeptide is altered by hybridization of an oligonucleoid encoding the desired mutation to a single-stranded DNA template. After hybridization, DNA polymerase synthesizes a complete second complementary strand of the template containing an oligonucleotide primer, which encodes the selected change in the DNA of the target polypeptide.

[0408] Suitable expression vectors typically replicate in host organisms as episomes or as part of the host's chromosomal DNA. Expression vectors typically contain selectable markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to enable detection of cells transformed with the desired DNA sequence.

[0409] Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding the antibody in question. Other suitable microbial hosts include bacilli such as Bacillus subtilis and other enterobacteria such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can be produced in these prokaryotic organisms. These vectors must contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of well-known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter, or the lambda phage promoters. Promoters will typically control expression, optionally with an operator sequence, and contain ribosome binding site sequences, etc., for initiation and termination of transcription and translation.

[0410] Other microorganisms, such as yeast, are also suitable for expression. Examples of suitable yeast host cells include Saccharomyces (e.g., S. cerevisiae) and Pichia, with appropriate vectors containing expression control sequences (e.g., promoters), an origin of replication, termination sequences, and other sequences, as required. Typical promoters include those of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, those of alcohol dehydrogenase, isocytochrome C, and the enzymes responsible for maltose and galactose utilization.

[0411] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in vitro in cell culture) can also be used to express and produce the anti-LAG-3 antibody of the present invention (e.g., polynucleotides encoding the anti-LAG-3 antibody). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may include expression control sequences such as an origin of replication, promoter, and enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.Examples of suitable expression control sequences include promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomavirus, cytomegalovirus, and others. See Co et al., J. Immunol. 148:1149 (1992). In other methods, the antibodies of the invention can be produced in mice (see, for example, Laffleur et al., "Production of human or humanized antibodies in mice," Methods Mol Biol. 2012; 901:149-59).

[0412] After synthesis (chemical or recombinant), whole antibodies, their dimers, individual light and heavy chains, or other forms of the antibody in question (e.g., scFv, etc.) can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography methods, gel electrophoresis, etc. (see, as a rule, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The antibody in question can be substantially purified, for example, by at least about 80-85%, at least about 85-90%, at least about 90-95%, or 98-99%, or more, for example, free from contaminants such as cellular debris, macromolecules other than the antibody in question, etc.

[0413] Nucleic acid molecules, expression vectors and host cells

[0414] The present invention also provides nucleic acid molecules comprising nucleotide sequences encoding an anti-LAG-3 antibody of the invention or an antigen-binding fragment thereof.

[0415] In some embodiments, a nucleic acid molecule of the present invention encodes an anti-LAG-3 antibody in question, comprising a heavy chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 17. In some embodiments, a nucleic acid molecule of the present invention encodes an anti-LAG-3 antibody in question, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 17.

[0416] In some embodiments, a nucleic acid molecule of the present invention encodes an anti-LAG-3 antibody in question, comprising a light chain variable region comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 18. In some embodiments, a nucleic acid molecule of the present invention encodes an anti-LAG-3 antibody in question, comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 18.

[0417] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0418] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0419] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0420] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0421] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the sequences of SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively.

[0422] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the sequences of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

[0423] In some embodiments, a nucleic acid molecule of the present invention encodes the subject anti-LAG-3 antibody comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the sequences of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, respectively.

[0424] In some embodiments, a nucleic acid molecule according to the present invention encodes the subject anti-LAG-3 antibody comprising a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the sequences of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0425] In some embodiments, the nucleic acid molecule of the present invention encodes the subject anti-LAG-3 antibody comprising a light chain variable region and a heavy chain variable region.

[0426] A nucleic acid molecule encoding the antibody in question may be operably linked to one or more regulatory elements, such as a promoter and enhancer, that enable expression of the nucleotide sequence in intended target cells (e.g., a cell that is genetically modified to synthesize the encoded antibody).

[0427] Suitable promoter and enhancer elements are known in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; the T3 promoter; the T5 promoter; the lambda P promoter; the trp promoter; the lac operon promoter; a hybrid promoter, such as a hybrid lac / tac promoter, a hybrid tac / trc promoter, a trp / lac promoter, a T7 / lac promoter; the trc promoter; the tac promoter, etc.; the gpt promoter; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or a related promoter (see, e.g., US Patent Publication 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), the nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), etc. (See, for example, Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888–892); the sigma 70 promoter, such as the sigma 70 consensus promoter (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); the stationary phase promoter, such as the dps promoter, the spv promoter, etc.; the SPI-2 pathogenicity island-derived promoter (see, e.g., WO 96 / 17951); the actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087–1096); the rpsM promoter (see, e.g., Valdivia and Falkow (1996) Mol. Microbiol. 22:367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); the SP6 promoter (see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to, Trc, Tac, T5, T7, and P. Lambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the LacI repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21–25).

[0428] In some embodiments, for example, for expression in yeast cells, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter, and the like; or a regulated promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and an AOX1 promoter (e.g., for use in Pichia).

[0429] For expression in a eukaryotic cell, suitable promoters include, but are not limited to, the immunoglobulin light and / or heavy chain promoter and enhancer elements; the cytomegalovirus immediate early response gene promoter; the herpes simplex virus thymidine kinase; the SV40 early and late promoters; the promoter present in the long terminal repeats of a retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0430] The selection of a suitable vector and promoter is within the competence of a person skilled in the art.

[0431] A nucleic acid molecule encoding the antibody in question may be present in an expression vector and / or a cloning vector. The present invention provides a recombinant vector that contains a nucleic acid molecule encoding the antibody in question in a cloning vector. The present invention also provides a recombinant molecule that contains a nucleic acid molecule encoding the antibody in question operably linked to appropriate regulatory sequence(s) in an expression vector to ensure expression of the encoded antibody. When the antibody in question comprises two separate polypeptides, the nucleic acid molecules encoding the two polypeptides can be cloned into the same or separate vectors to form one or more recombinant molecules.The recombinant molecule may include a selectable marker, an origin of replication, and other distinguishing features that ensure replication and / or maintenance of the recombinant molecule.

[0432] A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for generating the recombinant molecule in question. The following vectors are provided as examples. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0433] Expression vectors typically have restriction sites located near the promoter sequence to allow insertion of nucleotide sequences encoding heterologous proteins. A selectable marker functional in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors. Examples of viral vectors include, but are not limited to, viral vectors based on: vaccinia virus; poliovirus; adenovirus (see, for example, Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 19191; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, for example, Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683-690, 1997, Rolling et al., Hum Gene Ther 10:641-648, 1999; Ali et al., Hum Mol Genet 5:591-594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelsohn et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey murine sarcoma virus, avian leukemia virus, human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999), myeloproliferative murine sarcoma virus, and mammary tumor virus); etc.

[0434] As noted above, the subject nucleic acid molecule comprises a nucleotide sequence encoding an anti-LAG-3 antibody according to the present invention. In some embodiments, the subject nucleic acid molecule comprises a nucleotide sequence encoding the CDR regions of the heavy and light chains of the subject antibody 13E2 or 34F4. In some embodiments, the subject nucleic acid molecule comprises a nucleotide sequence encoding the CDR regions of the heavy and light chains of the subject antibody, wherein the CDR-coding sequences are interspersed with FR-coding nucleotide sequences. In some embodiments, the FR-coding nucleotide sequences are human FR-coding nucleotide sequences.

[0435] Host cells

[0436] The present invention provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with the subject nucleic acid molecule. In some embodiments, the subject isolated genetically modified host cell can produce the subject antibody. Such a cell is called a recombinant cell. A recombinant cell comprises a recombinant molecule encoding the subject antibody.

[0437] Suitable host cells include eukaryotic host cells, such as mammalian cells, insect host cells, and yeast cells; and prokaryotic cells, such as bacterial cells. Introduction of the nucleic acid in question into the host cell can be achieved, for example, by calcium phosphate precipitation, dextran-mediated DEAE transfection, liposome-mediated transfection, electroporation, or other known methods.

[0438] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. In some cases, the cells are HEK cells.In some cases, the cells are CHO cells, such as CHO-K1 cells (ATCC No. CCL-61), CHO-M cells, CHO-DG44 cells (ATCC No. PTA-3356), and the like. In some embodiments, the host cell is a COS cell. In some embodiments, the host cell is a 293 cell. In some embodiments, the host cell is a CHO cell.

[0439] Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, etc. In some embodiments, the host cell is Saccharomyces. In some embodiments, the host cell is Pichia.

[0440] Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Bacillus (e.g., B. subtilis), Lactobacillus sp., and the like. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Patent 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Typically, the laboratory strain is a strain that is non-pathogenic. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is Bacillus subtilis.

[0441] Compositions

[0442] The present disclosure provides compositions comprising the antibody of the invention. The composition of the antibody of the invention may, along with the antibody of the invention, comprise one or more of the following: a salt, such as NaCl, MgCl2, KCl, MgSO4, etc.; a buffering agent, such as a phosphate buffer, citrate buffer, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; detergent, such as non-ionic detergent such as Tween-20, etc.; protease inhibitor; glycerin; etc.

[0443] Pharmaceutical compositions

[0444] The present description provides compositions, including pharmaceutical compositions, containing the antibody in question. Generally, a pharmaceutical composition is also referred to herein as a dosage form containing an effective amount of the antibody in question. "Effective amount" means a dose sufficient to achieve a desired result, such as reducing an adverse symptom associated with an immune disorder, alleviating a symptom of an immune disorder, slowing the progression of an immune disorder, etc. In general, the desired result is at least a reduction in the symptom of an immune disorder compared to a control.

[0445] Dosage forms

[0446] In the methods under consideration, the antibody can be administered to the body by any standard route capable of producing the desired therapeutic effect or diagnostic effect. Thus, it can be included in a variety of dosage forms for therapeutic administration. In particular, the antibody in question can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, or other pharmaceutically acceptable excipients, and can be included in preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, and aerosols. In some embodiments, the pharmaceutical composition comprises the antibody in question and a pharmaceutically acceptable excipient.

[0447] In pharmaceutical dosage forms, the antibody in question can be administered in the form of its pharmaceutically acceptable salts, which can be used alone or in suitable combinations, as well as in combination with other pharmaceutically active compounds. The following methods and excipients are provided for example only and are not limiting in any way.

[0448] For oral preparations, the antibody in question can be used alone or in combination with appropriate additives to produce tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrants such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0449] The antibody in question can be formulated into injectable preparations by dissolving, suspending, or emulsifying the antibody in an aqueous or non-aqueous solvent such as vegetable or other similar oils, propylene glycol, synthetic glycerides of aliphatic acids, injectable organic esters (e.g., ethyl oleate), esters of higher aliphatic acids, or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as Ringer's dextrose supplements), and the like.In addition, the pharmaceutical composition according to the present invention may contain additional agents such as dopamine or psychopharmacological drugs depending on the intended use of the pharmaceutical composition.

[0450] Pharmaceutical compositions containing the antibody in question were prepared by mixing the antibody in question to the required degree of purity with optional physiologically acceptable carriers, other excipients, stabilizers, surfactants, buffers and / or isotonic agents. Acceptable carriers, other excipients and / or stabilizers should not be toxic to the recipient at the doses and concentrations used, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl parabens, benzalkonium chloride or combinations thereof);Amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X or polyethylene glycol (PEG).;

[0451] The pharmaceutical composition may be in liquid form, lyophilized form, or a liquid form reconstituted from a lyophilized form, provided that the lyophilized preparation is reconstituted with a sterile solution before administration. The standard technique for reconstituting a lyophilized composition is to add a volume of pure water (usually equivalent to the volume removed during lyophilization); however, in the preparation of pharmaceutical compositions for parenteral administration, solutions containing antibacterial agents may be used; see also Chen (1992) Drag Dev Ind Pharm 18, 1311-54.

[0452] Typical concentrations of the antibody in the pharmaceutical composition under consideration may be from about 1 mg / ml to 200 mg / ml, or from about 50 mg / ml to 200 mg / ml, or from about 150 mg / ml to 200 mg / ml.

[0453] Aqueous antibody formulations can be prepared in a buffered solution, for example, to a pH in the range of about 4.0 to 7.0, about 5.0 to 6.0, or about 5.5. Examples of buffers suitable for pH in this range include phosphate, histidine, citrate, succinate, acetate, and other organic acid buffers. The buffer concentration can be from about 1 mM to 100 mM or from about 5 mM to 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.

[0454] The antibody dosage form may include a substance to modulate the tonicity of the formulation. Typical substances to maintain tonicity include sodium chloride, potassium chloride, glycerol, and any other components from the group of amino acids, sugars, and combinations thereof. In some embodiments, aqueous forms are isotonic, although hypertonic or hypotonic solutions may also be suitable. The term "isotonic" means a solution having the same tonicity as another solution to which it is being compared, such as physiological saline or serum. Substances to maintain tonicity can be used in an amount of from about 5 mM to 350 mM, for example, in an amount of from 100 mM to 350 nM.

[0455] The antibody dosage form may also contain a surfactant to reduce aggregation of the antibodies contained therein and / or minimize the formation of solid particles and / or reduce adsorption. Typical surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), polyoxyethylene alkylphenyl ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (poloxamer, pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters include polysorbate 20 (marketed under the trade name Tween 20™) and polysorbate 80 (marketed under the trade name Tween 80™). Examples of suitable polyethylene-polypropylene copolymers include those. which are sold under the name Pluronic® F68 or Poloxamer 188™. Examples of suitable polyoxyethylene alkyl ethers are those sold under the trade name Brij™.Typical surfactant concentrations can range from approximately 0.001% to 1% w / v.

[0456] Lyoprotectants can also be added to protect a labile active ingredient (e.g., protein) from destabilizing conditions during lyophilization. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants can be included in amounts ranging from approximately 10 mM to 500 nM.

[0457] In some embodiments, the subject dosage form comprises the subject antibody and one or more of the above-mentioned substances (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl- or propylparabens, benzalkonium chloride, and combinations thereof. In other embodiments, the preservative is included in the formulation, for example, at a concentration of from about 0.001% to about 2% (w / v).

[0458] For example, the dosage form in question may be a liquid or lyophilized form suitable for parenteral administration and may contain: from about 1 mg / mL to 200 mg / mL of the antibody in question; from about 0.001% to 1% of at least one surfactant; from about 1 mM to 100 mM of a buffer; optionally, from about 10 mM to 500 mM of a stabilizer; and from about 5 mM to 305 mM of a tonicity agent; and has a pH of from about 4.0 to 7.0.

[0459] As another example, the parenteral dosage form in question is a liquid or lyophilized form comprising: about 1 mg / mL to 200 mg / mL of the antibody in question; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5.

[0460] As another example, the parenteral dosage form in question is a lyophilized form comprising: 1) 15 mg / mL of the antibody in question; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 2) 75 mg / mL of the antibody in question; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 3) 75 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 4) 75 mg / mL of the antibody in question; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 5) 75 mg / ml of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

[0461] As another example, the parenteral dosage form in question is a liquid form comprising: 1) 7.5 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 120 mM L-histidine; and 250 mM sucrose; and has a pH of 5.5; or 2) 37.5 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg / mL of the antibody in question; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 4) 37.5 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 10 mM L-histidine; 125 mM trehalose; and has a pH of 5.5; or 5) 37.5 mg / mL of the antibody in question; 0.01% Tween 20 w / v; 10 mM L-histidine; and 125 mM trehalose; and has a pH of 5.5; or 6) 5 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 7) 75 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol;and has a pH of 5.5; or 8) 75 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 9) 150 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM trehalose; and has a pH of 5.5; or 10) 150 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM mannitol; and has a pH of 5.5; or 11) 150 mg / mL of the antibody in question; 0.02% Tween 20 w / v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 12) 10 mg / ml of the antibody in question; 0.01% Tween 20 w / v; 20 mM L-histidine; and 40 mM sodium chloride; and has a pH of 5.5.

[0462] The antibody in question can be formulated as an aerosol for administration by inhalation. The antibody in question can be formulated with suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Aerosol dosage forms, such as nasal spray dosage forms, comprise purified aqueous or other solutions of the active agent with preservatives and isotonic agents. Such formulations are pH-adjusted and are isotonic to a level compatible with the mucous membranes of the nasal cavity.

[0463] Furthermore, the antibody in question can be formulated into suppositories by mixing with various bases, such as emulsifying bases or water-soluble bases. The antibody in question can be administered rectally via a suppository. The suppository may include carriers such as cocoa butter, carbowax, and polyethylene glycols, which melt at body temperature but solidify at room temperature.

[0464] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, such as a teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the antibody in question in the composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0465] The term "unit dosage form" as used herein refers to physically discrete units suitable as single doses for humans and animals, each unit containing a predetermined quantity of the anti-LAG-3 antibody of the present invention, calculated in an amount sufficient to produce the desired effect, together with a pharmaceutically acceptable diluent, carrier, or excipient. Specifications for a given antibody may depend on the particular antibody used and the desired effect, as well as the pharmacodynamics of each antibody in the body.

[0466] Other routes of administration are also suitable for the present method. For example, the antibody in question can be formulated into suppositories, and in some cases, into aerosol and intranasal formulations. For suppositories, the filler should include conventional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories can be prepared from mixtures containing the active ingredient in a range of approximately 0.5% to 10% (w / w), for example, approximately 1% to 2%.

[0467] Intranasal formulations typically include excipients that do not irritate the nasal mucosa or significantly impair ciliary function. Diluents such as water, aqueous saline, or other known substances may be used. Nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may also be present to enhance absorption of the antibody in question from the nasal mucosa.

[0468] The antibody in question can be administered as an injectable formulation. Injectable compositions are typically prepared as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The preparation can also be emulsified, or the antibody can be encapsulated in liposomal carriers.

[0469] Suitable auxiliary excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents. Specific methods for preparing such dosage forms are known or will be obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The administered composition or formulation, in any case, will contain an amount of the antibody in question sufficient to achieve the desired condition in the subject to be treated.

[0470] Pharmaceutically acceptable excipients such as fillers, adjuvants, carriers, or diluents are readily available. Furthermore, pharmaceutically acceptable excipients such as pH adjusting agents, buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc. are also readily available.

[0471] In some embodiments, the antibody in question is formulated as a controlled-release formulation. Sustained-release preparations can be manufactured using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, wherein the matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamine, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable copolymers of lactic acid and glycolic acid, and poly-D-(-)-3-hydroxybutyric acid.Possible loss of biological activity and possible changes in the immunogenicity of antibodies contained in sustained-release preparations can be prevented by using suitable additives, by controlling the moisture content and by developing specific polymer matrix compositions.

[0472] Controlled release within the scope of the present invention may mean any of a variety of sustained release dosage forms. The following terms may be considered as substantially equivalent to controlled release for the purposes of the present invention: continuous release, controlled release, delayed release, depot, extended release, stepped release, immediate release, sustained release, programmed release, prolonged release, uniform release, sustained release, depot, retard, slow release, interval release, extended release, time-coated, time-release, delayed action, extended action, layered action, long-acting, prolonged action, repeated action, delayed action, delayed action and sustained-action drugs.Further descriptions of these terms are contained in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0473] Various controlled release technologies cover a very wide range of dosage forms. Controlled release technologies include, but are not limited to, physical systems and chemical systems.

[0474] Physical systems include, but are not limited to, reservoir systems with rate-controlling membranes, such as microencapsulation, macroencapsulation, and membrane systems; reservoir systems without rate-controlling membranes, such as hollow fibers, ultraporous cellulose triacetate, and porous polymeric substrates and foams; monolithic systems, including systems physically dissolved in nonporous, polymeric, or elastomeric matrices (e.g., non-erodible, erodible, permeable to surrounding substances, and degradable), and materials physically dispersed in nonporous, polymeric, or elastomeric matrices (e.g., non-erodible, erodible, permeable to surrounding substances, and degradable); laminated structures, including reservoir layers that are chemically similar or dissimilar to external control layers; and other physical methods, such as osmotic pumps or adsorption onto ion-exchange resins.

[0475] Chemical systems include, but are not limited to, chemical erosion of polymer matrices (e.g., heterogeneous or homogeneous erosion) or biological erosion of the polymer matrix (e.g., heterogeneous or homogeneous). Additional discussion of various controlled release systems can be found in Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory, and Applications, 1980 (CRC Press, Inc.).

[0476] There are a number of controlled-release dosage forms designed for oral administration. These include, but are not limited to, osmotic pressure-controlled gastrointestinal delivery systems; hydrodynamic pressure-controlled gastrointestinal delivery systems; permeability-controlled membrane gastrointestinal delivery systems, which include permeability-controlled microporous membrane gastrointestinal delivery devices; gastric acid-resistant, gut-targeted controlled-release gastrointestinal delivery devices; gel diffusion-controlled gastrointestinal delivery systems; and ion exchange-controlled gastrointestinal delivery systems, which include cationic and anionic drugs. Additional information on controlled-release drug delivery systems is provided in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).

[0477] Dosage

[0478] The appropriate dosage can be determined by the attending physician or other qualified personnel based on various clinical factors. As is well known in the medical field, the dosage for any patient depends on many factors, including the patient's size, body surface area, age, the specific compound administered, the patient's gender, the time and route of administration, the general health condition, and other drugs administered concurrently. The antibody in question can be administered in an amount of from 1 ng / kg body weight to 20 mg / kg body weight per dose, for example, from 0.001 to 10, from 0.01 to 10, from 0.1 to 10, from 1 to 10, from 0.001 to 1, from 0.01 to 1, from 0.1 to 1, from 0.05 to 5, from 0.05 to 0.5, or from 0.5 to 5 mg / kg body weight. However, doses below or above this typical range are also possible, especially taking into account the factors mentioned above. If the regimen involves continuous infusion, the dose may range from 1 mcg to 10 mg / kg body weight per minute.

[0479] In some embodiments, the dose of the subject anti-LAG-3 antibody is in the range of from 0.001 μg to 100 mg, such as from 0.001 μg to 10 mg, from 0.001 μg to 1 mg, from 0.001 μg to 0.1 mg, from 0.01 μg to 10 mg, from 0.1 μg to 1 mg, or from 0.1 μg to 0.1 mg, or from 0.01 mg to 100 mg, from 0.01 mg to 10 mg, from 0.01 mg to 1 mg, from 0.01 mg to 0.1 mg, from 0.1 mg to 100 mg, from 0.1 mg to 10 mg, from 0.1 mg to 1 mg.

[0480] In some embodiments, the dose may vary, for example, from about 0.0001 to 100 mg / kg, or from about 0.01 to 5 mg / kg (e.g., from 0.02 to 5 mg / kg, from 0.25 to 5 mg / kg, from 0.5 to 5 mg / kg, from 0.75 to 5 mg / kg, from 1 to 5 mg / kg, from 2 to 5 mg / kg, etc.) of body weight. For example, the doses may be 0.1, 1, or 10 mg / kg of body weight, or be within the range of 0.01-10 mg / kg, or be at least 0.1 mg / kg.

[0481] In specific embodiments, the dosage of the anti-LAG-3 antibody of the invention or fragment thereof is up to 0.5 mg / kg body weight, such as in the range of 0.0001 to 0.5 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.5 mg / kg, 0.1 to 0.5 mg / kg, 0.0001 to 0.1 mg / kg, 0.001 to 0.1 mg / kg, 0.01 to 0.1 mg / kg body weight.

[0482] In some embodiments, the subject anti-LAG-3 antibody is administered in an amount that provides a maximum serum concentration of from about 0.001 μg / mL to about 1 mg / mL, such as from 0.0001 μg / mL to about 1 μg / mL, from 0.001 μg / mL to about 1 μg / mL, from 0.001 μg / mL to about 0.1 μg / mL, from about 0.01 to about 1 μg / mL, or from about 0.005 μg / mL to about 1 μg / mL, or from about 0.1 μg / mL to about 1 μg / mL, or from about 1 μg / mL to about 2.5 μg / mL, from about 2.5 mcg / ml to 5 mcg / ml, about 5 mcg / ml to 7.5 mcg / ml, about 7.5 mcg / ml to 10 mcg / ml, about 10 mcg / ml to 25 mcg / ml, about 25 mcg / ml to 50 mcg / ml, about 50 mcg / ml to 100 mcg / ml, about 100 mcg / ml to 250 mcg / ml, about 250 mcg / ml to 500 mcg / ml, about 500 mcg / ml to 750 mcg / ml, or about 750 mcg / ml to 1000 mcg / ml.In some embodiments, the subject anti-LAG-3 antibody is administered in an amount that provides a maximum serum concentration of greater than 1 mg / mL, such as from about 1 mg / mL to 2 mg / mL, from about 2 mg / mL to 5 mg / mL, or from about 5 mg / mL to 10 mg / mL. In other embodiments, an anti-LAG-3 antibody of the invention or fragment thereof is administered in an amount that provides a peak serum concentration of up to 1 μg / ml, such as in the range of 0.0001 μg / ml to 1 μg / ml, 0.001 μg / ml to 1 μg / ml, 0.01 μg / ml to 1 μg / ml, 0.1 μg / ml to 1 μg / ml, 0.0001 μg / ml to 0.1 μg / ml, 0.001 μg / ml to 0.1 μg / ml, or 0.01 μg / ml to 0.1 μg / ml. Suitably, such administration is performed by subcutaneous injection.

[0483] Individuals may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Illustrative treatment involves administering multiple doses over an extended period, for example, at least six months. Additional illustrative treatment regimens include once every two weeks, once monthly, or once every 3-6 months.

[0484] Illustrative dosage regimens include 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg, 0.1 to 1 mg / kg, 1 to 10 mg / kg, or 15 mg / kg on subsequent days, 0.02 to 20 mg / kg, such as 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg every other day, or 0.1 to 100 mg / kg, such as 1 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or 60 mg / kg once a week. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each administered antibody falls within the specified ranges. Progress can be monitored through periodic assessment.

[0485] The number of CD4+ and / or CD8+ T cells expressing LAG-3 in a subject is relatively low. It is contemplated that a single administration of the antibody of the invention (in particular, an antibody of the invention that has a serum half-life of at least two weeks and that lacks significant CDC and ADCC activity, such as a human IgG isotype (which lacks CDC and ADCC activity), or an antibody that contains one or more mutations to reduce or eliminate CDC and ADCC activity) can be effective in inhibiting antigen-induced CD4+ and / or CD8+ T cell proliferation for at least several weeks. In view of this, a suitable treatment regimen may comprise administering the antibody of the invention (e.g., 0.01 to 1 mg / kg of antibody) once every four, six, eight or ten weeks, or once every two or three months.Such treatment may be provided for a period of at least six months or at least one, two, three, four or five years, or longer, such as for the duration of the disease being treated by administration or for the life of the subject.

[0486] Persons skilled in the art will appreciate that dosage levels and administration schedules may vary depending on the specific antibody, the severity of symptoms, and the subject's susceptibility to side effects. Those skilled in the art will readily be able to determine preferred dosages and administration schedules for this compound using a variety of methods.

[0487] Methods of administration

[0488] The antibody in question is administered to an individual by any available method or route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and local administration routes.

[0489] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraperitoneal, intraarterial (e.g., via the carotid artery), spinal or intracerebral delivery, rectal, nasal, oral and other enteral and parenteral routes of administration. The routes of administration can be combined, as desired, or selected depending on the antibody and / or the desired effect. The composition of the subject antibody can be administered as a single dose or multiple doses. In some embodiments, the composition of the subject antibody is administered orally. In some embodiments, the composition of the subject antibody is administered by inhalation. In some embodiments, the composition of the subject antibody is administered intranasally. In some embodiments, the composition of the subject antibody is administered locally.In some embodiments, the antibody composition is administered intracranially. In some embodiments, the antibody composition is administered intravenously. In some embodiments, the antibody composition is administered intrathecally.

[0490] The antibody of the present invention can be administered to the body by any standard route and method suitable for the delivery of conventional drugs, including systemic or local routes. In general, envisaged routes of administration include, but are not particularly limited to, enteral, parenteral, or inhalational administration.

[0491] Parenteral routes of administration, in addition to inhalation, include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intrathecal, and intravenous administration—that is, any route of administration other than via the gastrointestinal tract. Parenteral administration may be for systemic or local delivery of the antibody in question. If systemic delivery is required, administration typically involves invasive, topical, or mucosal administration of systemically acting pharmaceuticals.

[0492] The antibody in question may also be administered to a subject enterally. Enteral routes of administration include, but are not limited to, oral and rectal (e.g., via suppository) administration.

[0493] By "treatment" is meant at least the alleviation of symptoms associated with a pathological condition in the host, where "alleviation" is used in a broad sense and means at least a reduction in the magnitude of a parameter, for example, a symptom associated with the pathological condition being treated, such as an immune disorder. Thus, treatment also encompasses situations in which the pathological condition or at least the symptoms associated with it are completely suppressed, for example, do not occur, or interrupted, for example, ceased, so that the organism no longer suffers from the pathological condition or at least from the symptoms that characterize the pathological condition.

[0494] In some embodiments, the antibody in question is administered by injection and / or puncture, for example, into a cerebral artery or directly into brain tissue. The antibody in question can also be administered directly to the target site, for example, via biolistic delivery to the target site.

[0495] A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject," "individual," and "patient") can be treated in accordance with the disclosed methods. Typically, such hosts are "mammals" or "mammalian," with these terms being used broadly to refer to organisms from the class Mammalia, including the orders Carnivora (e.g., cats), Herbivores (e.g., cattle, horses, and sheep), Omnivores (e.g., dogs, goats, and pigs), Rodents (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the host is an individual that has a complement system, such as a mammal, fish, or vertebrate.In some embodiments, the host is a mammal containing the complement system, a fish or invertebrate, a domestic animal, a farm animal, a working animal, a zoo animal, or a laboratory animal. In some embodiments, the host is a human.

[0496] Embodiments include compositions contained in a suitable container comprising the subject anti-LAG-3 antibody for administration to an individual. For example, the subject antibody may be within a container suitable for containing a pharmaceutical composition. The container may be, for example, a bottle (e.g., with a closure such as a lid), a blister pack (e.g., which may contain one or more doses per blister), a vial, flexible packaging (e.g., sealed Mylar or plastic bags), an ampoule (for single doses in solution), a pipette, a syringe, a thin film, a test tube, and the like. In some embodiments, a container, such as a sterile container, contains the subject pharmaceutical composition. In some embodiments, the container is a bottle or a syringe. In some embodiments, the container is a bottle.In some embodiments, the container is a syringe.

[0497] Kits containing single doses of the antibody in question, for example, in oral or injectable doses, are provided. In addition to the containers containing the single doses, such kits also include an information leaflet describing the use and associated benefits of the antibody in the treatment of the pathological condition of interest. Preferred compounds and single doses are those described above.

[0498] Treatment methods

[0499] According to the invention, there is also provided an antibody according to the invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the invention for use as a medicine.

[0500] The antibody, fragment or composition according to the present invention can be used in any therapy in which it is desirable to enhance the effects of LAG-3 in a subject.

[0501] The term "subject" includes any human being or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses.

[0502] The antibody, fragment or composition can be used in any therapy where it is desired to negatively regulate the proliferation and / or function of T cells.

[0503] Also, according to the invention, there is provided an antibody of the invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the invention for use in the treatment of a disorder associated with the proliferation or activity of CD4+ and / or CD8+ T cells in a subject, or a disorder associated with reduced expression and / or activity of LAG-3 in a subject.

[0504] Also, according to the invention, there is provided the use of an antibody of the invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a disorder associated with the proliferation or activity of CD4+ and / or CD8+ T cells in a subject, or a disorder associated with reduced expression and / or activity of LAG-3 in a subject.

[0505] Furthermore, according to the invention, there is provided a method for treating a disorder associated with the proliferation or activity of CD4+ and / or CD8+ T cells in a subject, or a disorder associated with reduced expression and / or activity of LAG-3 in a subject, which comprises administering an effective amount of an antibody according to the invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the invention to a subject in need of such treatment.

[0506] A disorder associated with the proliferation or activity of CD4+ and / or CD8+ T cells may be an immune disorder, in particular a T cell-mediated immune disorder such as an inflammatory disease or an autoimmune disorder.

[0507] The antibody, fragment, or composition can be used to reduce inflammation or prevent the onset of inflammation. In one embodiment, it is intended to reduce the proliferation or activation of T cells in vivo, particularly those involved in inappropriate inflammatory immune responses, such as those recruited to the vicinity / location of such a response.

[0508] A reduction in T cell proliferation or activation, as used herein, may be a reduction of 10, 20, 30, 40, 50, 60, 70, 80, 90 or more percent compared to the pre-treatment or no-treatment condition.

[0509] Preferably, treatment with an antibody, fragment, or composition according to the present invention can reduce the proliferation or activation of T cells without reducing the patient's total T cell count (inactivated T cells). This can result in fewer side effects and prevent T cell depletion in the patient.

[0510] The immune disorder may, for example, be selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, sepsis, arthritis, rheumatoid arthritis, asthma, COPD (chronic obstructive pulmonary disease), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, postoperative adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus erythematosus (such as systemic lupus erythematosus) and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy,Idiopathic thrombocytopenic purpura, Meniere's syndrome, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's syndrome, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, cardiac disease including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria or infertility associated with fetal-maternal intolerance.

[0511] The antibody, fragment or composition of the invention can be used in any therapy where it is desired to inhibit the binding of LAG-3 to MHC class II molecules in order to antagonize the MHC class II activating signal in antigen-presenting cells (APCs), or to inhibit LAG-3-induced activation of APCs.

[0512] Also according to the invention, there is provided an antibody of the invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the invention for use in the treatment of a disorder associated with APC activation in a subject.

[0513] Also, according to the invention, there is provided the use of an antibody according to the invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a disorder associated with the activation of APC in a subject.

[0514] Furthermore, according to the invention, there is provided a method of treating a disorder associated with APC activation in a subject, which comprises administering an effective amount of an antibody according to the invention or an antigen-binding fragment thereof, or a pharmaceutical composition according to the invention to a subject in need of such treatment.

[0515] The terms "therapy," "treatment," "treat," and the like, as used herein, refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the onset of a disease or a symptom thereof, and / or may be therapeutic in terms of partially or completely curing the disease and / or an adverse effect caused by the disease. "Therapy," as used herein, encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of a disease in a subject who may be predisposed to the disease but who has not yet been diagnosed with it; (b) inhibiting the disease, i.e., stopping its progression; and (c) alleviating the disease, i.e., causing regression of the disease.

[0516] The terms "individual," "subject," "host," and "patient," when used interchangeably herein, include a human or a non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses.

[0517] "Therapeutically effective amount" or "effective amount" refers to the amount of an anti-LAG-3 antibody that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to treat the disease. "The therapeutically effective amount" will vary depending on the anti-LAG-3 antibody, the disease and its severity, as well as the age, weight, etc., of the patient being treated.

[0518] All publications cited herein are incorporated by reference for the disclosure and description of the methods and / or materials with respect to which these publications are cited. Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0519] Figure 1 shows the amino acid sequence (SEQ ID NO: 27) of mature human LAG-3 protein. The four extracellular domains of the Ig superfamily are located at amino acid residues: 1-149 (D1); 150-239 (D2); 240-330 (D3); and 331-412 (D4). The amino acid sequence of the outer loop structure of the D1 domain of human LAG-3 protein is shown underlined and in bold (SEQ ID NO: 40);

[0520] Figure 2 shows a graphic representation of the CDR V loops H monoclonal antibody 13E2 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003));

[0521] Figure 3 shows the amino acid sequence of domain V Hmonoclonal antibody 13E2 aligned with the coding nucleic acid sequence;

[0522] Figure 4 shows a graphic representation of the CDR V loops L monoclonal antibody 13E2 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003));

[0523] Figure 5 shows the amino acid sequence of domain V L monoclonal antibody 13E2 aligned with the coding nucleic acid sequence;

[0524] Figure 6 shows a graphic representation of the CDR V loops H monoclonal antibody 34F4 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003));

[0525] Figure 7 shows the amino acid sequence of domain V H monoclonal antibody 34F4, aligned with the coding nucleic acid sequence;

[0526] Figure 8 shows a graphic representation of the CDR V loops Lmonoclonal antibody 34F4 (Lefranc, M.-P. et al, Dev. Comp. Immunol., 27, 55-77 (2003));

[0527] Figure 9 shows the amino acid sequence of domain V L monoclonal antibody 34F4, aligned with the coding nucleic acid sequence;

[0528] Figure 10 shows the best BLAST alignments of the VDJ germline for the nucleotide sequence encoding the V region H monoclonal antibody 13E2;

[0529] Figure 11 shows the best BLAST alignments of the VJ germline for the nucleotide sequence encoding the V region L monoclonal antibody 13E2;

[0530] Figure 12 shows the best BLAST alignments of the VDJ germline for the nucleotide sequence encoding the V region H monoclonal antibody 34F4;

[0531] Figure 13 shows the best BLAST alignments of the VJ germline for the nucleotide sequence encoding the V region L monoclonal antibody 34F4;

[0532] Figure 14 shows the results of binding of different concentrations of agonist anti-LAG-3 monoclonal antibodies 13E2 and 34F4, and antagonist anti-LAG-3 monoclonal antibody 17B4 to Chinese hamster ovary (CHO) cells transfected with LAG-3, compared with an isotype control antibody (mIgG1);

[0533] Figure 15 shows the results of binding of different concentrations of agonist anti-LAG-3 monoclonal antibodies 13E2 and 34F4, and antagonist anti-LAG-3 monoclonal antibody 17B4 to CD4 + and CD8 + primary cells (SEB-stimulated PBMCs) compared with an isotype control antibody (mIgG1) from a healthy donor (donor 1);

[0534] Figure 16A shows the results of inhibition of IMP321 (LAG-3Ig, 1 μg / mL) binding to MHC class II-positive B cells by different concentrations of agonist anti-LAG-3 monoclonal antibodies 13E2 and 34F4, and antagonist anti-LAG-3 monoclonal antibody 17B4, compared with an isotype control antibody (mIgG1). Figure 16B shows the results of inhibition of THP-1 cell activation by IMP321 (20 ng / mL) in the presence of different concentrations of agonist anti-LAG-3 monoclonal antibodies 13A2 and 34F4, and antagonist anti-LAG-3 monoclonal antibody 17B4, compared with an isotype control antibody (mIgG1);

[0535] Figure 17 (A) shows CMV-induced CD8 cell proliferation profiles +T cells from a single donor in the presence of mIgG1, 17B4, 13E2 or 34F4 analyzed by flow cytometry and the gating strategy used for the assay described in Example 10. Figure 17(B) shows the results of the CD8 proliferation inhibition assay. + T cells with 13E2, 34F4, and 17B4 antibodies compared to an isotype control antibody (mIgG1) for the same donor. Baseline proliferation (No Stim) is also shown;

[0536] Figure 18 shows the results of CD4 cell proliferation inhibition assay + or CD8 + T cells with 13E2 or 34F4 antibodies compared with an isotype control antibody (mIgG1) in several different donors;

[0537] Figure 19 shows the effect of different concentrations of agonistic anti-LAG-3 monoclonal antibodies 13E2 and 34F4 on CD8 proliferation + T cells;

[0538] Figure 20 shows the amino acid sequence of the heavy chain of the chimeric 13E2-human IgG4 Fc antibody (the amino acid sequence of the heavy chain of the chimeric 13E2-human IgG4 Fc is designated as 13E2IgG4mut in the figure), and the amino acid sequence of the light chain of the chimeric 13E2-human IgK antibody (the amino acid sequence of the light chain of the chimeric 13E2-human IgK is designated as 13E2IgK in the figure);

[0539] Figure 21 shows the different effects on T cells of depleting, antagonist and agonist anti-LAG-3 antibodies;

[0540] Figure 22 shows the alignment of the variable regions of humanized variants 1-4 V H(VH1, VH2, VH3, and VH4) and alignment of the variable regions of the humanized VL variants 1–4 (VL1, VL2, VL3, and VL4) with the corresponding sequence of the parental murine monoclonal antibody 13E2 (VH 13E2 and VL 13E2, respectively). The CDR sequences are highlighted in gray. Changes in the humanized framework sequences of the variants compared to the parental murine sequence are shown underlined and in bold;

[0541] Figure 23 shows the amino acid sequence of the heavy chain of the humanized 13E2-human IgG4 Fc antibody (IMP761) aligned with the amino acid sequence of the chimeric 13E2-human IgG4 Fc heavy chain (13E2IgG4mut) antibody Chim13E2IgG4. Region V His shown in bold, and the Fc region is highlighted. Amino acid residues of the humanized IMP761 sequence, which differ from the corresponding residues of the chimeric 13E2IgG4mut sequence, are underlined with a single line. CDR sequences (based on the combined identification of IMGT / Kabat CDR sequences) are underlined with a double line;

[0542] Figure 24 shows the amino acid sequence of the light chain of the humanized 13E2-human IgE antibody (IMP761) aligned with the chimeric 13E2-human IgK (13E2IgK) light chain amino acid sequence of the Chim13E2IgG4 antibody. Region V Lis shown in bold, and the IgK region is highlighted. Amino acid residues of the humanized IMP761 sequence that differ from the corresponding residues of the chimeric 13E2Ig sequence are single-underlined. CDR sequences (based on the combined identification of IMGT / Kabat CDR sequences) are shown as double-underlined;

[0543] Figure 25 shows the results of the assay performed to test the binding of chimeric 13E2-human IgG4 Fc antibody (Chim13E2IgG4) and IMP761 to CHO-LAG-3 cells + ;

[0544] Figure 26 shows the results of the assay conducted to test the effect of IMP761 and Chim13E2IgG4 on: (a) antigen-induced CD8 proliferation + T cells; and (b) expression of CD25 in CD8 + T cells;

[0545] Figure 27 shows the effect of IMP761 and Chim13E2IgG4 on antigen-induced CD8 proliferation +T cells and CD25 expression as a graph: (a) percentage inhibition of CD8 proliferation + T cells; and (b) percentage inhibition of CD25 expression in CD8 + T cells compared to isotype-matched controls;

[0546] Figure 28 shows the effect of different concentrations of IMP761 and Chim13E2IgG4 on antigen-induced CD8 proliferation + T cells;

[0547] Figure 29 shows the results of ADCC assays performed to determine whether IMP761 has cytotoxic activity against LAG-3-expressing cells using: ADCC Reporter Bioassay available from Promega (a); and an ADCC assay using IL-2-stimulated PBMC as effector cells - results are plotted as percentage: CD4 + and CD8 + T cells (b); or LAG-3 + CD4 + and LAG-3 + CD8 + T cells (c) in the PBMC population;

[0548] Figure 30 shows the results of CDC assays using rabbit complement to determine whether IMP761 has cytotoxic activity against LAG-3-expressing cells - results are plotted as a percentage: (a) CD4 + and CD8 + T cells; or (b) LAG-3 + CD4 + and LAG-3 + CD8 + T cells in the PBMC population; and

[0549] Figure 31 shows the results of the assay to determine whether IMP761 has cytotoxic activity against LAG-3-expressing cells after culturing antigen-stimulated PBMCs with the antibody for 3 days. The results are plotted as: (a) CD4 percentage + and CD8 + T cells in the PBMC population; and (b) the percentage of LAG-3 + cells in the CD4 subpopulation + and CD8 + T cells.

[0550] Example 1

[0551] Production of anti-LAG-3 monoclonal antibody 13E2

[0552] To obtain anti-LAG-3 antibodies, 15 Balb / c mice (referred to as mice No. 1-15 below) were immunized according to the immunization protocol described below.

[0553] Thirteen mice were each immunized by subcutaneous (sc) injections of 100 μg IMP321 (LAG-3Ig), clinical use lot S017 / LC1 / 041011 (hereinafter referred to as “LC1”): 3 times (mouse number 12); 4 times (mouse number 9); 5 times (mice numbers 5 and 14); or 6 times (mice numbers 3 and 11) on Day 0, Day 15, Day 30, Day 50, Day 67, and Day 108. Mice numbers 1, 2, 4, 8, 10, 13, and 15 were immunized up to 4 additional times. In parallel, two mice (mouse number 6 and mouse number 7), used as controls, were immunized with 10 μg LC1 in complete Freund's adjuvant (CFA) once on Day 0 and with the same dose of antigen in incomplete Freund's adjuvant (IFA) on Day 15, Day 30, Day 50, Day 67, and Day 108.

[0554] Twelve days after the 6th immunization, sera were collected from mice numbered 1, 2, 3, 4, 8, 10, 11, 13, and 15 (mice numbered 5, 9, 12, and 14 had already been sacrificed) and analyzed in an enzyme-linked immunosorbent assay (ELISA) using D1-D4 LAG-3 as the coated antigen, purified anti-LAG-3 mouse monoclonal antibody 17B4 as the reference, and goat anti-mouse Ig-HRP as the labeled secondary antibody to determine the concentration of anti-LAG-3 antibodies present in the sera. The results are shown in Table 2 below:

[0555]

[0556] After six immunizations, sera from several mice (including mouse number 3) unexpectedly performed better than sera from two positive control mice (numbers 6 and 7), even though these mice were immunized with IMP321 in the absence of CFA or IFA as an adjuvant. This unconventional method (i.e., immunization with IMP321 in PBS without CFA or IFA as an adjuvant) also yielded good results compared to other experiments conducted using LAG-3-expressing CHO cells in IFA (data not shown). Without being bound by theory, it is believed that this may be due to the fact that IMP321 itself is an adjuvant, meaning it directly triggers the activation and maturation of dendritic cells.

[0557] The same serum samples were assessed for their ability to inhibit IMP321 binding to its ligand, MHC class II, expressed on Raji B cells. Ten microliters of Alexa Plio488-conjugated IMP321 at a concentration of 10 μg / mL were preincubated with 5 μL of serum collected from each mouse, or without serum, or native serum for 30 min at 4°C. Raji cells were then added to a final volume of 50 μL and incubated for 30 min at 4°C. Cell-bound fluorescence was analyzed by flow cytometry.

[0558] Mouse number 3 was selected because the serum from this mouse showed a high titer (423 μg / mL) in the D1-D4 serum ELISA assay (Table 1) and a high ability to inhibit IMD321 binding to MHC class II + - Raji B. cells.

[0559] Twelve days after the sixth immunization, mouse number 3 was intravenously (iv) stimulated with 10 μg of D1-D4 LAG-3 (without Fc tail) recombinant protein (produced in CHO cells and purified). Three days after the iv stimulating injection, mouse number 3 was sacrificed and the spleen was removed. Splenocytes were isolated by squeezing pieces of spleen with a rubber plunger of a 5 ml syringe in a Petri dish containing complete serum-free DMEM medium. Splenocytes and Sp2 / 0 myeloma cells (cultured in RPMI 1640 medium + 10% FCS + 2 mM glutamine + 0.5% P / S) were washed in serum-free medium. The two cell types were mixed together in a 5:1 ratio of splenocytes to myeloma cells and then pelleted by centrifugation. Fusion agent (PEG-1500, polyethylene glycol solution, 50% w / v in PBS, Roche 10783641001, 1 ml per 10 8Cells were pre-warmed at 37°C and added dropwise to the cell pellet. The cells were carefully resuspended and diluted by doubling the volume after 90 seconds. The cells were further diluted at regular intervals to a final dilution of 1 in 15 over approximately 5 minutes. The cells were then centrifuged and resuspended in medium containing 10% FBS and incubated for approximately one hour at 37°C. Cells (10,000 cells / well) were then seeded in 46 96-well plates in complete RPMI medium containing 10% Ultralow Ig FBS (Gibco 16250), 2% HAT (Gibco 21060) and supplemented with 10% BM Condimed H1 (a culture medium supplement to support B-cell hybridoma growth after fusion and during cloning, Roche 11088947001) and cultured until screening.

[0560] Screening was performed by cytometry using CHO cells expressing membrane-bound LAG-3 to analyze the binding capacity of antibodies present in the supernatants of growing hybridomas, detected with FITC-conjugated goat anti-mouse Ig. Positive hybridomas were expanded and rescreened on LAG-3 cells. + CHO and wild-type CHO cells. A total of 632 wells from this fusion were screened by FACS analysis on LAG-3-expressing CHO cells (14% yield). Four hybridoma clones were found to stably express the anti-LAG-3 antibody, including 13E2. The hybridoma was then subcloned by limiting dilution.

[0561]

[0562] Example 2

[0563] Amino acid sequence of the variable region of antibody 13E2

[0564] Amino acid sequence V H :

[0565] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGLGWIRQPSGKGLEWLTHIWWDDIKRYNPDLRSRLTISKDTSSSOIFLKIASVDTADTATYYCARIVEGSYSSSYFDVWGAGTTVTVSS (SEQ ID NO: 7).

[0566] SEQ ID NO: 7 is the amino acid sequence of the heavy chain variable domain (V H ) 13E2 antibody. Complementarity-determining regions (CDRs) defined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)) are underlined. CDRs defined according to the Kabat numbering system are shown in bold.

[0567] Figure 2 shows a graphic representation of the CDR V loops Hmonoclonal antibody 13E2 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). The shaded circles (residues 4, 12, 13, 19, 21, 23, 25, 41, 50, 52, 53, 71, 76, 78, 87, 89, 91, 94, 100) represent hydrophobic (non-polar) residues in framework regions 1-3 at sites that are hydrophobic in most antibodies. The squares represent key residues at the beginning and end of each CDR region. Residues 23, 41, 89, 104, 118 in the framework region represent structurally conserved amino acids;

[0568] Amino acid sequence V L :

[0569] DIVMTQPHKFMSTSVEDRVTITCKASQDVIFDVAWYQQKPGQSPKLLIYSASSRVSGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTTLEIK (SEQ ID NO: 8).

[0570] SEQ ID NO: 8 is the amino acid sequence of the light chain variable domain (V L) 13E2 antibody. Complementarity-determining regions (CDRs) defined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)) are underlined. CDRs defined according to the Kabat numbering system are shown in bold.

[0571]

[0572] Figure 4 shows a graphic representation of the CDR V loops Lmonoclonal antibody 13E2 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). The shaded circles (residues numbered 4, 1 1, 19, 21, 23, 25, 40, 41, 52, 53, 54, 71, 76, 87, 89, 91, 94, 96, 100, 101) represent hydrophobic (non-polar) residues in framework regions 1-3 at sites that are hydrophobic in most antibodies. The squares represent key residues at the beginning and end of each CDR. Residues numbered 23, 41, 89, 104, 118 in the framework region represent structurally conserved amino acids.

[0573] Example 3

[0574] Nucleic acid sequence encoding the variable domains of antibody 13E2

[0575] The nucleic acid sequence encoding the VH domain of monoclonal antibody 13E2 is shown in Figure 3 and below:

[0576]

[0577] BLAST alignment of nucleic acid sequences revealed that the nucleic acid sequence encoding the VH domain of monoclonal antibody 13E2 shares significant identity with the following germline genes: IGHV8-8*01, IGHV8-11*01, IGHV8-12*01, IGHD2-12*01, IGHD1-1*01, IGHJ1*01, IGHJ1*02, IGHJ1*03.

[0578] Figure 10 shows the alignment of the nucleic acid sequence encoding the VH domain of the 13E2 antibody with its best match to the germline gene. Figure 10 shows that the portion containing nucleotides 1-301 of the 13E2 VH region (which encompasses the FR1, FR2, and FR3 heavy chain framework regions) has a nucleic acid sequence that is 92.7% identical to the nucleotide sequence of the V gene of IGHV8-8*01.

[0579]

[0580] The nucleic acid sequence encoding the VL domain of monoclonal antibody 13E2 is shown in Figure 5 and below:

[0581]

[0582] BLAST alignment of nucleic acid sequences revealed that the nucleic acid sequence encoding the VL domain of monoclonal antibody 13E2 has significant identity with the following germline genes: IGKV6-17*01, IGKV6-25*01, IGKV6-23*01, IGKJ2*01, IGKJ2*03, IGKJ2*02.

[0583] Figure 11 shows the alignment of the nucleic acid sequence encoding the VL domain of the 13E2 antibody with its best match to germline genes. Figure 11 shows that the portion containing nucleotides 1–284 of the 13E2 VL region (which encompasses the FR1, FR2, and FR3 framework regions of the light chain) has a nucleic acid sequence that is 94.7% identical to the nucleotide sequence of the IGKV6-17*01 V gene.

[0584]

[0585] Example 4.

[0586] Creation of anti-LAG-3 monoclonal antibody 34F4

[0587] Each of 4 Balb / c mice (referred to as mice 1-4 below) were immunized with four (mice 2 and 4) or five (mice 1 and 3) subcutaneous (sc) injections of 100 μg IMP321 (LAG-3Ig), clinical use lot S017 / LC1 / 041011 (referred to as “LC1” below), on Day 0, Day 14, Day 28, Day 43, and Day 70. An additional Balb / c mouse (referred to as mouse 5 below) was immunized with three subcutaneous (sc) injections of D1-D4 LAG-3.

[0588] Two weeks after the third injection, serum from each mouse was tested for anti-LAG-3 antibody content in an ELISA assay (as described in Example 1). The results are presented below in Table 7:

[0589]

[0590] The ability of serum from each mouse to inhibit IMP321 binding to Raji B cells was determined by FACS analysis (as described in Example 1).

[0591] As shown in Table 7, antibody titers were low in all mice. None of the sera inhibited IMP321 binding to MHC class II. + Raji B cells after three immunizations. No further selections were performed to test serum titers and inhibitory capacity. The immunization process continued, and mouse number 3, which had received five sc injections of LC1, was stimulated with 10 μg of D1-D4 LAG-3 intravenously on Day 92.

[0592] Three days after the stimulating intravenous (iv) injection, 73 million splenocytes from mouse number 3 were fused with 15 million Sp2 / 0 myeloma cells following the same procedure as described in Example 1. Approximately 25,500 cells per well were seeded into 40 wells of 96-well plates and then cultured with the addition of culture medium containing 10% BM Condimed H1. 2,256 wells were screened by FACS analysis for LAG-3-expressing CHO cells (59% yield). Two resistant anti-LAG-3 hybridomas were selected, including 34F4 (see Table 8 below).

[0593]

[0594] Example 5

[0595] Amino acid sequence of the variable region of antibody 34F4

[0596] Amino acid sequence V H ;

[0597]

[0598] SEQ ID NO: 17 is the amino acid sequence of the heavy chain variable domain (VH ) 34F4 antibodies. Complementarity-determining regions (CDRs) defined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)) are underlined, CDRs defined according to the Kabat numbering system are shown in bold.

[0599] Figure 6 shows a graphic representation of V loops H CDRs of monoclonal antibody 34F4 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). The filled circles (residues 4, 12, 13, 19, 21, 23, 25, 41, 50, 52, 53, 71, 76, 78, 87, 89, 91, 94, 100) represent hydrophobic (non-polar) residues in framework regions 1-3 at sites that are hydrophobic in most antibodies. Squares represent key residues at the beginning and end of each CDR. Residues 23, 41, 89, 104, 118 in the framework region are structurally conserved amino acids.

[0600] Amino acid sequence VL :

[0601]

[0602] SEQ ID NO: 18 is the amino acid sequence of the light chain variable domain (V L ) 34F4 antibodies. Complementarity-determining regions (CDRs) defined according to the IMGT numbering system (Lefranc, M.-P. et al., Nucleic Acids Research, 27, 209-212 (1999)) are underlined, CDRs defined according to the Kabat numbering system are shown in bold.

[0603]

[0604]

[0605] Figure 8 shows a graphic representation of the CDR V loops Lmonoclonal antibody 34F4 (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)). The filled circles (residues numbered 4, 11, 19, 21, 23, 25, 40, 41, 52, 53, 54, 71, 76, 87, 89, 91, 94, 96, 100, 101) represent hydrophobic (non-polar) residues in framework regions 1-3 at sites that are hydrophobic in most antibodies. The squares represent key residues at the beginning and end of each CDR. Residues numbered 23, 41, 89, 104, 118 in the framework region represent structurally conserved amino acids.

[0606] Example 6.

[0607] Nucleic acid sequence encoding the variable domains of the 34F4 antibody

[0608] The nucleic acid sequence encoding the VH domain of monoclonal antibody 34F4 is shown in Figure 7 and below:

[0609]

[0610] BLAST alignment of the nucleic acid sequence shows that the nucleic acid sequence encoding the VH domain of monoclonal antibody 34F4 shares significant identity with the following germline genes: IGHV8-8*01, IGHV8-12*01, IGHV8-11*01, IGHD1-1*01, IGHD1-2*01, IGHD2-3*01, IGHJ2*01, IGHJ2*02, IGHJ2*03. Figure 12 shows the alignment of the nucleic acid sequence encoding the VH domain of antibody 34F4 with its best match to the germline genes. Figure 12 shows that the portion containing nucleotides 1-301 of the VH region of 34F4 (which encompasses the heavy chain framework regions FR1, FR2 and FR3) has a nucleic acid sequence that is 94.4% identical to the nucleotide sequence of the V gene IGHV8-8*01.

[0611]

[0612] The nucleic acid sequence encoding the VL domain of monoclonal antibody 34F4 is shown in Figure 9 and below:

[0613]

[0614] BLAST alignment of the nucleic acid sequences indicates that the nucleic acid sequence encoding the VL domain of monoclonal antibody 34F4 shares significant identity with the following germline genes: IGKV6-17*01, IGKV6-25*01, IGKV6-23*01, IGKJ1*01, IGKJ1*02, IGKJ2*01.

[0615] Figure 13 shows the alignment of the nucleic acid sequence encoding the VL domain of the 34F4 antibody with its best match to germline genes. Figure 13 shows that the portion containing nucleotides 1–284 of the 34F4 VL region (which encompasses the FR1, FR2, and FR3 framework regions of the light chain) has a nucleic acid sequence that is 94.7% identical to the nucleotide sequence of the V gene of IGKV6-17*01.

[0616]

[0617]

[0618] Example 7.

[0619] Binding of agonistic monoclonal antibodies 13E2 and 34F4 to LAG-3 + transfected and primary cells compared with the antagonist monoclonal antibody 17B4

[0620] LAG-3 + -transfected CHO cells or SEB-stimulated PBMCs from a healthy donor were incubated with anti-LAG-3 monoclonal antibody or isotype control (mIgG1) for 30 min in PBS, 0.5% BSA, 0.1% azide at 4°C. Cells were washed, and cell-bound antibody was detected with FITC-conjugated goat F(ab')2-anti-mouse Ig (H+L) (Coulter). The secondary antibody was washed out, and CHO cells were directly analyzed by flow cytometry. PBMCs were phenotyped using CD4-PE-Cy7 and CD8-APC-Cy7. Binding to LAG-3 + CHO cells

[0621] The results are presented as mean fluorescence intensity (MFI) of CHO cells transfected with a plasmid encoding human LAG-3, depending on the antibody concentration. The results are shown in Table 12 below and Figure 14.

[0622]

[0623] Based on the results shown in Table 12, the EC value 50 The binding ELISA of each antibody to LAG-3-expressing CHO cells was: 17B4: 0.7 nM; 13E2: 0.3 nM; 34F4: 0.5 nM.

[0624] EU average values 50 from four independent experiments (data not shown) for the binding of each antibody to LAG-3-expressing CHO cells are: 17B4: 0.7 nM; 13E2: 0.4 nM; 34F4: 0.5 nM. The average EC 50 13E2 from four independent experiments is 2.7 times higher than the average EC value 50 17B4.

[0625] EU average 5034F4 from four independent experiments is 1.6 times the EU average 50 17B4.

[0626] Binding to SEB-stimulated PBMCs.

[0627] Results are presented as mean fluorescence intensity on CD4+ or CD8+ cells from donor PBMCs (Donor 1) stimulated for three days with 0.5 μg / ml SEB depending on the antibody concentration. CD4 binding results + and CD8 + cells for Donor 1 are shown in Table 13 below and Figure 15.

[0628]

[0629] Based on the results shown in Table 13, the EC50 value for binding of each antibody to CD4 + cells was: 17B4: 0.5 nM; 13E2: 0.1 nM; 34F4: 0.1 nM.

[0630] EU average 50 from three donors (data not shown) for the binding of each antibody to CD4 + cells was: 17B4: 0.8 nM; 13E2: 0.2 nM; 34F4: 0.2 nM.

[0631] EU average 50 CD4 + 13E2 and 34F4 from three donors were 3.8 times higher than the EU average 50 17B4.

[0632] Based on the results shown in Table 13, the EC50 value for binding of each antibody to CD8 + cells was: 17B4: 0.7 nM; 13E2: 0.3 nM; 34F4: 0.2 nM.

[0633] EU average 50 from three donors (data not shown) for binding of each antibody to CD8 + cells was: 17B4: 1 nM; 13E2: 0.4 nM; 34F4: 0.5 nM.

[0634] Average EC50 CD8 value + 13E2 and 34F4 from three donors were 2.5 times higher than the EU average 50 17B4.

[0635] The results showed that monoclonal antibodies 13E2 and 34F4 each bind to CHO cells expressing LAG-3 + , and with CD4 + -T cells and CD8 + -T cells with higher affinity than 17B4.

[0636] Biacore assay with LAG-3Ig on-chip and 17B4 antibody in running buffer showed the following results:

[0637]

[0638] Biacore assay with 17B4 antibody on chip and LAG-3lg in running buffer gave the following results:

[0639]

[0640] Example 8

[0641] Inhibition of IMP321 (LAG-3Ig) binding to MHC class H-positive cells by 13E2 and 34F4

[0642] Binding of IMP321 (LAG-3Ig-Alexa 488) conjugate to MHC class II-positive B cells (Raji cells) was determined after pre-incubation of the conjugate (1 μg / ml at 4°C) with anti-LAG-3 monoclonal antibody (13E2, 34F4, or 17B4) or isotype control (mIgG1). Cell-associated fluorescence analysis was performed using fluorescence-activated cell sorting (FACS).

[0643] The mean fluorescence intensity (MFI) corresponding to cell-bound LAG-3Ig depending on the antibody concentration is shown in Table 14 below and Figure 16A.

[0644]

[0645]

[0646] The results showed that IMP321 binding to Raji cells was inhibited by pre-incubation with each of the LAG-3-specific monoclonal antibodies.

[0647] Example 9

[0648] Inhibition of IMP321 (LAC-3Ig)-induced monocyte activation by 13E2 and 34F4

[0649] IMP321 (20 ng / ml) was pre-incubated with anti-LAG-3 monoclonal antibody 13E2, 34F4, or 17B4, or isotope control (mIgG1) for 5 min at 37°C before incubation of the mixture with THP-1 cells for 4 h at 37°C. The amount of CCL4 secreted by THP-1 cells was used to determine the level of monocyte activation.

[0650] The concentration of CCL4 (expressed in pg / mL) depending on the concentration of Ab is shown in Table 15 below and Figure 16B.

[0651]

[0652] The results showed that IMP321-induced monocyte activation was inhibited by pre-incubation of IMP321 with the antagonist anti-LAG-3 monoclonal antibody 17B4, as well as by pre-incubation with the agonist monoclonal antibodies 13E2 and 34F4.

[0653] Based on these results and the results obtained in Example 8, it can be concluded that the agonist monoclonal antibodies 13E2 and 34F4, like the antagonist monoclonal antibody 17B4, interact with or near the MHC class II binding site of LAG-3, as evidenced by their ability to block the binding and activity of LAG-3Ig (IMP321).

[0654] Example 10

[0655] Inhibition of T cell proliferation by 13E2 and 34F4 compared with 17B4

[0656] PBMC from 3 healthy donors (0.2×10 6 cells / well, at 1×10 6 / ml in complete RPMI medium + 10% FBS) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and incubated with a pool of peptides covering the CMV pp35 sequence in the presence of monoclonal anti-LAG-3 antibody 13E2, 34F4, 17B4 or isotype control (mIgG1) (supra-optimal dose, 300 ng / ml for donor #1 and #2, 100 ng / ml for donor #3).

[0657] T cell response was examined by measuring CD4 proliferation + or CD8 + CFSE-based T cells at day 5. FACS profiles for CD8 + Donor #1 T cell counts in the presence of each antibody, as well as the gating strategy, are shown in Figure 17(A). Figure 17(B) shows the percentage of CD8 +T cells under each division peak depending on cell division for the same donor. The results for 3 donors are shown in Table 16 below. Baseline proliferation without antigen peptides (no stimulus) was also measured (see Figure 17(A), lower panel, and Table 16). CD4 + Donor #1 T cells did not show any CMV-specific proliferation, so results for this population are not included.

[0658]

[0659] Proliferation Index (PI) (calculated as the sum of: CD4 percentage + or CD8 + The number of T cells under each division peak, multiplied by the number of divisions, is shown in Table 17. This index represents the percentage of cells that have experienced multiple rounds of division. Table 17 also shows the percentage inhibition for each antibody compared to the isotype control (mIgG1) based on PL values.

[0660]

[0661] The results showed that monoclonal antibodies 13E2 and 34F4 comparablely inhibit CD4 proliferation + and CD8 + T cells induced by antigenic peptides, whereas the monoclonal antibody 17B4 had a slight positive effect at the concentration tested.

[0662] Example 11

[0663] Inhibition of T cell proliferation by 13E2 and 34F4

[0664] PBMC from 12 healthy donors (0.2×10 6 cells / well, at 1×10 6 / ml in complete RPMI medium+10% FBS) were labeled with CFSE and incubated with a pool of peptides covering the CMV pp35 sequence in the presence of monoclonal anti-LAG-3 antibody 13E2, 34F4 or isotype control (mIgG1).

[0665] T cell response was examined on day 5 by measuring CD4 proliferation + or CD8 + CFSE-based T cells. CD4 percentage + or CD8 +T cells under each division peak were calculated as a function of cell division using the gating strategy shown in Figure 17(A). Baseline proliferation without antigen peptides (no stimulus) was also measured. CD4 + T cells from donors #1, #5, and #12 did not show any CMV-specific proliferation, so results for these samples were not included.

[0666] Proliferation Index (PI) (calculated as the sum of: CD4 percentage + or CD8 + The number of T cells under each division peak multiplied by the number of divisions) for each donor is shown in Table 18, and the results are plotted in Figure 18. Table 18 also shows the percentage inhibition for each antibody compared to the isotype control (mIgG1) based on PI values.

[0667]

[0668]

[0669] Donor #1, 2, 4, 5, 6, 7, 8, 9: 300 ng / ml; Donor #3: 10 ng / ml; Donor #10, 11, 12: 1000 ng / ml

[0670] The average values ​​of these results are shown in Table 19.

[0671]

[0672] The results showed that monoclonal anti-LAG-3 antibodies 13E2 and 34F4 inhibit CD4 proliferation + and CD8 + T cells induced by antigenic peptides. The results suggest that the inhibitory effect of each antibody may be more pronounced for CD8 + T cells than CD4 + T cells. In most donors tested, the effects of the 13E2 and 34F4 antibodies were highly similar, indicating that the antibodies likely have comparable activity.

[0673] Example 12

[0674] Dose-dependent effect of agonist antibody on CP8 proliferation + T cells

[0675] CFSE-labeled PBMCs were stimulated with CMV peptide as described above in the presence of different concentrations of agonist anti-LAG-3 monoclonal antibody 13E2, 34F4, or isotype control (mIgG1).

[0676] T cell response was examined by measuring CD8 proliferation on day 5 + T cells based on CFSE. Proliferation index (calculated as the sum of the percentage of CD8 + T cells under each division peak, multiplied by the number of divisions) is shown in Table 20.

[0677]

[0678] Table 21 below shows the CD8 proliferation index + T cells depending on antibody concentration. The results in Table 21 are plotted in Figure 19.

[0679]

[0680] The results showed that a dose of only 30 ng / ml of monoclonal anti-LAG-3 antibody 13E2 or 34F4 caused maximal inhibition of CD8 proliferation +T cells. The results also showed that the effects of antibodies are very similar.

[0681] Example 13

[0682] CD8 cell proliferation inhibition + T cells are altered by 34F4 by pre-incubation with IMP321

[0683] CFMS-labeled PBMCs from 2 donors were stimulated with CMV peptide as described above in the presence of varying concentrations of 34F4 antibody. A dose of 1 μg / ml 34F4 was also assessed after neutralization with a 10-fold excess of IMP321.

[0684] T cell response was examined by measuring CFSE-based CD8 proliferation on day 5 + T cells. Percentage of CD8 proliferation inhibition + T cells were calculated based on the percentage of dividing cells observed in the presence of 34F4 antibody or 34F4 antibody and IMP321 (LAG-3lg) compared to controls with or without IMP321.

[0685] The results are shown in Table 22 below.

[0686]

[0687] The results showed that pre-incubation of 34F4 antibody with IMP321 reversed the inhibitory effect of 34F4 antibody on CD8 proliferation + T cells. This shows that inhibition of CD8 proliferation + 34F4-mediated T cell activation depends on the binding of the 34F4 antibody to LAG-3.

[0688] Example 14

[0689] CD8 cell proliferation inhibition + T-cell antibodies 13E2 and 34F4 are not affected by IL-2

[0690] CFSE-labeled PBMCs were stimulated with CMV peptides as described above in the presence of 13E2 or 34F4 antibody with or without IL-2.

[0691] T cell response was examined on day 5 by measuring CFSE-based CD8 proliferation + T cells. Percentage of CD8 proliferation inhibition + T cells were calculated based on the percentage of dividing cells observed in the presence of 13E2 or 34F4 antibody with or without IL-2 compared to isotype control with or without IL.

[0692] The results are shown in Table 23 below.

[0693]

[0694] The results showed that the addition of exogenous IL-2 could not overcome the inhibitory effect of 13E2 or 34F4 antibody on CD8 proliferation. + T cells. From these results, it can be concluded that antibodies 13E2 and 34F4 each directly inhibit signal 1 (response to CMV antigen, a T-cell receptor-dependent pathway), but not signal 2 (response to IL-2, which is help from CD4 cells) in CD8 + T cells.

[0695] Example 15

[0696] Effect of 13E2 on the secretion of T cell activation marker

[0697] Peripheral blood mononuclear cells (PBMCs) include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. IFN-γ is predominantly secreted by activated CD4 + and CD8 +Memory and effector T cells and NK cells upon activation. Following re-stimulation with a specific antigen in vitro, IFN-γ secretion is induced.

[0698] PBMCs from four healthy donors (0.2×10 6 cells / well at 1×10 6 / ml in complete RPMI medium + 10% FBS) were labeled with CFSE and incubated with a pool of peptides covering the CMV pp35 sequence in the presence of the monoclonal anti-LAG-3 antibody 13E2 or an isotopic control (mIgG1). The T cell response was examined by measuring the release of IFN-γ in the cell supernatant on day 2. The concentration of IFN-γ and the percentage inhibition of IFN-γ secretion by the 13E2 antibody are shown below in Table 24.

[0699]

[0700] The results showed that monoclonal antibody 13E2 inhibited IFN-γ secretion in each of the tested donors. This indicates that monoclonal antibody 13E2 inhibits T cell activation.

[0701] Example 16

[0702] Effect of 13E2 and 34F4 on the expression of T cell activation marker

[0703] PBMCs from four healthy donors (0.2×10 6 cells / well, at 1×10 6 / ml in complete RPMI medium+10% FBS) were labeled with CFSE and incubated with a pool of peptides covering the CMV pp35 sequence in the presence of monoclonal anti-LAG-3 antibody 13E2 or 34F4, or isotype control (mIgG1).

[0704] T cell response was examined by measuring the expression of CD25, as an activation marker, on CD8 at day 5 + T cells. CD8 percentage + CD25-expressing T cells and the percentage inhibition of CD25 expression by 13E2 or 34F4 are presented in Table 25 below.

[0705]

[0706] The results showed that each monoclonal antibody, 13E2 and 34F4, significantly inhibited the expression of CD25 on CD8 +T cells. This indicates that each antibody inhibits CD8 activation. + T cells.

[0707] Example 17

[0708] Sequences of chimeric human antibody 13E2

[0709] The nucleotide sequences encoding the variable regions of the mouse 13E2 heavy and light chains were fused with the constant regions of the human IgG4 heavy and kappa light chains, respectively. These synthetic chimeric sequences were subcloned into an expression vector and expressed in CHO cells grown in suspension.

[0710] The amino acid sequence of the heavy chain of the chimeric human antibody 13E2 to the Fc fragment of IgG4 is shown below and in Figure 20(A). The antibody contains the V domain. H mouse monoclonal antibody 13E2 and the Fc fragment of human IgG4 with the S228P mutation (to abolish the exchange of Fab fragments) (13E2IgG4mut). In the figure, region V Hshown in bold and the Fc region shown in color.

[0711] 13E2IgG4mut

[0712]

[0713] The amino acid sequence of the light chain of the chimeric human IgK antibody 13E2 is shown below and in Figure 20(B). The antibody contains the V domain. L monoclonal antibody 13E2 and the C region of the wild-type human IgK kappa chain (13E2IgK). In the figure, region V L is shown in bold and the IgK region is shown in color.

[0714] 13E2IgK

[0715]

[0716] Chimeric human antibody 13E2 (referred to as Chim13E2IgG4) contains chimeric heavy and light chains: 13E2IgG4mut; and 13E2IgK.

[0717] Example 18

[0718] Sequences of humanized monoclonal antibody 13E2 (IMP761)

[0719] To optimally maintain the CDR loop conformation, a combined IMGT / Kabat CDR sequence identification was used to graft the CDRs of the murine 13E2 antibody onto human scaffolds to generate a humanized version of 13E2. These synthetic chimeric sequences were subcloned into an expression vector and expressed in CHO cells grown in suspension.

[0720] The amino acid sequences of the heavy and light chains of the humanized monoclonal antibody 13E2 (also known as IMP761) are shown below. The variable domains are shown in bold, and the CDR sequences are shown underlined.

[0721] Amino acid sequence of the heavy chain of IMP761

[0722]

[0723] The alignment of this sequence (IMP761 heavy chain) with the chimeric heavy chain sequence, 13E2IgG4mut, from Example 17 is shown in Figure 23. In this figure, region V His shown in bold and the Fc region is shown in color. Amino acid residues of the humanized IMP761 sequence that differ from the corresponding residues of the chimeric 13E2IgG4mut sequence are underlined with a single line. The CDR sequences (based on the combined identification of CDR sequences according to IMGT / Kabat) are underlined with a double line. The substituted residues in the humanized sequence are also listed below in Table 26 (as VH variant 4, VH4, as well as the substituted residues in three other humanized variants of the original 13E2 heavy chain sequence: VH variants 1, 2, and 3, VH1, VH2, and VH3).

[0724]

[0725] The framework sequences of the heavy chain of the humanized antibody (IMP761 antibody) are as follows:

[0726] VH FR1: QITLKESGPTLVKPTQTLTLTCTFS (SEQ ID NO: 64);

[0727] VH FR2: WIRQPPGKTLEWLT (SEQ ID NO: 65);

[0728] VH FR3: RLSITKDTSKNQVVLTMTNMDPLDTGTYYC (SEQ ID NO: 66); And

[0729] VH FR4: WGQGTLVTVSS (SEQ ID NO: 67).

[0730] Amino acid sequence of the light chain of IMP761

[0731]

[0732] The alignment of this sequence (IMP761 light chain) with the chimeric light chain sequence, 13E2Ig, from Example 17 is shown in Figure 24. In this figure, region V Lis shown in bold, and the IgK region is highlighted in color. Amino acid residues of the humanized IMP761 sequence that differ from the corresponding residues of the chimeric 13E2Ig sequence are underlined with a single line. CDR sequences (based on the combined identification of CDR sequences according to IMGT / Kabat) are underlined with a double line. The substituted residues in the humanized sequence are shown below in Table 27 (as VL variant 3, VH3, as well as the substituted residues in three other humanized variants of the original 13E2 light chain sequence: VL variants 1, 2, and 4, VL1, VL2, and VL4).

[0733]

[0734] The framework sequences of the light chain of the humanized antibody (IMP761 antibody) are:

[0735] VL FR1: DIVMTQTPSSLSASVGDRVTITC (SEQ ID NO: 64);

[0736] VL FR2: WYQQRPGQAPKLLIY (SEQ ID NO: 65);

[0737] VL FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 66); And

[0738] VL FR4: FGQGTRLDIK (SEQ ID NO: 67)

[0739] Example 19

[0740] Binding of chimeric 13E2-human antibody (Chim13E2IgG4) and humanized 13E2 antibody (IMP761) to CHQ-LAG-3 + cells

[0741] CHO cells expressing LAG-3 on their surface (0.05×10 6 cells / well in PBS, 0.5% BSA, 0.1% azide) were incubated with various concentrations of the chimeric 13E2-human antibody (referred to as Chim13E2IgG4) containing the chimeric heavy and light chains described in Example 17 (heavy chain: 13E2IgG4mut; light chain: 13E2IgK), IMP761, or human IgG4 (as an isotype-matched negative control). A secondary goat anti-human IgG-FITC antibody was used to detect the presence of antibodies on the surface of LAG-3 +CHO cells. The mean fluorescence intensity (MFI) of FITC was determined after flow cytometric analysis.

[0742] The results are shown below in Table 28 and Figure 25.

[0743]

[0744] The results showed that the humanized monoclonal antibody IMP761 binds to CHO cells expressing LAG-3 on their surface in a very similar manner to the chimeric antibody.

[0745] Example 20

[0746] Binding affinity of chimeric 13E2-human antibody (Chim13E2IgG4) and humanized 13E2 antibody (IMP761) to human LAG-3Ig protein

[0747] Biacore™ surface plasmon resonance analysis was performed using the chimeric antibody Chim13E2IgG4 (containing the chimeric heavy chain 13E2IgG4mut and the chimeric light chain 13E2IgK described in Example 17) or the humanized 13E2 antibody (IMP761) described in Example 18, covalently immobilized on the C1 sensor chip. Coating was performed in 10 mM sodium acetate, pH 5.0, to reach 13±1 RU. Recombinant human LAG-3Ig protein (IMP321) was then run over captured antibodies at six different concentrations ranging from 0.078 to 2.5 nM in assay buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) at 25°C with regeneration after each run. Analysis was performed on a Biacore™ T200 instrument, and the data were fitted using a kinetic global fit model (Langmuir 1:1). Kinetic parameters are shown in Table 29 and represent the average of three runs.

[0748]

[0749] The results showed that the humanized monoclonal antibody IMP761 had the same affinity for the human LAG-3Ig protein as the chimeric antibody. Both antibodies exhibited a very high association rate, explaining the high affinity of 13E2-derived antibodies for LAG-3.

[0750] Example 21

[0751] Effect of humanized antibody 13E2 (IMP761) on CD8 cell proliferation + T cells and CD25 expression induced by antigen stimulation

[0752] CFMS-labeled PBMCs from healthy donors (0.2×10 6 cells / well in complete RPMI+10%FBS) were incubated with a pool of peptides covering the CMV pp35 sequence in triplicate with 300 ng / ml human IgG4 (isotype control), Chim13E2IgG4 or IMP761. The T-cell response was assessed by measuring proliferation assessed using the proliferation index (calculated as the sum of the percentage of CD8 +T cells under each division peak, multiplied by the number of divisions) and CD25 expression on day 5 by flow cytometry. Percent inhibition for each antibody compared to the isotype-matched negative control (huIgG4) was calculated based on the proliferation index or CD25 percentage values. + T cells in the CD8 population + T cells.

[0753] The results are shown below in Tables 30 and 31, and Figures 26 and 27.

[0754]

[0755]

[0756] The results showed that the humanized monoclonal antibody IMP761 had an effect on inhibiting antigen-induced CD8 proliferation + T cells and CD25 expression + T cells similar to the chimeric antibody Chim13E2IgG4. Both antibodies caused, on average, approximately 60% inhibition of antigen-induced CD8 proliferation. + T cells and approximately 45% inhibition of CD25+ T cells in the CD8+ T cell population.

[0757] Example 22

[0758] Effect of different doses of chimeric 13E2-human antibody (Chim13E2IgG4) and humanized 13E2 antibody (IMP761) on the CP8 response + T cells

[0759] CFMS-labeled PBMCs from healthy donors (0.2×10 6 cells / well in complete RPMI+10%FBS) were incubated with a pool of peptides covering the CMV pp35 sequence in triplicate with different doses of Chim13E2IgG4, IMP761, or human IgG4 (isotype-matched negative control). T cell responses were assessed by measuring proliferation (CFSE dilution) on day 5 using flow cytometry. The percentage of CD8 + T cells for each division number were calculated for the different antibody doses used.

[0760] The results are shown below in Table 32 and Figure 28.

[0761]

[0762] The results showed that the inhibitory effect of IMP761 and Chim13E2IgG4 on antigen-induced CD8 proliferation + T-cell inhibition was dose-dependent. Specifically, the inhibitory effect of each antibody increased with increasing dose from 10 ng / ml to 100 ng / ml. At 300 ng / ml, the inhibitory effect was similar to that of 100 ng / ml. The inhibitory effect of IMP761 was similar to that of Chim13E2IgG4 at all doses tested.

[0763] Example 23

[0764] Humanized antibody 13E2 (IMP761) does not have cytotoxic activity against LAG-3-expressing cells

[0765] Several types of assays were used to confirm that the humanized 13E2 antibody (IMP761) does not have cytotoxic activity against LAG-3-expressing cells.

[0766] 1) ADCC Reporter Bioassay (Promega, G7015)

[0767] In this assay, primary donor PBMCs or NK cells were replaced with Jurkat cells stably expressing human FcγRIIIa (the high-affinity V158 receptor) and an NFAT-responsive element, which stimulates expression of the luciferase reporter gene. If the test antibody has ADCC activity, it will bind the target cell and the Jurkat cell's FcγRIIIa receptor. The resulting activation of the FcγRIIIa downstream receptor signaling leads to activation of the NFAT pathway, thereby inducing expression of the luciferase reporter gene. Luciferase activity was quantified by luminescence reading.

[0768] LAG-3-transfected CHO and Jurkat cells and PBMCs stimulated with SEB for 2 days to induce LAG-3 expression (55% of PBMCs were LAG-3 +) were used as target cells to analyze the ADCC activity of IMP761 compared with an isotype-matched negative control antibody, hIgG4 (a recombinant mAb from BioRad). Anti-CD20 antibody and Raji cells provided in the assay kit were used as positive controls. Anti-CD20 antibody was also tested on SEB-stimulated PBMCs. Assays were performed according to the manufacturer's instructions using 75,000 effector cells with 12,500 target cells. After incubation for 6 hours at 37°C, the Bio-Glo luciferase assay system was used according to the manufacturer's instructions to measure luminescence using a PerkinElmer En Vision 2103 luminometer (integration time 0.5 sec / well).

[0769] The results are shown below in Table 33 and Figure 29(a). The results are presented as the fold change in luminescence intensity (RLU), calculated by dividing the RLU value obtained in the presence of the test antibody (at the maximum concentration recommended by the manufacturer, 3 μg / mL) by the RLU value obtained without the antibody.

[0770]

[0771] The results showed that the RLU fold change for the IMP761 antibody was approximately 1-fold for each of the different target cells tested, regardless of whether the target cell expressed LAG-3. The RLU fold change obtained for the isotype-matched negative control antibody, hIgG4, was slightly higher and ranged from 1.4- to 2.5-fold for different target cells. The positive control anti-CD20 antibody showed significant ADCC activity against Raji cells (a B cell line) and against SEB-stimulated PBMCs containing a negligible percentage of B cells.

[0772] From these results, it was concluded that the IMP761 antibody does not have any ADCC activity against LAG-3-expressing cells.

[0773] 2) Conventional ADCC Assay

[0774] This assay used PBMC stimulated for one day in X-Vivo 10 medium (Lonza) with 100 IU / ml IL-2 (Roche) and CFSE-labeled PBMC stimulated with SEB for two days to promote LAG-3 expression on T cells. The assay was performed in X-Vivo 10 medium at an effector:target ratio of 50:1, with a high dose (3 μg / ml) of IMP761 or an isotype-matched negative control antibody, hIgG4. After 4 hours, cell mixtures were harvested and stained for CD4, CD8, CD25, and LAG-3 using fluorochrome-conjugated antibodies. Cell viability in each blood cell population was assessed by flow cytometry after excluding cells that were positive for staining with 7-amino-actinomycin D (7-AAD), a fluorescent dye that labels cells that have lost their membrane integrity, a phenomenon that occurs rapidly after cell death.

[0775] The results are shown in Table 34 and Figure 29(b) and (c). The results are presented as the percentage of live CD4 + or CD8 + cells in the PBMC population (b), and the percentage of living LAG-3 + CD4 + or LAG-3 + CD8 + cells in the PBMC population (c).

[0776]

[0777] The results showed that the IMP761 antibody did not reduce the percentage of CD8 + or CD4 + T cells in the PBMC population, or the percentage of LAG-3 + CD8 + cytotoxic T cells or LAG-3 + CD4 + helper T cells in the PBMC population. An isotype-matched negative control antibody, hIgG4, caused a slight decrease in T cell viability in the PBMC population, particularly in activated T cells expressing LAG-3.

[0778] From these results, it can be concluded that the IMP761 antibody does not have any ADCC activity against LAG-3-expressing T cells. 3) CDC analysis.

[0779] For CDC testing, SEB-stimulated cells used as target cells were incubated with 3 μg / ml IMP761, an isotype-matched negative control antibody, hIgG4, a CDC-positive anti-CD3 control antibody (clone MEM-57, Cerdalane), or an isotype-matched negative control mouse antibody, mlgG2a, for 45 minutes in PBS, 0.5% BSA. Unbound antibodies were then washed away, and the cells were incubated with rabbit complement diluted with 3 volumes in RPMI medium for 1 hour at 37°C. Cells were stained for CD4, CD8, CD25, and LAG-3 using fluorochrome-conjugated antibodies. Cell viability in each blood cell population was assessed by flow cytometry after exclusion of 7-AAD-labeled cells.

[0780] The results are shown in Table 35 and Figure 30. The results are presented as the percentage of live CD4 + or CD8 + cells in the PBMC population (a), and the percentage of living LAG-3 + CD4 + or LAG-3 + CD8 + cells in the PBMC population (b).

[0781]

[0782] The results showed that the IMP761 antibody did not reduce the percentage of CD8 + or CD4 + T cells in the PBMC population, or the percentage of LAG-3 + CD8 + cytotoxic T cells, or LAG-3 + CD4 + helper T cells in the PBMC population. As expected, the anti-CD3-positive control antibody did indeed cause a decrease in the percentage of T cells in the PBMC population and activated T cells expressing LAG-3.

[0783] Based on the obtained results, it can be concluded that the IMP761 antibody does not have any CDC activity against LAG-3-expressing T cells.

[0784] 4) Evaluation of cytotoxicity in T cell proliferation assay

[0785] The IMP761 antibody did not show cytotoxic activity in any of the short cytotoxicity assays described above in (1)-(3). The cytotoxicity of IMP761 against LAG-3-expressing T cells was also assessed after culturing antigen-stimulated PBMCs for several days. Similar to the proliferation assays described in the previous examples, PBMCs from healthy donors (0.2×10 6 cells / well in complete RPMI medium + 10% FBS) were incubated in triplicate with a pool of peptides covering the CMV pp35 sequence in the presence of 300 ng / ml IMP761 or human IgG4 (as an isotype-matched negative control). After three days, the percentage of CD8 + and CD4 +T cells gated on live lymphocytes, as well as the percentage of LAG-3 + cells in these T cell subsets were measured using flow cytometry.

[0786] The results are shown in Table 36 and Figure 31.

[0787]

[0788] The results showed that the IMP761 antibody did not reduce the percentage of CD8 + or CD4 + T cells in the lymphocyte population, or the percentage of LAG-3 + CD8 + cytotoxic T cells or LAG-3 + CD4 + helper T cells in the lymphocyte population.

[0789] Based on these results, it can be concluded that IMP761 antibody does not exhibit any cytotoxic activity against LAG-3-expressing T cells in this proliferation assay.

[0790] Based on the results presented in this example, it can be concluded that the IMP761 antibody does not have cytotoxic activity, thus the inhibition of antigen-induced T cell proliferation and activation by this antibody is not due to any cytotoxic activity against activated T cells.

[0791] --->

[0792] SEQUENCE LISTING

[0793] <110> IMMUTEP S.A.S.

[0794] <120> Anti-LAG-3 antibodies

[0795] <130> P / 73968.WO01

[0796] <150> GB 1515572.4

[0797] <151> 2015-09-02

[0798] <150> GB 1612437.2

[0799] <151> 2016-07-18

[0800] <160> 85

[0801] <170> PatentIn version 3.5

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[0849] Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

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[0851] Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

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[0853] Gly Met Gly Leu Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

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[0855] Trp Leu Thr His Ile Trp Trp Asp Asp Ile Lys Arg Tyr Asn Pro Asp

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[0857] Leu Arg Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Ser Gln Ile

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[0859] Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

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[0861] Cys Ala Arg Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr Phe Asp Val

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[0863] Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

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[0872] Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ile Phe Asp

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[0874] Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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[0876] Tyr Ser Ala Ser Ser Arg Val Ser Gly Val Pro Asp Arg Phe Thr Gly

[0877] 50 55 60

[0878] Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

[0879] 65 70 75 80

[0880] Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr

[0881] 85 90 95

[0882] Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys

[0883] 100 105

[0884] <210> 9

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[0886] <212> DNA

[0887] <213> Balb / c mouse

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[0889] caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

[0890] acttgttctt tctctgggtt ttcactgagc acttctggta tgggtctagg ctggattcgt 120

[0891] cagccatcag ggaagggtct ggagtggctg acacacattt ggtgggatga tatcaagcgc 180

[0892] tataacccag acctgaggag ccgactgact atctccaagg atacctccag cagccagatt 240

[0893] ttcctcaaga tcgccagtgt ggacactgca gatactgcca catattactg tgctcgaata 300

[0894] gtggagggtt catacagtag tagttacttc gatgtctggg gcgcagggac cacggtcacc 360

[0895] gtctcctcag 370

[0896] <210> 10

[0897] <211> 322

[0898] <212> DNA

[0899] <213> Balb / c mouse

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[0902] atcacctgca aggccagtca ggatgtgatt tttgatgtag cctggtatca acagaaacca 120

[0903] ggacaatctc ctaaattact gatttactcg gcatcctccc gggtcagtgg agtccctgat 180

[0904] cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagtag tgtgcaggct 240

[0905] gaagacctgg cagtttatta ctgtcagcaa cactatagta ctccgtacac gttcggaggg 300

[0906] gggaccacgc tggaaataaa ac 322

[0907] <210> 11

[0908] <211> 10

[0909] <212> PRT

[0910] <213> Balb / c mouse

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[0912] Gly Phe Ser Leu Asn Thr Ser Gly Met Gly

[0913] 1 5 10

[0914] <210> 12

[0915] <211> 7

[0916] <212> PRT

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[0919] Ile Trp Trp Asp Asp Val Lys

[0920] 1 5

[0921] <210> 13

[0922] <211> 15

[0923] <212> PRT

[0924] <213> Balb / c mouse

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[0926] Ala Arg Ile Glu Gly Asp Thr Tyr Tyr Asp Tyr Tyr Phe Asp Tyr

[0927] 1 5 10 15

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[0931] <213> Balb / c mouse

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[0933] Gln Asp Val Ser Ile Ala

[0934] 1 5

[0935] <210> 15

[0936] <211> 3

[0937] <212> PRT

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[0940] Ser Ala Ser

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[0947] Gln Gln His Tyr Ser Ile Pro Trp Thr

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[0951] <212> PRT

[0952] <213> Balb / c mouse

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[0954] Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

[0955] 1 5 10 15

[0956] Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Ser

[0957] 20 25 30

[0958] Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

[0959] 35 40 45

[0960] Trp Leu Thr His Ile Trp Trp Asp Asp Val Lys Arg Tyr Asn Pro Ala

[0961] 50 55 60

[0962] Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Ser Gln Val

[0963] 65 70 75 80

[0964] Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

[0965] 85 90 95

[0966] Cys Ala Arg Ile Glu Gly Asp Thr Tyr Tyr Asp Tyr Tyr Phe Asp Tyr

[0967] 100 105 110

[0968] Trp Gly Gln Gly Val Thr Leu Thr Val Ser Ser

[0969] 115 120

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[0975] Asp Ile Val Met Thr Gln Ser His Lys Leu Met Ser Thr Ser Val Gly

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[0977] Asp Gly Leu Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

[0978] 20 25 30

[0979] Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

[0980] 35 40 45

[0981] Tyr Ser Ala Ser Phe Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

[0982] 50 55 60

[0983] Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

[0984] 65 70 75 80

[0985] Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Trp

[0986] 85 90 95

[0987] Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

[0988] 100 105

[0989] <210> 19

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[0994] caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

[0995] acttgttctt tctctgggtt ttcactgaac acttctggta tgggtgtagg ctggattcgt 120

[0996] cagccatcag ggaagggtct ggagtggctg acacacattt ggtgggatga tgtcaagcgc 180

[0997] tataatccag ccctgaagag ccgactgact atctccaagg atacctccag cagccaggta 240

[0998] ttcctcaaga tcgccagtgt ggacactgca gatactgcca catactactg tgctcgaata 300

[0999] gagggggata cttactacga ctattacttt gactactggg gccaaggcgt cactctcaca 360

[1000] gtctcctcag 370

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[1002] <211> 322

[1003] <212> DNA

[1004] <213> Balb / c mouse

[1005] <400> 20

[1006] gacattgtga tgacccagtc tcacaaactc atgtccacat cagttggaga cgggctcagc 60

[1007] atcacctgca aggccagtca ggatgtgagc attgctgtag tctggtatca acagaaacca 120

[1008] ggacaatctc ctaaactgct gatttactcg gcatccttcc ggtacactgg agtccctgat 180

[1009] cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 240

[1010] gaagacctgg cagtttatta ctgtcagcaa cattatagta ttccgtggac gttcggtgga 300

[1011] ggcaccaagc tggaaatcaa ac 322

[1012] <210> 21

[1013] <211> 7

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[1015] <213> Balb / c mouse

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[1017] Thr Ser Gly Met Gly Leu Gly

[1018] 1 5

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[1024] His Ile Trp Trp Asp Asp Ile Lys Arg Tyr Asn Pro Asp Leu Arg Ser

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[1038] Lys Ala Ser Gln Asp Val Ile Phe Asp Val Ala

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[1045] Ser Ala Ser Ser Arg Val Ser

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[1059] Leu Gln Pro Gly Ala Glu Val Pro Val Val Trp Ala Gln Glu Gly Ala

[1060] 1 5 10 15

[1061] Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser

[1062] 20 25 30

[1063] Leu Leu Arg Arg Ala Gly Val Thr Trp Gln His Gln Pro Asp Ser Gly

[1064] 35 40 45

[1065] Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His Pro

[1066] 50 55 60

[1067] Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu

[1068] 65 70 75 80

[1069] Ser Val Gly Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro

[1070] 85 90 95

[1071] Arg Val Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu

[1072] 100 105 110

[1073] Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala

[1074] 115 120 125

[1075] Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu

[1076] 130 135 140

[1077] Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser

[1078] 145 150 155 160

[1079] Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala

[1080] 165 170 175

[1081] Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly Arg Val Pro Val Arg

[1082] 180 185 190

[1083] Glu Ser Pro His His His Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln

[1084] 195 200 205

[1085] Val Ser Pro Met Asp Ser Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg

[1086] 210 215 220

[1087] Asp Gly Phe Asn Val Ser Ile Met Tyr Asn Leu Thr Val Leu Gly Leu

[1088] 225 230 235 240

[1089] Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala Gly Ala Gly Ser Arg Val

[1090] 245 250 255

[1091] Gly Leu Pro Cys Arg Leu Pro Ala Gly Val Gly Thr Arg Ser Phe Leu

[1092] 260 265 270

[1093] Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Asp Leu Leu Val Thr

[1094] 275 280 285

[1095] Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala

[1096] 290 295 300

[1097] Gln Ala Gly Thr Tyr Thr Cys His Ile His Leu Gln Glu Gln Gln Leu

[1098] 305 310 315 320

[1099] Asn Ala Thr Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe

[1100] 325 330 335

[1101] Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val

[1102] 340 345 350

[1103] Ser Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln

[1104] 355 360 365

[1105] Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu

[1106] 370 375 380

[1107] Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly

[1108] 385 390 395 400

[1109] Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser

[1110] 405 410 415

[1111] Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His Leu Leu Leu Phe Leu

[1112] 420 425 430

[1113] Thr Leu Gly Val Leu Ser Leu Leu Leu Leu Val Thr Gly Ala Phe Gly

[1114] 435 440 445

[1115] Phe His Leu Trp Arg Arg Gln Trp Arg Pro Arg Arg Phe Ser Ala Leu

[1116] 450 455 460

[1117] Glu Gln Gly Ile His Pro Gln Ala Gln Ser Lys Ile Glu Glu Leu Glu

[1118] 465 470 475 480

[1119] Gln Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu

[1120] 485 490 495

[1121] Pro Glu Pro Glu Gln Leu

[1122] 500

[1123] <210> 28

[1124] <211> 149

[1125] <212> PRT

[1126] <213> Homo sapiens

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[1128] Leu Gln Pro Gly Ala Glu Val Pro Val Val Trp Ala Gln Glu Gly Ala

[1129] 1 5 10 15

[1130] Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser

[1131] 20 25 30

[1132] Leu Leu Arg Arg Ala Gly Val Thr Trp Gln His Gln Pro Asp Ser Gly

[1133] 35 40 45

[1134] Pro Pro Ala Ala Ala Pro Gly His Pro Leu Ala Pro Gly Pro His Pro

[1135] 50 55 60

[1136] Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu

[1137] 65 70 75 80

[1138] Ser Val Gly Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro

[1139] 85 90 95

[1140] Arg Val Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu

[1141] 100 105 110

[1142] Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala

[1143] 115 120 125

[1144] Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu

[1145] 130 135 140

[1146] Gly Gln Ala Ser Met

[1147] 145

[1148] <210> 29

[1149] <211> 90

[1150] <212> PRT

[1151] <213> Homo sapiens

[1152] <400> 29

[1153] Thr Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu

[1154] 1 5 10 15

[1155] Asn Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe

[1156] 20 25 30

[1157] Arg Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His

[1158] 35 40 45

[1159] His Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp

[1160] 50 55 60

[1161] Ser Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val

[1162] 65 70 75 80

[1163] Ser Ile Met Tyr Asn Leu Thr Val Leu Gly

[1164] 85 90

[1165] <210> 30

[1166] <211> 469

[1167] <212> PRT

[1168] <213> Artificial

[1169] <220>

[1170] <223> VH domain of mouse monoclonal antibody 13E2, and a human IgG4 Fc

[1171] portion with an S228P mutation

[1172] <400> 30

[1173] Met Gly Trp Thr Leu Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly

[1174] 1 5 10 15

[1175] Val His Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln

[1176] 20 25 30

[1177] Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu

[1178] 35 40 45

[1179] Ser Thr Ser Gly Met Gly Leu Gly Trp Ile Arg Gln Pro Ser Gly Lys

[1180] 50 55 60

[1181] Gly Leu Glu Trp Leu Thr His Ile Trp Trp Asp Asp Ile Lys Arg Tyr

[1182] 65 70 75 80

[1183] Asn Pro Asp Leu Arg Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser

[1184] 85 90 95

[1185] Ser Gln Ile Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala

[1186] 100 105 110

[1187] Thr Tyr Tyr Cys Ala Arg Ile Val Glu Gly Ser Tyr Ser Ser Ser Tyr

[1188] 115 120 125

[1189] Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser

[1190] 130 135 140

[1191] Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

[1192] 145 150 155 160

[1193] Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

[1194] 165 170 175

[1195] Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

[1196] 180 185 190

[1197] His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

[1198] 195 200 205

[1199] Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

[1200] 210 215 220

[1201] Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

[1202] 225 230 235 240

[1203] Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

[1204] 245 250 255

[1205] Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

[1206] 260 265 270

[1207] Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

[1208] 275 280 285

[1209] Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

[1210] 290 295 300

[1211] Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

[1212] 305 310 315 320

[1213] Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

[1214] 325 330 335

[1215] Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

[1216] 340 345 350

[1217] Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

[1218] 355 360 365

[1219] Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

[1220] 370 375 380

[1221] Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

[1222] 385 390 395 400

[1223] Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

[1224] 405 410 415

[1225] Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

[1226] 420 425 430

[1227] Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

[1228] 435 440 445

[1229] Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

[1230] 450 455 460

[1231] Leu Ser Leu Gly Lys

[1232] 465

[1233] <210> 31

[1234] <211> 7

[1235] <212> PRT

[1236] <213> Balb / c mouse

[1237] <400> 31

[1238] Thr Ser Gly Met Gly Val Gly

[1239] 1 5

[1240] <210>...

Claims

1. An isolated agonist antibody to lymphocyte activation gene-3 (LAG-3) or antigen-binding fragment thereof that binds to LAG-3 and inhibits antigen-induced CD4 proliferation + and / or CD8 + T cells and / or antigen-induced CD4 activation + and / or CD8 + T cells, where the antibody or its antigen-binding fragment contains: i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR1) comprising SEQ ID NO: 1, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 1; (b) a VH CDR2 region comprising SEQ ID NO: 2, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 2; and (c) a VH CDR3 region comprising SEQ ID NO: 3, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 3; and a light chain variable region (VL) containing: (a) a VL CDR1 region comprising SEQ ID NO: 4, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 4; (b) a VL CDR2 region comprising SEQ ID NO: 5, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 5; and (c) a VL CDR3 region comprising SEQ ID NO: 6, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 6; or ii) a heavy chain variable region comprising: (a) a VH CDR1 region comprising SEQ ID NO: 21, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 21; (b) a VH CDR2 region comprising SEQ ID NO: 22, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 22; and (c) a VH CDR3 region comprising SEQ ID NO: 23, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 23; and a light chain variable region comprising: (a) a VL CDR1 region comprising SEQ ID NO: 24, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 24; (b) a VL CDR2 region comprising SEQ ID NO: 25, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO: 25; and (c) a VL CDR3 region comprising SEQ ID NO: 26, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO:

26.

2. An isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is obtained by immunization with IMP321 in the absence of complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA).

3. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: (i) the isolated antibody or antigen-binding fragment thereof inhibits antigen-induced CD4 proliferation + T cells and antigen-induced CD8 proliferation + T cells, and / or (ii) the isolated antibody or antigen-binding fragment thereof inhibits antigen-induced CD8 proliferation + T cells are greater than antigen-induced CD4 proliferation + T cells, and / or (iii) inhibition of antigen-induced CD8 proliferation + T cell activation is LAG-3 dependent and IL-2 independent.

4. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that: inhibits the binding of LAG-3 or a chimeric protein consisting of the extracellular domain of human LAG-3 fused to human IgG1 Fc (IMP321) to MHC class II-positive cells; and / or inhibits LAG-3-induced activation of antigen-presenting cells (APCs) or IMP321-induced activation of monocytes; and / or binds to an epitope of LAG-3 that overlaps with the MHC class II binding site of LAG-3.

5. An isolated antibody or antigen-binding fragment thereof according to any preceding claim, which comprises one, two or three complementarity determining regions (CDRs) of a heavy chain variable region (VH) of an antibody comprising the amino acid sequence of SEQ ID NO: 7, and one, two or three CDRs of a light chain variable region (VL) of an antibody comprising the amino acid sequence of SEQ ID NO:

8.

6. The isolated antibody or antigen-binding fragment thereof according to claim 5, characterized in that the CDR regions of the VH region of the antibody are selected from the CDR regions of the amino acid sequence of SEQ ID NO: 1, 2, 3, 21, 22 and 23 and the CDR regions of the VL region of the antibody are selected from the CDR regions of the amino acid sequence of SEQ ID NO: 4, 5, 6, 24, 25 and 26.

7. An isolated antibody or antigen-binding fragment thereof according to claim 5 or 6, which comprises a VH region of an antibody comprising CDR1 VH, CDR2 VH and CDR3 VH, wherein CDR1 VH has an amino acid sequence selected from SEQ ID NO: 1 and 21, and / or CDR2 VH has an amino acid sequence selected from SEQ ID NO: 2 and 22, and / or CDR3 VH has an amino acid sequence selected from SEQ ID NO: 3 and 23.

8. The isolated antibody or antigen-binding fragment thereof according to claim 7, characterized in that: CDR1 VH has an amino acid sequence selected from SEQ ID NO: 1 and 21, and CDR2 VH has an amino acid sequence selected from SEQ ID NO: 2 and 22; CDR1 VH has an amino acid sequence selected from SEQ ID NO: 1 and 21, and CDR3 VH has an amino acid sequence selected from SEQ ID NO: 3 and 23; CDR2 of VH has an amino acid sequence selected from SEQ ID NO: 2 and 22, and CDR3 of VH has an amino acid sequence selected from SEQ ID NO: 3 and 23; or CDR1 VH has an amino acid sequence selected from SEQ ID NO: 1 and 21, CDR2 VH has an amino acid sequence selected from SEQ ID NO: 2 and 22, and CDR3 VH has an amino acid sequence selected from SEQ ID NO: 3 and 23.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 5-8, which comprises a VL region of an antibody comprising CDR1 VL, CDR2 VL and CDR3 VL, characterized in that CDR1 VL has an amino acid sequence selected from SEQ ID NO: 4 and 24, and / or CDR2 VL has an amino acid sequence selected from SEQ ID NO: 5 and 25, and / or CDR3 VL has an amino acid sequence selected from SEQ ID NO: 6 and 26.

10. The isolated antibody or antigen-binding fragment thereof according to claim 9, characterized in that: CDR1 VL has an amino acid sequence selected from SEQ ID NO: 4 and 24, and CDR2 VL has an amino acid sequence selected from SEQ ID NO: 5 and 25; CDR1 VL has an amino acid sequence selected from SEQ ID NO: 4 and 24, and CDR3 VL has an amino acid sequence selected from SEQ ID NO: 6 and 26; CDR2 VL has an amino acid sequence selected from SEQ ID NO: 5 and 25, and CDR3 VL has an amino acid sequence selected from SEQ ID NO: 6 and 26; or CDR1 VL has an amino acid sequence selected from SEQ ID NO: 4 and 24, CDR2 VL has an amino acid sequence selected from SEQ ID NO: 5 and 25, and CDR3 VL has an amino acid sequence selected from SEQ ID NO: 6 and 26.

11. An isolated antibody or antigen-binding fragment thereof according to any of the preceding claims, which comprises: The VH region of an antibody with CDR regions comprising the amino acid sequences of SEQ ID NOs: 1, 2 and 3, or the VL region of an antibody with CDR regions comprising the amino acid sequences of SEQ ID NOs: 4, 5 and 6; or The VH region of an antibody with CDR regions comprising the amino acid sequences of SEQ ID NO: 21, 22 and 23, or the VL region of an antibody with CDR regions comprising the amino acid sequences of SEQ ID NO: 24, 25 and 26.

12. An isolated antibody or antigen-binding fragment thereof according to any preceding paragraph, which comprises a VH region of an antibody comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 7, and a VL region of an antibody comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO:

8.

13. An isolated antibody or antigen-binding fragment thereof according to claim 12, which contains: a VH region of an antibody comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 7, and a VL region of an antibody comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 8; a VH region of an antibody comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 7, and a VL region of an antibody comprising the amino acid sequence of SEQ ID NO: 8; an antibody VH region comprising the amino acid sequence of SEQ ID NO: 7 and an antibody VL region comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 8; or The VH region of the antibody comprising the amino acid sequence of SEQ ID NO: 7, and the VL region of the antibody comprising the amino acid sequence of SEQ ID NO:

8.

14. An isolated antibody or antigen-binding fragment thereof according to any preceding claim, which is a humanized monoclonal antibody or antigen-binding fragment thereof.

15. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that comprises a humanized light chain framework region.

16. The isolated antibody or antigen-binding fragment thereof according to claim 15, wherein the humanized light chain framework region comprises an amino acid sequence with any of the amino acid substitutions shown for VL1, VL2, VL3, or VL4 in Table 27 below:

17. The isolated antibody or antigen-binding fragment thereof according to claim 15 or 16, wherein the humanized light chain framework region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 68-83.

18. An isolated antibody or antigen-binding fragment thereof according to claim 17, which contains: framework region 1 of VL (FR1 VL) of the sequence SEQ ID NO: 68; FR2 VL of the sequence SEQ ID NO: 69; FR3 VL of the sequence SEQ ID NO: 70; and FR4 VL of the sequence SEQ ID NO: 71; framework region 1 of VL (FR1 VL) of the sequence SEQ ID NO: 72; FR2 VL of the sequence SEQ ID NO: 73; FR3 VL of the sequence SEQ ID NO: 74; and FR4 VL of the sequence SEQ ID NO: 75; framework region 1 of VL (FR1 VL) of the sequence SEQ ID NO: 76; FR2 VL of the sequence SEQ ID NO: 77; FR3 VL of the sequence SEQ ID NO: 78; and FR4 VL of the sequence SEQ ID NO: 79; or framework region 1 VL (FR1 VL) of the sequence SEQ ID NO: 80; FR2 VL of the sequence SEQ ID NO: 81; FR3 VL of the sequence SEQ ID NO: 82; and FR4 VL of the sequence SEQ ID NO:

83.

19. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that comprises a humanized heavy chain framework region.

20. The isolated antibody or antigen-binding fragment thereof according to claim 19, wherein the humanized heavy chain framework region comprises an amino acid sequence with any of the amino acid substitutions shown for VH1, VH2, VH3, or VH4 in Table 26 below:

21. The isolated antibody or antigen-binding fragment thereof according to claim 19 or 20, wherein the humanized heavy chain framework region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 52-67.

22. An isolated antibody or antigen-binding fragment thereof according to claim 21, which contains: framework region 1 VH (FR1 VH) of the sequence SEQ ID NO: 52; FR2 VH of the sequence SEQ ID NO: 53; FR3 VH of the sequence SEQ ID NO: 54; and FR4 VH of the sequence SEQ ID NO: 55; framework region 1 VH (FR1VH) of the sequence SEQ ID NO: 56; FR2 VH of the sequence SEQ ID NO: 57; FR3 VH of the sequence SEQ ID NO: 58; and FR4 VH of the sequence SEQ ID NO: 59; framework region 1 VH (FR1 VH) of the sequence SEQ ID NO: 60; FR2 VH of the sequence SEQ ID NO: 61; FR3 VH of the sequence SEQ ID NO: 62; and FR4 VH of the sequence SEQ ID NO: 63; or framework region 1 VH (FR1 VH) of the sequence SEQ ID NO: 64; FR2 VH of the sequence SEQ ID NO: 65; FR3 VH of the sequence SEQ ID NO: 66; and FR4 VH of the sequence SEQ ID NO:

67.

23. An isolated antibody or antigen-binding fragment thereof according to any preceding claim, which is a chimeric antibody molecule or antigen-binding fragment thereof.

24. An isolated antibody or antigen-binding fragment thereof according to claim 23, which comprises the amino acid sequence of the variable region of the antibody according to any one of claims 1-22 and the amino acid sequence of a human constant region.

25. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that lacks complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

26. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that binds to human LAG-3 protein or human LAG-3Ig protein with a dissociation constant K D, which is not more than 100 pM, not more than 90 pM, not more than 80 pM, not more than 70 pM, not more than 60 pM, not more than 50 pM, not more than 40 pM, not more than 30 pM, or not more than 25 pM, for example, as determined by Biacore analysis.

27. An isolated antibody or antigen-binding fragment thereof according to any preceding claim that does not bind to the 30-amino acid outer loop sequence (SEQ ID NO: 40) of the first N-terminal domain of D1 of human LAG-3 protein.

28. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any preceding claim.

29. The nucleic acid of claim 28, comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 9 and 10.

30. A recombinant expression vector containing a nucleic acid according to claim 28 or 29.

31. A recombinant cell for producing an antibody or an antigen-binding fragment thereof, containing a nucleic acid according to claim 28 or 29 or a recombinant vector according to claim 30.

32. A pharmaceutical composition for the treatment of a T-cell mediated immune disorder selected from an inflammatory disease or an autoimmune disorder, comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-27 and a pharmaceutically acceptable carrier, excipient or diluent.

33. Use of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-27 or a pharmaceutical composition according to claim 32 in the treatment of a T-cell-mediated immune disorder selected from an inflammatory disease and an autoimmune disorder.

34. Use of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-27 or a pharmaceutical composition according to claim 32 in the manufacture of a medicament for the treatment of a T-cell-mediated immune disorder selected from an inflammatory disease and an autoimmune disorder.

35. A method for treating a T-cell-mediated immune disorder selected from an inflammatory disease and an autoimmune disorder, which comprises administering an effective amount of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-27 or a pharmaceutical composition according to claim 32 to a subject in need of such treatment.

36. The use according to claim 33 or 34, wherein the T-cell-mediated immune disorder is selected from the group consisting of infections, infection-associated endotoxic shock, sepsis, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, post-surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus erythematosus and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's syndrome, pemphigus,Primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's syndrome, other autoimmune disorders, pancreatitis, trauma, graft-versus-host disease, transplant rejection, cardiac disease including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria or infertility associated with fetal-maternal intolerance.

37. The use according to claim 36, wherein the infection is a viral, bacterial, fungal or parasitic infection, or where the lupus erythematosus is systemic lupus erythematosus, or where the injury is a surgical intervention.

38. The method of claim 35, wherein the T cell-mediated immune disorder is selected from the group consisting of infections, infection-associated endotoxic shock, sepsis, arthritis, rheumatoid arthritis, asthma, COPD, pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, post-surgical adhesions, stroke, type I diabetes, Lyme disease, arthritis, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous system such as multiple sclerosis, lupus erythematosus and Guillain-Barré syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's syndrome, pemphigus, primary biliary cirrhosis,sarcoidosis, scleroderma, Wegener's syndrome, other autoimmune disorders, pancreatitis, trauma, graft-versus-host disease, transplant rejection, cardiac disease including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria or infertility associated with fetal-maternal intolerance.

39. The method of claim 38, wherein the infection is a viral, bacterial, fungal or parasitic infection, or where the lupus erythematosus is systemic lupus erythematosus, or where the injury is a surgical intervention.

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