Anti-LAG3 antibodies and uses thereof

Abstract

To provide antibodies that bind to lymphocyte activation gene 3 (LAG3) protein, which is a T cell co-inhibitor, and methods of use.SOLUTION: In various embodiments of the invention, the antibodies are fully human antibodies that specifically bind to LAG3. In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing LAG3 activity, thus providing means of treating a disease or disorder such as cancer or viral infection. The antibodies of the present invention are useful, inter alia, for targeting immune cells expressing LAG3 and for modulating LAG3 activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to isolated antibodies or antigen-binding fragments thereof that specifically bind to the immunoregulatory receptor lymphocyte activation gene 3 (LAG3) protein, and to therapeutic and diagnostic methods using these antibodies.

[0002] Sequence Listing An official copy of the Sequence Listing is being submitted contemporaneously herewith electronically via EFS-Web as an ASCII formatted Sequence Listing with the filename "2016_10_07_10176WO01_SEQ_LIST_ST25.txt", a creation date of October 7, 2016, and a size of approximately 240 kilobytes. The Sequence Listing contained in this ASCII formatted document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] T cell costimulatory and T cell co-inhibitory molecules (collectively termed co-signaling molecules) play pivotal roles in regulating T cell activation, subset differentiation, effector function, and survival (Chen et al., 2013, Nature Rev. Immunol., 13:227-242). After the T cell receptor recognizes the cognate peptide-MHC complex on an antigen-presenting cell, co-signaling receptors co-localize with the T cell receptor at the immune synapse, where they synergize with signaling by the TCR to promote or inhibit T cell activation and function (Flies et al., 2011, Yale J. Biol. Med., 84:409-421). The final immune response is regulated by the balance between costimulatory and co-inhibitory signals ("immune checkpoints") (Pardoll, 2012, Nature Reviews Cancer, 12:252-264). Lymphocyte activation gene 3 (LAG3) functions as one such "immune checkpoint" in mediating peripheral T cell tolerance.

[0004] LAG3 (also called CD223) is a 503 amino acid transmembrane protein receptor expressed on activated CD4 and CD8 T cells, gamma delta T cells, natural killer T cells, B cells, natural killer cells, plasmacytoid dendritic cells, and regulatory T cells. LAG3 is a member of the immunoglobulin (Ig) superfamily. The primary function of LAG3 is to dampen immune responses. Binding of LAG3 to MHC class II molecules results in the delivery of a negative signal to LAG3-expressing cells, downregulating antigen-dependent CD4 and CD8 T cell responses. LAG3 negatively regulates the ability of T cells to proliferate, produce cytokines, and lyse target cells, a process referred to as T cell "exhaustion." LAG3 has also been reported to play a role in enhancing regulatory T (Treg) cell function (Pardoll, 2012, Nature Reviews Cancer, 12:252-264).

[0005] LAG3 plays an important role in tumor immunity and infection immunity, making it an ideal target for immunotherapy. Blockade of LAG3 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections (Turnis et al., 2015, Eur. J. Immunol., 45:1892-1905).

[0006] Monoclonal antibodies against LAG3 are known in the art and are described, for example, in U.S. Patent / Publication Nos. 5,976,877, 6,143,273, 6,197,524, 8,551,481, 20110070238, 20110150892, 20130095114, 20140093511, 20140127226, 20140286935, and WO 97 / 023666. No. 5 / 30750, WO97 / 03695, WO98 / 58059, WO2004 / 078928, WO2008 / 132601, WO2010 / 019570, WO2014 / 008218, EP0510079B1, EP0758383B1, EP0843557B1, EP0977856B1, EP1897548B2, EP2142210A1, and EP2320940B1. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Pat. No. 5,976,877 [Patent Document 2] U.S. Patent No. 6,143,273 [Patent Document 3] U.S. Patent No. 6,197,524 Summary of the Invention [Problem to be solved by the invention]

[0008] When developing immunotherapies for treating humans, antibodies exhibiting properties such as low immunogenicity, suitable binding kinetic parameters, cross-reactivity with monkey targets, suitable in vitro activity and / or suitable in vivo activity are needed. [Means for solving the problem]

[0009] The present invention provides antibodies and antigen-binding fragments thereof that bind to LAG3. The antibodies of the present invention are useful, inter alia, for targeting immune cells expressing LAG3 and modulating LAG3 activity. In certain embodiments, the antibodies of the present invention are useful for inhibiting or neutralizing LAG3 activity and / or stimulating T cell activation, for example, in situations where T cell-mediated killing is beneficial or desirable. In certain embodiments, the antibodies are useful for inhibiting regulatory T cell function and / or reversing the anergic state of exhausted T cells. The anti-LAG3 antibodies of the present invention or antigen-binding portions thereof can be included as part of a multispecific antigen-binding molecule, for example, to modulate immune responses and / or target the antibodies to specific cell types, such as tumor cells or virus-infected cells. The antibodies are useful in treating diseases or disorders, such as cancer and virus infections.

[0010] The antibodies of the invention may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, e.g., to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.

[0011] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to LAG3. In certain embodiments, the antibody is a fully human antibody.

[0012] Exemplary anti-LAG3 antibodies of the present invention are listed in Tables 1-3 herein. Table 1 sets forth amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-LAG3 antibodies. Table 2 sets forth nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-LAG3 antibodies. Table 3 sets forth amino acid sequence identifiers for the heavy and light chain sequences of exemplary anti-LAG3 antibodies.

[0013] The present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR comprising an amino acid sequence selected from any of the amino acid sequences of the HCVRs listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0014] The present invention also provides antibodies or antigen-binding fragments thereof comprising an LCVR comprising an amino acid sequence selected from any of the amino acid sequences of the LCVRs listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0015] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the amino acid sequences of the HCVRs listed in Table 1 paired with any of the amino acid sequences of the LCVRs listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pairs are SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 300 / 301, 302 / 303, 304 / 305, 306 / 307, 308 / 309, 310 / 311, 312 / 313, 314 / 315, 316 / 317, 318 / 318, 318 / 319, 320 / 321, 322 / 323, 324 / 325, 326 / 326, 328 / 329, 330 / 332, 334 / 335, 336 / 337, 338 / 339, 340 / 340, 342 / 345, 346 / 347, 348 / 349, 350 / 351, 352 / 353, 354 / 355, 356 / 357, 358 / 359, 360 / 361, 362 / 363, 364 / 365, 366 / 367, 368 / 369, 370 / 371, 372 / 373, 374 / 375 06 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 386 / 394 (e.g., H4sH15479P), 418 / 426 (e.g., H4sH15482P), or 538 / 546 (e.g., H4sH14813N). In certain embodiments, the present invention provides anti-LAG3 antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 with no more than five amino acid substitutions, and the LCVR comprises an amino acid sequence listed in Table 1 with no more than five amino acid substitutions.For example, the invention provides an anti-LAG3 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 418 with no more than five amino acid substitutions, and the LCVR comprises the amino acid sequence of SEQ ID NO: 426 with no more than five amino acid substitutions. In another exemplary embodiment, the invention provides an anti-LAG3 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 418 with at least one amino acid substitution, and the LCVR comprises the amino acid sequence of SEQ ID NO: 426 with one amino acid substitution.

[0016] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the amino acid sequences of HCDR1 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the amino acid sequences of HCDR2 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the amino acid sequences of HCDR3 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] The present invention also provides antibodies or antigen-binding fragments thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the amino acid sequences of LCDR1 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the amino acid sequences of LCDR2 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0021] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the amino acid sequences of LCDR3 listed in Table 1, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] The present invention also provides antibodies or antigen-binding fragments thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 392 / 400 (e.g., H4sH15479P), 424 / 432 (e.g., H4sH15482P), and 544 / 552 (e.g., H4sH14813N).

[0023] The present invention also provides antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an HCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an HCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. In certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an LCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an LCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. For example, the present invention provides an anti-LAG3 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 420 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 420, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 422 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 422, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 424 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 424. In another exemplary embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 428 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 428, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 430 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 430, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 432 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 432.

[0024] The present invention provides antibodies or antigen-binding fragments thereof comprising a heavy chain comprising an amino acid sequence selected from any of the amino acid sequences of the HCs listed in Table 3, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] The present invention also provides antibodies or antigen-binding fragments thereof comprising a light chain comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Table 3, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0026] The present invention also provides antibodies or antigen-binding fragments thereof comprising an HC and LC amino acid sequence pair (HC / LC) comprising any of the HC amino acid sequences listed in Table 3 paired with any of the LC amino acid sequences listed in Table 3. According to certain embodiments, the present invention provides antibodies or antigen-binding fragments thereof comprising an HC / LC amino acid sequence pair contained within any of the exemplary anti-LAG3 antibodies listed in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 577 / 578, 579 / 578, and 580 / 581.

[0027] The present invention also provides antibodies or antigen-binding fragments thereof that include a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-LAG3 antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected from the group consisting of SEQ ID NOs: 388-390-392-396-398-400 (e.g., H4sH15479P), 420-422-424-428-430-432 (e.g., H4sH15482P), and 540-542-544-548-550-552 (e.g., H4sH14813N).

[0028] In related embodiments, the present invention provides antibodies or antigen-binding fragments thereof that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-LAG3 antibodies listed in Table 1. For example, the present invention includes antibodies or antigen-binding fragments thereof that comprise a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386 / 394 (e.g., H4sH15479P), 418 / 426 (e.g., H4sH15482P), and 538 / 546 (e.g., H4sH14813N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the amino acid sequences of the specified HCVRs and / or LCVRs disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition terms are based on sequence variability, the Chothia definition terms are based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol., 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0029] The present invention includes anti-LAG3 antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites may be useful, for example, to increase antibody-dependent cell-mediated cytotoxicity (ADCC) function, and antibodies lacking fucose moieties present in the oligosaccharide chains may be useful for this purpose (see Shield et al. (2002), JBC 277:26733). In other applications, galactosylation modifications may be made to modify complement-dependent cytotoxicity (CDC).

[0030] The present invention includes anti-LAG3 antibodies comprising an Fc domain, where the Fc domain comprises an IgG1 or IgG4 isotype, as described elsewhere herein. In certain embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 569, 570, 571, 572, and 573.

[0031] The present invention also provides antibodies or antigen-binding fragments thereof that compete for specific binding to LAG3 with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR, respectively, have an amino acid sequence selected from the HCVR sequences and LCVR sequences listed in Table 1.

[0032] The present invention also provides isolated antibodies and antigen-binding fragments thereof that block the binding of LAG3 to MHC class II. In some embodiments, the antibodies or antigen-binding fragments thereof that block the binding of LAG3 may bind to the same epitope on LAG3 as MHC class II, or may bind to a different epitope on LAG3 than MHC class II.

[0033] The present invention also provides antibodies and antigen-binding fragments thereof that specifically bind to LAG3 from humans or other species. In certain embodiments, the antibodies may bind to human LAG3 and / or monkey LAG3.

[0034] The present invention also provides antibodies or antigen-binding fragments thereof that cross-compete for binding to LAG3 with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR, respectively, have an amino acid sequence selected from the HCVR sequences and LCVR sequences listed in Table 1.

[0035] The present invention also provides a reference antibody or antigen-binding fragment thereof comprising a CDR of an HCVR and a CDR of an LCVR, wherein the HCVR and LCVR, respectively, bind to the same epitope as an antibody or antigen-binding fragment thereof having an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1. In certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising a reference antibody or antigen-binding fragment thereof comprising a CDR of an HCVR and a CDR of an LCVR, wherein the HCVR / LCVR amino acid sequence pair binds to the same epitope as an antibody or antigen-binding fragment thereof having SEQ ID NO: 418 / 426.

[0036] The present invention also includes anti-LAG3 antibodies that interact with one or more amino acids contained within the extracellular domain of human LAG3 (SEQ ID NO:588). In certain embodiments, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with an amino acid sequence selected from the group consisting of: (a) amino acids 28-69 of SEQ ID NO:588; (b) amino acids 28-71 of SEQ ID NO:588; (c) amino acids 31-52 of SEQ ID NO:588; and (d) amino acids 32-69 of SEQ ID NO:588. In certain embodiments, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with one or more amino acids contained within SEQ ID NO:589, for example, the present invention provides anti-LAG3 antibodies and antigen-binding fragments thereof that interact with at least 5 amino acids, at least 10 amino acids, or at least 20 amino acids contained within SEQ ID NO:589. In certain embodiments, the invention provides anti-LAG3 antibodies, and antigen-binding fragments thereof, that interact with the amino acid sequence of SEQ ID NO:589 (corresponding to amino acids 28-71 of SEQ ID NO:588).

[0037] In one embodiment, the present invention provides a recombinant human monoclonal antibody or antigen-binding fragment having one or more of the following characteristics: (a) specifically binding to human LAG3 and / or cynomolgus monkey LAG3; (b) blocking the binding of LAG3 to MHC class II; (c) blocking LAG3-induced T cell downregulation and rescue T cell signaling; and (d) inhibiting tumor growth and increasing survival in subjects with cancer.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to LAG3 in an agonistic manner, i.e., may enhance or stimulate the binding and / or activity of LAG3, and in other embodiments, the antibody may specifically bind to LAG3 in an antagonistic manner, i.e., may block LAG3 from binding to its ligand.

[0039] In certain embodiments, the antibodies or antigen-binding fragments of the invention are bispecific, comprising a first binding specificity to LAG3 and a second binding specificity to a second target epitope. The second target epitope may be another epitope on LAG3 or another epitope on a different protein. In certain embodiments, the second target epitope may be an epitope on a different cell, including a different T cell, B cell, tumor cell, or virus-infected cell.

[0040] In a second aspect, the invention provides nucleic acid molecules encoding anti-LAG3 antibodies or portions thereof. For example, the invention provides nucleic acid molecules encoding any of the amino acid sequences of the HCVRs listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of the HCVRs listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0041] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of the LCVRs listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of the LCVRs listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0042] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of HCDR1 listed in Table 1, however, in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR1 listed in Table 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0043] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of HCDR2 listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR2 listed in Table 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0044] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of HCDR3 listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of HCDR3 listed in Table 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0045] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of LCDR1 listed in Table 1, although in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of LCDR1 listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0046] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of LCDR2 listed in Table 1, although in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of LCDR2 listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0047] The present invention also provides nucleic acid molecules encoding any of the amino acid sequences of LCDR3 listed in Table 1, although in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of LCDR3 listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0048] The present invention also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and the set of amino acid sequences of HCDR1-HCDR2-HCDR3 is defined by any of the exemplary anti-LAG3 antibodies listed in Table 1.

[0049] The present invention also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of amino acid sequences of LCDR1-LCDR2-LCDR3 is defined by any of the exemplary anti-LAG3 antibodies listed in Table 1.

[0050] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises any of the amino acid sequences of the HCVRs listed in Table 1, and the LCVR comprises any of the amino acid sequences of the LCVRs listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences of the HCVRs listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the nucleic acid sequences of the LCVRs listed in Table 2, or sequences substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, both of which are derived from the same anti-LAG3 antibody, as listed in Table 1.

[0051] The present invention provides nucleic acid molecules that encode any of the heavy chain amino acid sequences listed in Table 3. The present invention also provides nucleic acid molecules that encode any of the light chain amino acid sequences listed in Table 3.

[0052] The present invention also provides nucleic acid molecules encoding both a heavy chain (HC) and a light chain (LC), wherein the HC comprises any of the HC amino acid sequences listed in Table 3, and the LC comprises any of the LC amino acid sequences listed in Table 3.

[0053] In a related aspect, the invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-LAG3 antibody. For example, the invention includes recombinant expression vectors comprising a nucleic acid molecule encoding any of the above-mentioned nucleic acid molecules, i.e., any of the HCVR, LCVR, and / or CDR sequences set forth in Table 1. The invention also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain of an anti-LAG3 antibody. For example, the invention includes recombinant expression vectors comprising a nucleic acid molecule encoding any of the above-mentioned nucleic acid molecules, i.e., any of the heavy or light chain sequences set forth in Table 3. Also included within the scope of the invention are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions allowing for the production of the antibodies or antibody fragments and recovering the antibodies and antibody fragments thus produced.

[0054] In a third aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to LAG3 and a pharma- ceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-LAG3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-LAG3 antibody. Exemplary agents that may be advantageously combined with the anti-LAG3 antibody include, without limitation, other agents that bind to LAG3 and / or modulate signaling by LAG3 (including other antibodies or antigen-binding fragments thereof, etc.), and / or agents that do not directly bind to LAG3 but nevertheless modulate immune cell activation. Additional combination therapies and co-formulations involving the anti-LAG3 antibodies of the invention are disclosed elsewhere herein.

[0055] In a fourth aspect, the present invention provides a method of modulating an immune response in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the present invention provides a method of enhancing an immune response in a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof of the present invention, the antibody or fragment thereof binding to LAG3. In one embodiment, the present invention provides a method of stimulating or enhancing activation of T cells in a subject. In certain embodiments, the present invention provides a method of rescuing activity of T cells, comprising contacting the T cells with an effective amount of an antibody of the present invention, such that activity of the T cells is rescued. In one embodiment, the present invention provides a method of inhibiting regulatory T (Treg) cells in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the subject in need thereof may suffer from a disease or disorder, such as cancer or a viral infection. In certain embodiments, the invention provides a method of rescuing LAG3-mediated inhibition of T cell activity, comprising contacting a T cell with an effective amount of an antibody of the invention.

[0056] In a fifth aspect, the present invention provides a method of treatment for a disease or disorder, such as cancer or a viral infection in a subject, using an anti-LAG3 antibody or antigen-binding portion of the present invention, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antibody fragment of the present invention. The disorder to be treated is any disease or condition that is ameliorated, inhibited, or prevented by stimulating or inhibiting LAG3 activity or signaling. In certain embodiments, the antibody or antigen-binding fragment of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent can be selected from the group consisting of an antibody against another T cell co-inhibitor, an antibody against a tumor cell antigen, an antibody against a T cell receptor, an antibody against an epitope on a virus-infected cell, a cytotoxic agent, an anti-cancer drug, an anti-viral drug, an anti-inflammatory drug (e.g., a corticosteroid), a chemotherapeutic agent, radiation therapy, an immunosuppressant, and any other drug or treatment known in the art. In certain embodiments, the second therapeutic agent may be an agent that helps antagonize or reduce any possible side effects associated with the antibody or antigen-binding fragment thereof of the present invention, if such side effects occur.

[0057] In certain embodiments, the present invention provides a method for suppressing tumor growth. For example, the present invention provides suppression of tumor growth in a subject, resulting from a primary tumor or a metastatic tumor. In certain embodiments, the present invention provides a method for enhancing the survival (e.g., progression-free survival or overall survival) of a subject with cancer. Examples of cancer include, but are not limited to, primary and / or recurrent cancers, including blood cancer (e.g., hematological malignancies such as lymphoma, myeloma, or leukemia), brain cancer (e.g., glioblastoma multiforme), lung cancer (e.g., non-small cell lung cancer), head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, melanoma, mesothelioma, ovarian cancer, bladder cancer, breast cancer, bone cancer, colorectal cancer, prostate cancer, and colon cancer. In certain embodiments, the present invention provides a method for inhibiting or suppressing the growth of an established tumor. The method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-LAG3 antibody of the present invention. In certain embodiments, the antibody is administered in combination with a second therapeutic agent selected from the group consisting of a programmed cell death 1 (PD-1) inhibitor (e.g., an anti-PD-1 antibody such as nivolumab or REGN2810), a programmed cell death ligand 1 (PD-L1) inhibitor (e.g., an anti-PD-L1 antibody), a vascular endothelial growth factor (VEGF) antagonist (e.g., aflibercept, bevacizumab), an angiopoietin 2 (Ang2) inhibitor (e.g., an anti-Ang2 antibody such as nesvacumab), a cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitor (e.g., ipilimumab), a CD20xCD3 bispecific antibody, a cytotoxin, a chemotherapeutic agent, and a radiotherapy. Further examples of additional therapies / therapeutic agents that can be used in combination with the anti-LAG3 antibody of the present invention in the use for treating cancer are described elsewhere herein.

[0058] The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. The antibody or fragment thereof may be administered at a dose of about 0.1 mg to about 100 mg per kg of the subject's body weight.

[0059] The present invention also includes the use of an anti-LAG3 antibody, or antigen-binding fragment thereof, of the present invention in the manufacture of a medicament for treating a disease or disorder that benefits from blocking LAG3 binding and / or signaling, such as cancer. The present invention provides, for example, the following items. (Item 1) An isolated antibody or antigen-binding fragment thereof that specifically binds to human lymphocyte-activation gene 3 (LAG3) protein, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within the extracellular domain of LAG3 (amino acids 28-71 of SEQ ID NO:588) as determined by hydrogen / deuterium exchange. (Item 2) 2. The isolated antibody or antigen-binding fragment thereof of claim 1, which interacts with one or more amino acids contained within SEQ ID NO:589 as determined by hydrogen / deuterium exchange. (Item 3) 2. The isolated antibody or antigen-binding fragment thereof according to item 1, which interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 28 to 69 of SEQ ID NO: 588; (b) amino acids 28 to 71 of SEQ ID NO: 588; (c) amino acids 31 to 52 of SEQ ID NO: 588; and (d) amino acids 32 to 69 of SEQ ID NO: 588. (Item 4) 3. The isolated antibody or antigen-binding fragment thereof of item 2, which interacts with at least 10 amino acids contained within SEQ ID NO:589. (Item 5) 5. The isolated antibody or antigen-binding fragment thereof of item 2 or 4, which interacts with at least 20 amino acids contained within SEQ ID NO:589. (Item 6) 6. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 5, which interacts with the amino acid sequence of SEQ ID NO: 589. (Item 7) The antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVRs are (i) the amino acid sequence of SEQ ID NO: 418; (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 418; (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 418; or (iv) the amino acid sequence of SEQ ID NO: 418, having no more than five amino acid substitutions said LCVR comprising: (i) the amino acid sequence of SEQ ID NO: 426; (ii) an amino acid sequence having at least 90% identity to SEQ ID NO: 426; (iii) an amino acid sequence having at least 95% identity to SEQ ID NO: 426; or (iv) the amino acid sequence of SEQ ID NO: 426, having no more than five amino acid substitutions 7. The isolated antibody of any one of items 1 to 6, comprising: (Item 8) (a) a binding dissociation equilibrium constant (K) of less than about 1.5 nM as measured in a surface plasmon resonance assay at 25° C. D ) that binds to monomeric human LAG3; (b) a K of less than about 2 nM as measured in a surface plasmon resonance assay at 37° C. D and binding to monomeric human LAG3; (c) a K of less than about 20 pM as measured in a surface plasmon resonance assay at 25° C. D and binding to dimeric human LAG3; (d) a K of less than about 90 pM as measured in a surface plasmon resonance assay at 37° C. D and binding to dimeric human LAG3; (e) an EC of less than about 2 nM as measured in a flow cytometry assay 50 and binding to hLAG3-expressing cells; (f) an EC of less than about 2.3 nM as measured in a flow cytometry assay 50 , binding to mfLAG3-expressing cells; (g) IC of less than about 20 nM as determined by cell adhesion assay 50 by blocking the binding of hLAG3 to human MHC class II; (h) IC of less than about 15 nM as determined by cell adhesion assay 50 to block the binding of hLAG3 to mouse MHC class II; (i) blocking hLAG3 binding to MHC class II by >90%, as determined by cell adhesion assays; (j) an EC of less than about 2.5 nM as determined in a luciferase reporter assay 50 rescue LAG3-mediated inhibition of T cell activity; and (k) an EC of less than about 1.2 nM as determined in a fluorescent assay 50 and binds to activated CD4+ and CD8+ T cells 8. The isolated antibody or antigen-binding fragment thereof of any one of items 1 to 7, having one or more properties selected from the group consisting of: (Item 9) 9. The isolated antibody or antigen-binding fragment thereof of item 7 or 8, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 418, with no more than five amino acid substitutions. (Item 10) 10. The isolated antibody or antigen-binding fragment thereof of any one of items 7 to 9, comprising an LCVR comprising the amino acid sequence of SEQ ID NO: 426, with no more than five amino acid substitutions. (Item 11) 10. The isolated antibody or antigen-binding fragment thereof of any one of items 7 to 9, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 418, with not more than five amino acid substitutions, and an LCVR comprising the amino acid sequence of SEQ ID NO: 426, with not more than five amino acid substitutions. (Item 12) 9. The isolated antibody or antigen-binding fragment thereof according to item 7 or 8, comprising an HCDR1 comprising an amino acid sequence of SEQ ID NO: 420 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 420, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 422 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 422, an HCDR3 comprising an amino acid sequence of SEQ ID NO: 424 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 424, an LCDR1 comprising an amino acid sequence of SEQ ID NO: 428 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 428, an LCDR2 comprising an amino acid sequence of SEQ ID NO: 430 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 430, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 432 or an amino acid sequence that differs by one amino acid from SEQ ID NO: 432. (Item 13) 13. The isolated antibody or antigen-binding fragment thereof of any one of items 7 to 12, comprising an HCVR having the amino acid sequence of SEQ ID NO: 418. (Item 14) 14. The isolated antibody or antigen-binding fragment thereof of any one of items 7 to 13, comprising an LCVR having the amino acid sequence of SEQ ID NO: 426. (Item 15) 9. The isolated antibody or antigen-binding fragment thereof according to item 7 or 8, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, comprising the amino acid sequence of SEQ ID NOs: 420-422-424-428-430-432. (Item 16) 16. The isolated antibody or antigen-binding fragment thereof of item 15, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 418 / 426. (Item 17) 17. The isolated antibody of claim 16, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 579. (Item 18) 17. The isolated antibody of claim 16, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 578. (Item 19) 19. The isolated antibody or antigen-binding fragment of item 17 or 18, comprising the heavy chain / light chain amino acid sequence pair of SEQ ID NOs: 579 / 578. (Item 20) An antibody or antigen-binding fragment thereof that competes with an antibody comprising the three CDRs of HCVR having the amino acid sequence of SEQ ID NO: 418 and the three CDRs of LCVR having the amino acid sequence of SEQ ID NO: 426 for binding to LAG3. (Item 21) An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody comprising the three CDRs of HCVR having the amino acid sequence of SEQ ID NO: 418 and the three CDRs of LCVR having the amino acid sequence of SEQ ID NO: 426 on LAG3. (Item 22) (a) a binding dissociation equilibrium constant (K) of less than about 1.5 nM as measured in a surface plasmon resonance assay at 25° C. D ) that binds to monomeric human LAG3; (b) a K of less than about 2 nM as measured in a surface plasmon resonance assay at 37° C. D and binding to monomeric human LAG3; (c) a K of less than about 20 pM as measured in a surface plasmon resonance assay at 25° C. D and binding to dimeric human LAG3; (d) a K of less than about 90 pM as measured in a surface plasmon resonance assay at 37° C. D and binding to dimeric human LAG3; (e) an EC of less than about 2 nM as measured in a flow cytometry assay 50 and binding to hLAG3-expressing cells; (f) an EC of less than about 2.3 nM as measured in a flow cytometry assay. 50 , binding to mfLAG3-expressing cells; (g) IC of less than about 20 nM as determined by cell adhesion assay 50 by blocking the binding of hLAG3 to human MHC class II; (h) IC of less than about 15 nM as determined by cell adhesion assay 50 to block the binding of hLAG3 to mouse MHC class II; (i) blocking hLAG3 binding to MHC class II by >90%, as determined by cell adhesion assays; (j) an EC of less than about 2.5 nM as determined in a luciferase reporter assay 50 rescue LAG3-mediated inhibition of T cell activity; and (k) an EC of less than about 1.2 nM as determined in a fluorescent assay 50 and binds to activated CD4+ and CD8+ T cells 22. The antibody or antigen-binding fragment thereof according to item 20 or 21, having one or more properties selected from the group consisting of: (Item 23) 23. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of items 1 to 22 and a pharma- ceutically acceptable carrier or diluent. (Item 24) 23. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of items 1 to 22. (Item 25) 23. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody of any one of items 1 to 22. (Item 26) 26. A vector comprising the polynucleotide sequence according to item 24 or 25. (Item 27) A cell expressing the vector according to item 26. (Item 28) 23. A method for inhibiting tumor or tumor cell growth in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of items 1 to 22. (Item 29) 29. The method of claim 28, wherein the tumor is a primary tumor or a recurrent tumor. (Item 30) 29. The method of claim 28, wherein the tumor is an established tumor. (Item 31) 31. The method of any one of items 28 to 30, wherein the tumor is present in a subject with a disease or disorder selected from the group consisting of blood cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B cell lymphoma, and melanoma. (Item 32) 32. The method of any one of items 28 to 31, wherein the antibody or antigen-binding fragment thereof is administered as an initial dose followed by one or more secondary doses, each secondary dose being administered 1 to 4 weeks after the immediately preceding dose. (Item 33) 33. The method of claim 32, wherein the antibody or antigen-binding fragment is administered at a dose of about 1, 3, or 10 mg / kg of the subject's body weight. (Item 34) 34. The method of any one of items 28 to 33, wherein the antibody or antigen-binding fragment thereof is administered to the subject in combination with a second therapeutic agent. (Item 35) 35. The method of claim 34, wherein the second therapeutic agent is selected from the group consisting of CTLA inhibitors, antibodies against tumor specific antigens, antibodies against viral infected cell antigens, PD-L1 inhibitors, CD20 inhibitors, bispecific antibodies against CD20 and CD3, dietary supplements such as antioxidants, VEGF antagonists, chemotherapeutic agents, cytotoxic agents, radiation, NSAIDs, corticosteroids, and any other treatment useful in ameliorating at least one symptom associated with the disease or disorder. (Item 36) 35. The method of item 34, wherein the second therapeutic agent is a PD-1 inhibitor. (Item 37) 37. The method of claim 36, wherein the PD-1 inhibitor is REGN2810, nivolumab, or pembrolizumab. (Item 38) 38. The method of claim 36 or 37, wherein the PD-1 inhibitor is administered at a dose of 1, 3, or 10 mg / kg of the subject's body weight. (Item 39) 39. The method of any one of items 28 to 38, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially. (Item 40) 23. A method for treating a disease or disorder treatable by antagonizing LAG3, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of items 1 to 22. (Item 41) 41. The method of claim 40, wherein the disease or disorder is a chronic viral infection caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV). (Item 42) 41. The method of claim 40, wherein the disease or disorder is selected from the group consisting of blood cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B-cell lymphoma, and melanoma.

[0060] Other embodiments will become apparent from review of the following detailed description. [Brief description of the drawings]

[0061] [Figure 1]Figure 1 is a schematic diagram of the luciferase-based LAG3 bioassay described in Example 8 herein. Panel (A): "Bispecific" format: resting Jurkat-derived T cells are activated by T cell receptor (TCR) clustering via CD3xCD20 bispecific antibody. Panel (B): "Peptide" format: In this format, resting Jurkat-derived T cells are activated by a specific MHC / peptide complex (Ob2F3 TCR heterodimer with HLA-DR2AB, MHCII protein complexed with MBP85-99 peptide). Binding of LAG3 to DR2 attenuates the response in activated Jurkat-derived T cells. LAG3 blocking antibody rescues the response in activated Jurkat-derived T cells.

[0062] [Figure 2A] Figures 2A-2B summarize the in vivo efficacy results (described in Example 10) of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody against established Colon 26 tumors. Figure 2A: Mean tumor volume (mm3 ± SEM) within each treatment group was measured at multiple time points after tumor implantation. Treatment began on day 14, when the mean tumor volume reached 50 mm3. Treatment days are indicated by arrows. All antibodies were administered intraperitoneally (ip) at 10 mg / kg. Figure 2B: Individual tumor volumes within each treatment group were measured on day 35 after implantation. Statistical significance was determined by one-way ANOVA with post-Dunnett's multiple comparison test (*p<0.05). Figures 2A-2B: Treatment groups: isotype control rat IgG2a + mouse IgG1 control antibody (circles); anti-mouse PD-1 antibody + mouse IgG1 control (upward triangles); anti-mouse LAG3 antibody + rat IgG2a control (squares); anti-mouse PD-1 antibody + anti-mouse LAG3 antibody (downward triangles). [Figure 2B]Figures 2A-2B summarize the in vivo efficacy results (described in Example 10) of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody against established Colon 26 tumors. Figure 2A: Mean tumor volume (mm3 ± SEM) within each treatment group was measured at multiple time points after tumor implantation. Treatment began on day 14, when the mean tumor volume reached 50 mm3. Treatment days are indicated by arrows. All antibodies were administered intraperitoneally (ip) at 10 mg / kg. Figure 2B: Individual tumor volumes within each treatment group were measured on day 35 after implantation. Statistical significance was determined by one-way ANOVA with post-Dunnett's multiple comparison test (*p<0.05). Figures 2A-2B: Treatment groups: isotype control rat IgG2a + mouse IgG1 control antibody (circles); anti-mouse PD-1 antibody + mouse IgG1 control (upward triangles); anti-mouse LAG3 antibody + rat IgG2a control (squares); anti-mouse PD-1 antibody + anti-mouse LAG3 antibody (downward triangles).

[0063] [Figure 3A] Figures 3A-3B summarize the established MC38 in vivo efficacy results (described in Example 11) of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody. Figure 3A: Mean tumor volume (mm3 ± SEM) within each treatment group was measured at multiple time points after tumor implantation. Treatment began on day 8, when the mean tumor volume reached 45 mm3. Treatment days are indicated by arrows. Figure 3B: Individual tumor volumes within each treatment group were measured on day 23 after implantation. Statistical significance was determined by one-way ANOVA with Dunnett's post-test multiple comparisons (****p<0.0001). Figures 3A-3B: Treatment groups: isotype control rat IgG2a + mouse IgG1 (circles), anti-mouse PD-1 antibody + mIgG1 control (upward triangles), anti-mouse LAG3 antibody + rat IgG2a control (squares), and anti-mouse PD-1 + anti-mouse LAG3 antibody (downward triangles). [Figure 3B]Figures 3A-3B summarize the established MC38 in vivo efficacy results (described in Example 11) of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody. Figure 3A: Mean tumor volume (mm3 ± SEM) within each treatment group was measured at multiple time points after tumor implantation. Treatment began on day 8, when the mean tumor volume reached 45 mm3. Treatment days are indicated by arrows. Figure 3B: Individual tumor volumes within each treatment group were measured on day 23 after implantation. Statistical significance was determined by one-way ANOVA with Dunnett's post-test multiple comparisons (****p<0.0001). Figures 3A-3B: Treatment groups: isotype control rat IgG2a + mouse IgG1 (circles), anti-mouse PD-1 antibody + mIgG1 control (upward triangles), anti-mouse LAG3 antibody + rat IgG2a control (squares), and anti-mouse PD-1 + anti-mouse LAG3 antibody (downward triangles).

[0064] [Figure 4A] Figures 4A-4B show expression levels of mouse IFNγ and CD8b genes (normalized to expression of mouse cyclophilin B) examined by Taqman analysis in the draining lymph nodes (DLN) (A) and spleens (B) harvested from tumor-bearing mice (described in Example 11) at the end of the experiment (*P<0.05, **P<0.01, ***P<0.001). [Figure 4B] Figures 4A-4B show expression levels of mouse IFNγ and CD8b genes (normalized to expression of mouse cyclophilin B) examined by Taqman analysis in the draining lymph nodes (DLN) (A) and spleens (B) harvested from tumor-bearing mice (described in Example 11) at the end of the experiment (*P<0.05, **P<0.01, ***P<0.001).

[0065] [Figure 5A]Figures 5A-5B summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") and anti-human PD-1 antibody (REGN2810) against MC38 tumors (described in Example 12). Figure 5A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated with arrows. Figure 5B: Individual tumor volumes within each group were monitored over a 24-day period. The number of tumor-free mice within each group at day 24 is shown. Figures 5A-5B: Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (squares); 10 mg / kg REGN2810 (anti-hPD-1) (upward pointing triangles), and 25 mg / kg human isotype control antibody (circles). [Figure 5B] Figures 5A-5B summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") and anti-human PD-1 antibody (REGN2810) against MC38 tumors (described in Example 12). Figure 5A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated with arrows. Figure 5B: Individual tumor volumes within each group were monitored over a 24-day period. The number of tumor-free mice within each group at day 24 is shown. Figures 5A-5B: Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (squares); 10 mg / kg REGN2810 (anti-hPD-1) (upward pointing triangles), and 25 mg / kg human isotype control antibody (circles).

[0066] [Figure 6A]Figures 6A-6C summarize the in vivo efficacy (described in Example 13) of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors. Figure 6A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 6B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with Tukey's post-test multiple comparisons (*p<0.05; **p<0.01). Figures 6A-6B: Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (squares); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward pointing triangles); 25 mg / kg human isotype control (circles). Figure 6C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (****p<0.0001). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg human isotype control (circles). [Figure 6B]Figures 6A-6C summarize the in vivo efficacy (described in Example 13) of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors. Figure 6A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 6B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with Tukey's post-test multiple comparisons (*p<0.05; **p<0.01). Figures 6A-6B: Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (squares); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward pointing triangles); 25 mg / kg human isotype control (circles). Figure 6C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (****p<0.0001). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg human isotype control (circles). [Figure 6C]Figures 6A-6C summarize the in vivo efficacy (described in Example 13) of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors. Figure 6A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 6B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with Tukey's post-test multiple comparisons (*p<0.05; **p<0.01). Figures 6A-6B: Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (squares); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward pointing triangles); 25 mg / kg human isotype control (circles). Figure 6C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (****p<0.0001). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg human isotype control (circles).

[0067] [Figure 7A]Figures 7A-7C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in the first experiment (described in Example 14). Figure 7A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 7B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with post-Tukey multiple comparisons (*p<0.05). Figure 7C: Percentage tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (*p<0.0197). Treatment groups: mAb1 (anti-hLAG3) at 25 mg / kg (diamonds); REGN2810 (anti-hPD-1) at 10 mg / kg (hexagons); REGN2810 (anti-hPD-1) at 1 mg / kg (upward triangles); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 10 mg / kg (squares); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 25 mg / kg (downward triangles); and human isotype control at 25 mg / kg (circles). [Figure 7B]Figures 7A-7C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in the first experiment (described in Example 14). Figure 7A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 7B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with post-Tukey multiple comparisons (*p<0.05). Figure 7C: Percentage tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (*p<0.0197). Treatment groups: mAb1 (anti-hLAG3) at 25 mg / kg (diamonds); REGN2810 (anti-hPD-1) at 10 mg / kg (hexagons); REGN2810 (anti-hPD-1) at 1 mg / kg (upward triangles); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 10 mg / kg (squares); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 25 mg / kg (downward triangles); and human isotype control at 25 mg / kg (circles). [Figure 7C]Figures 7A-7C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in the first experiment (described in Example 14). Figure 7A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Figure 7B: Individual tumor volumes within each treatment group were measured 22 days after implantation. Statistical significance was determined by one-way ANOVA with post-Tukey multiple comparisons (*p<0.05). Figure 7C: Percentage tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test (*p<0.0197). Treatment groups: mAb1 (anti-hLAG3) at 25 mg / kg (diamonds); REGN2810 (anti-hPD-1) at 10 mg / kg (hexagons); REGN2810 (anti-hPD-1) at 1 mg / kg (upward triangles); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 10 mg / kg (squares); mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) at 25 mg / kg (downward triangles); and human isotype control at 25 mg / kg (circles).

[0068] [Figure 8A]Figures 8A-8C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in a second experiment (described in Example 14). Figure 8A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparison test (*p<0.05). Figure 8B: Individual tumor volumes within each treatment group measured at day 23 after implantation. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test (**p<0.01). Figure 8C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test with Bonferroni correction for multiple comparisons (***p<0.001, **p<0.01). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 5 mg / kg mAb1 (anti-hLAG3) (hexagons); 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (squares); 5 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); and 25 mg / kg human isotype control (circles). [Figure 8B]Figures 8A-8C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in a second experiment (described in Example 14). Figure 8A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparison test (*p<0.05). Figure 8B: Individual tumor volumes within each treatment group measured at day 23 after implantation. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test (**p<0.01). Figure 8C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test with Bonferroni correction for multiple comparisons (***p<0.001, **p<0.01). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 5 mg / kg mAb1 (anti-hLAG3) (hexagons); 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (squares); 5 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); and 25 mg / kg human isotype control (circles). [Figure 8C]Figures 8A-8C summarize the in vivo efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with anti-human PD-1 antibody (REGN2810) against MC38 tumors in a second experiment (described in Example 14). Figure 8A: Mean tumor volume (mm3 ± SEM) within each treatment group at multiple time points after tumor implantation. Treatment days are indicated by arrows. Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparison test (*p<0.05). Figure 8B: Individual tumor volumes within each treatment group measured at day 23 after implantation. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test (**p<0.01). Figure 8C: Percentage of tumor-free survival in treated mice. Statistical significance was determined by log-rank (Mantel-Cox) test with Bonferroni correction for multiple comparisons (***p<0.001, **p<0.01). Treatment groups: 25 mg / kg mAb1 (anti-hLAG3) (diamonds); 5 mg / kg mAb1 (anti-hLAG3) (hexagons); 10 mg / kg REGN2810 (anti-hPD-1) (upward triangles); 25 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (squares); 5 mg / kg mAb1 (anti-hLAG3) in combination with 10 mg / kg REGN2810 (anti-hPD-1) (downward triangles); and 25 mg / kg human isotype control (circles).

[0069] [Figure 9] 9 shows the mean tumor volume (mm3±SEM) within each treatment group at multiple time points following tumor implantation in an experiment evaluating the efficacy of anti-human LAG3 antibody ("mAb1") alone and in combination with an anti-human PD-1 antibody (REGN2810) against established MC38 tumors (described in Example 15 herein). Days of treatment are indicated by arrows.

[0070] [Figure 10]Figure 10 shows individual tumor volumes within each treatment group measured 22 days after implantation in the experiment described in Example 15. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test.

[0071] [Figure 11] Figure 11 shows the percent tumor-free survival in mice treated in the experiment described in Example 15 herein. Statistical significance was determined by the log-rank (Mantel-Cox) test with Bonferroni correction for multiple comparisons (**p<0.01).

[0072] [Figure 12] FIG. 12 shows a graph of mean (+SD) functional mAb1 concentration in serum versus time following a single intravenous infusion of mAb1 in female cynomolgus monkeys (as described in Example 16). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Before describing the method, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0074] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out or testing the present invention, any methods and materials similar or equivalent to those described herein can be used, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0075] The term "LAG3" refers to lymphocyte activation gene 3 protein, also known as CD223, which is an immune checkpoint receptor or T cell co-inhibitor. The amino acid sequence of full-length LAG3 is provided in GenBank under Accession No. NP_002277.4, and is also referred to herein as SEQ ID NO: 582. The term "LAG3" also includes protein variants of LAG3 having the amino acid sequence of SEQ ID NO: 574, 575, or 576. The term "LAG3" includes recombinant LAG3 or fragments thereof. The term also encompasses LAG3 or fragments thereof coupled to a signal sequence, such as, for example, a histidine tag, mouse Fc or human Fc, or ROR1. For example, the term includes the sequence exemplified by SEQ ID NO: 575, which includes mouse Fc (mIgG2a) coupled at the C-terminus to amino acid residues 29-450 of full-length LAG3. The protein variant exemplified by SEQ ID NO:574 comprises a histidine tag coupled at the C-terminus to full length LAG3 amino acid residues 29 to 450. The term "LAG3" refers to human LAG3, unless specified to be derived from a non-human species.

[0076] LAG3 is a member of the immunoglobulin (Ig) superfamily. It is a 503 amino acid type 1 transmembrane protein with four extracellular Ig-like domains, D1-D4, and is expressed on activated T cells, natural killer cells, B cells, plasmacytoid dendritic cells, and regulatory T cells. The LAG3 receptor binds to MHC class II molecules present on antigen-presenting cells (APCs).

[0077] As used herein, the term "T cell co-inhibitor" refers to ligands and / or receptors that modulate the immune response through the activation or suppression of T cells. The term "T cell co-inhibitor", also known as T cell co-signaling molecule, includes but is not limited to programmed cell death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin 3 (TIM3), T cell immunoreceptor with immunoglobulin and ITIM (TIGIT; also known as VSIG9), leucocyte associated immunoglobulin-like receptor 1 (LAIR1; also known as CD305), inducible T cell costimulator (ICOS; also known as CD278), V-domain Ig suppressor of T cell activation (V-domain Ig suppressor of T cell activation), and VEGF receptor 1 (VEGF receptor 1). These include, but are not limited to, CD40, CD50+, CD60+, and CD80+.

[0078] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule (i.e., a "full-length antibody molecule") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM), or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (C H 1 domain, C H 2 domain, and C H Each light chain is composed of a light chain variable region (LCVR or VVR). L") and the light chain constant region (C L ) is composed of V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragments thereof) may be identical to human germline sequences, may be naturally modified, or may be artificially modified. A consensus sequence of amino acids may be defined based on matching analysis of two or more CDRs.

[0079] Substitution of one or more CDR residues or omission of one or more CDRs are also possible. In the scientific literature, antibodies with one or two CDRs are described that are dispensable for binding. Padlan et al. (1995, FASEB J., 9:133-139) analyzed the contact regions of antibodies with their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs have no amino acids that contact the antigen (see also Vajdos et al., 2002, J Mol Biol 320:415-428).

[0080] CDR residues that do not contact antigen can be identified by molecular modeling and / or empirical analysis from regions of the Kabat CDRs that lie outside the Chothia CDRs based on previous studies (e.g., residues H60-H65 in CDRH2 are often not required). When a CDR or its residues are omitted, they are typically replaced with an amino acid occupying the corresponding position in another human antibody sequence, or a consensus sequence for such sequences. The positions for substitution within the CDRs, and the replacing amino acids, can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0081] The fully human anti-LAG3 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody was derived, or to the corresponding residues in another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof. H Domain and / or V LAll of the framework and / or CDR residues in the domain are backmutated to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, for example, only residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4 are mutated, or only residues found in CDR1, CDR2, or CDR3 are mutated. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0082] The present invention also includes fully human anti-LAG3 monoclonal antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes anti-LAG3 antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0083] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the invention may include, for example, within the CDRs and in particular CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies that are recombinantly produced in or within the cells of a non-human mammal. The term is not intended to include antibodies that are isolated from or developed in a human subject.

[0084] The term "recombinant" as used herein refers to an antibody or antigen-binding fragment thereof of the present invention that is created, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammalian (including a transgenic non-human mammalian, e.g., a transgenic mouse) expression system, or a cellular (e.g., CHO cell) expression system, or isolated from a recombinant combinatorial human antibody library.

[0085] The term "multispecific antigen-binding molecule" as used herein refers to bispecific, trispecific, or multispecific antigen-binding molecules and their antigen-binding fragments. Multispecific antigen-binding molecules may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide. Multispecific antigen-binding molecules may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with each other. The term "multispecific antigen-binding molecule" includes the antibodies of the present invention that may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as a protein or fragment thereof, to create a bispecific or multispecific antigen-binding molecule with a second binding specificity. In accordance with the present invention, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In certain embodiments, an antibody of the present invention is operatively linked to another antibody or its antigen-binding fragment to create a bispecific antibody with a second binding specificity. Bispecific and multispecific antibodies of the present invention are described elsewhere herein.

[0086] The terms "specifically binds" or "binds specifically to" and the like mean that an antibody or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding is at least about 1×10 -8 Equilibrium dissociation constants at or below M (e.g., small K D The binding of an antibody to a LAG3 molecule can be characterized by a specific binding affinity (depicting tight binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to LAG3 have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, multispecific antibodies that bind to a domain within LAG3 and to one or more additional antigens, or bispecific antibodies that bind to two different regions of LAG3, are nevertheless considered to be antibodies that "specifically bind to" as used herein.

[0087] The term "high affinity" antibody refers to an antibody that has a binding affinity for LAG3 of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -8 M, preferably 10 -9 M, more preferably 10 -10 M, and even more preferably 10 -11 M, and even more preferably 10 -12 K of M D The term refers to mAbs having binding affinities expressed as

[0088] The term "slow off rate," "Koff," or "kd" refers to a slow off rate greater than 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 seconds -1 or less, preferably 1×10 -4 seconds -1It means an antibody that dissociates from LAG3 with a rate constant equal to or less than this rate constant.

[0089] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to LAG3.

[0090] In specific embodiments, the antibodies or antibody fragments of the invention can be conjugated to a moiety such as a ligand or a therapeutic moiety such as a cytotoxin, a second anti-LAG3 antibody, an antibody against a tumor-specific antigen, an anti-cancer drug, or any other therapeutic moiety useful for treating a disease or condition, including cancer or a viral infection, including a chronic viral infection (an "immunoconjugate").

[0091] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to LAG3 is substantially free of Abs that specifically bind to antigens other than LAG3).

[0092] As used herein, a "blocking antibody" or "neutralizing antibody" (or an "antibody that neutralizes LAG3 activity" or an "antagonist antibody") is intended to refer to an antibody whose binding to LAG3 results in the inhibition of at least one biological activity of LAG3. For example, an antibody of the invention may prevent or block the binding of LAG3 to MHC class II.

[0093] As used herein, an "activating antibody" or "enhancing antibody" (or "agonist antibody") is intended to refer to an antibody whose binding to LAG3 results in an increase or stimulation of at least one biological activity of LAG3. For example, an antibody of the invention may increase the binding of LAG3 to MHC class II.

[0094] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biomolecular interactions by detecting alterations in protein concentration in a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0095] As used herein, "K D The term "antibody-antigen interaction" is intended to refer to the equilibrium dissociation constant for a particular antibody-antigen interaction.

[0096] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may exert different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term "epitope" also refers to a region of an antigen to which an antibody binds. Epitopes can be defined as structural epitopes or functional epitopes. Functional epitopes are generally a subset of structural epitopes, with residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational epitopes, i.e., epitopes composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are groupings of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three dimensional structural characteristics, and / or specific charge characteristics.

[0097] The term "cross-compete" as used herein means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in either orientation, i.e., when a first antibody binds to and blocks the binding of a second antibody, and vice versa. In certain embodiments, the first and second antibodies may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that the binding of one antibody inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, real-time, label-free bio-layer interferometry assays. Cross-competition between two antibodies can be expressed as the binding of the second antibody below the background signal due to self-binding (in which case the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as the % binding of the second antibody that is less than the baseline self-background binding (in which case the first and second antibodies are the same antibody).

[0098] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof refers to nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99%, of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, as measured by any well-known algorithm for sequence identity, discussed below. In certain cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule encodes a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0099] Sequence identity can be calculated using algorithms such as the Needleman-Wunsch algorithm for global alignment (Needleman and Wunsch, 1970, J. Mol. Biol., 48:443-453), or the Smith-Waterman algorithm for local alignment (Smith and Waterman, 1981, J. Mol. Biol., 147:195-197). Another preferred algorithm is described by Dufresne et al., Nature Biotechnology, 2002 (20:1269-71) and is used in the GenePAST software (GQ Life Sciences, Inc. Boston, Mass.).

[0100] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the GAP or BESTFIT programs using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this correction are well known to those skilled in the art. See, e.g., Pearson (1994), Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992), Science 256:1443-45. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0101] Polypeptide sequence similarity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a measure of similarity that is assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program within GCG Version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity for the best overlap region between the query sequence and the search sequence (Pearson (2000), supra). Another preferred algorithm for comparing the sequences of the present invention with a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters.See, for example, Altschul et al. (1990), J. Mol. Biol., 215:403-410; and (1997), Nucleic Acids Res., 25:3389-3402, each of which is incorporated herein by reference.

[0102] The phrase "therapeutically effective amount" refers to an amount that produces the desired effect upon administration. The exact amount will depend on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques (see, for example, Lloyd (1999) "The Art, Science and Technology of Pharmaceutical Compounding").

[0103] The term "subject" as used herein refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a viral infection or cancer. The term includes human subjects having or at risk of having cancer, metastatic cancer, or a viral infection.

[0104] As used herein, "anticancer drug" refers to any agent useful for treating or ameliorating or inhibiting cancer, including, but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, cyclophosphamide, mytotane (O,P'-(DDD)), biologics (e.g., antibodies and interferons), and radioactive agents. As used herein, "cytotoxin or cytotoxic agent" also refers to chemotherapeutic agents, and any agent that is detrimental to cells. Examples include Taxol® (paclitaxel), temozolamide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, coichicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0105] The term "antiviral drug" as used herein refers to any drug or treatment used to treat, prevent or reverse viral infection in a host subject.The term "antiviral drug" includes, but is not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, analgesics, and corticosteroids.In the context of the present invention, viral infection includes long-term or chronic infection caused by viruses, including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV).

[0106] The term "enhancing immune response" as used herein refers to the increase in the activity of immune cells, such as T cells or NK cells, against tumor cells or virus-infected cells. In the context of the present invention, the term includes blocking the inhibition of T cell activity, or rescuing or reversing the exhausted state of T cells, mediated by LAG3. The term "enhancing immune response" also includes the inhibition of regulatory T cell activity. As used in the context of the present invention, enhancing immune response results in increased tumor cell killing and / or inhibition of tumor growth.

[0107] The antibodies and antigen-binding fragments of the present invention specifically bind to LAG3 and enhance T cell activation. Anti-LAG3 antibodies may bind to LAG3 with high or low affinity. In certain embodiments, the antibodies of the present invention may be blocking antibodies, where the antibodies may bind to LAG3 and inhibit signaling by LAG3. In some embodiments, the antibodies of the present invention block the binding of LAG3 to MHC class II and / or stimulate or enhance T cell activation. In some embodiments, the antibodies bind to LAG3 and reverse the anergic state of exhausted T cells. In certain embodiments, the antibodies bind to LAG3 and inhibit the activity of regulatory T cells. In some embodiments, the antibodies may be useful for stimulating or enhancing immune responses and / or treating subjects suffering from cancer or viral infections. When administered to a subject in need thereof, the antibodies may reduce chronic infection with a virus such as HIV, LCMV, or HBV in the subject. The antibodies can be used to inhibit the growth of tumor cells in a subject. The antibodies can be used alone or as adjunctive therapy with other therapeutic moieties or therapeutic modalities known in the art for treating cancer or viral infections.

[0108] In certain embodiments, the anti-LAG3 antibody can be a multispecific antigen-binding molecule, comprising a first binding specificity for LAG3 and a second binding specificity for an antigen selected from the group consisting of another T cell co-inhibitor and a different epitope of LAG3.

[0109] Antibodies to LAG3 can be generated using immunogens including any one of the following: In certain embodiments, the antibodies of the invention are obtained from mice immunized with full-length, native LAG3 (see NCBI Accession No.: NP_002277.4) (SEQ ID NO: 582), or recombinant LAG3 peptides. Alternatively, LAG3 or fragments thereof can be generated using standard biochemical methods, modified (SEQ ID NOs: 574-576) and used as immunogens.

[0110] In certain embodiments, the immunogen is one or more extracellular domains of LAG3. In one embodiment of the invention, the immunogen is a fragment of LAG3 ranging from about amino acid residues 29 to 450 of SEQ ID NO:582.

[0111] In some embodiments, the immunogen can be recombinant LAG3 peptide expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0112] In certain embodiments, antibodies that specifically bind to LAG3 can be prepared using fragments of the above-referenced regions, or peptides extending beyond the indicated regions by about 5 to about 20 amino acid residues from either or both of the N-terminus and C-terminus of the regions described herein. In certain embodiments, any combination of the above-referenced regions or fragments thereof can be used in the preparation of LAG3-specific antibodies.

[0113] Peptides can be modified to include the addition or substitution of certain residues for tagging or for conjugation to carrier molecules such as KLH. For example, cysteine ​​can be added to the N-terminus or C-terminus of the peptide, and linker sequences can be added to prepare peptides for conjugation to KLH for immunization, for example.

[0114] Certain anti-LAG3 antibodies of the invention are capable of binding to LAG3 and neutralizing its activity as determined by in vitro or in vivo assays. The ability of an antibody of the invention to bind to LAG3 and neutralize its activity can be measured using any standard method known to those of skill in the art, including the binding or activity assays described herein.

[0115] Non-limiting exemplary in vitro assays for measuring binding activity are illustrated in the Examples herein. In Example 3, the binding affinity and kinetic constant of human anti-LAG3 antibodies to human LAG3 were determined by surface plasmon resonance, and measurements were performed on a Biacore 4000 instrument or a Biacore T200 instrument. In Example 4, a blocking assay was used to determine cross-competition between anti-LAG3 antibodies. Examples 5 and 6 describe the binding of antibodies to cells overexpressing LAG3. In Example 7, a binding assay was used to determine the ability of anti-LAG3 antibodies to block the MHC class II binding ability of LAG3 in vitro. In Example 8, a luciferase assay was used to determine the ability of anti-LAG3 antibodies to antagonize signaling by LAG3 in T cells. In Example 9, a fluorescent assay was used to determine the ability of anti-LAG3 antibodies to bind to activated monkey CD4+ and CD8+ T cells.

[0116] In certain embodiments, the antibodies of the invention are capable of enhancing or stimulating T cell activity in vitro, in subjects with cancer, or in subjects infected with a virus such as LCMV, In certain embodiments, the antibodies of the invention are used in combination with a second therapeutic agent, such as an antibody against a second T cell co-inhibitor, to enhance the immune response and inhibit tumor growth in a subject.

[0117] The antibody specific for LAG3 may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) may determine the orientation of the peptide relative to the surface to which it is bound. For example, when the surface is coated with avidin, a peptide containing an N-terminal biotin is oriented so that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or a label for MRI detection. In certain embodiments, such labeled antibodies can be used in diagnostic assays, including imaging assays.

[0118] Exemplary embodiments of the present invention In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human lymphocyte activation gene 3 (LAG3) protein, the antibody or antigen-binding fragment thereof having (a) a binding dissociation equilibrium constant (K) of less than about 10 nM as measured in a surface plasmon resonance assay at 25° C. D ) to monomeric human LAG3; (b) a K of less than about 8 nM as measured in a surface plasmon resonance assay at 37° C. D (c) binds to monomeric human LAG3 with a K of less than about 1.1 nM as measured in a surface plasmon resonance assay at 25° C. D (d) binds to dimeric human LAG3 with a K of less than about 1 nM as measured in a surface plasmon resonance assay at 37° C. D (e) an EC50 of less than about 8 nM as measured in a flow cytometry assay; 50 (f) an EC50 of less than about 2.3 nM as measured in a flow cytometry assay; 50 (g) binds to mfLAG3-expressing cells with an IC of less than about 32 nM as determined by a cell adhesion assay. 50(h) blocks the binding of hLAG3 to human MHC class II with an IC of less than about 30 nM as determined by a cell adhesion assay; 50 (i) blocks hLAG3 binding to mouse MHC class II by greater than 90%, as determined by a cell adhesion assay; (j) has an EC of less than about 9 nM, as determined by a luciferase reporter assay. 50 and (k) rescues LAG3-mediated inhibition of T cell activity with an EC of less than about 1.2 nM as measured in a fluorescent assay. 50 and binding to activated CD4+ and CD8+ T cells.

[0119] In certain embodiments, an isolated antibody or antigen-binding fragment thereof of the present invention comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences listed in Table 1; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences listed in Table 1. In certain embodiments, an isolated antibody or antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1. In certain embodiments, an isolated antibody or antigen-binding fragment thereof comprises a LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0120] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540, and 556; (b) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22 , 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558; (c) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558; (d) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544, and 560; (e) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 526, 534, 550, and 566;and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552, and 568;

[0121] In certain embodiments, the isolated antibodies or antigen-binding fragments thereof of the present invention are selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 32 2 / 330, 338 / 346, 354 / 362, 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

[0122] In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof comprising a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386 / 394, 418 / 426, and 538 / 546.

[0123] In certain embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that blocks the binding of LAG3 to MHC class II, comprising three CDRs of HCVR, the three CDRs being: SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 506, 514, 538, and and 554; and three CDRs of the LCVR, the CDRs being selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562.

[0124] In certain embodiments, the isolated antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 386 / 394, 418 / 426, and 538 / 546.

[0125] In certain embodiments, the present invention provides antibodies to the antibodies of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 338 / 346, 354 / 362 for binding to LAG3. The present invention provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of 370 / 378, 386 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

[0126] In certain embodiments, the present invention relates to nucleic acids selected from SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, 306 / 314, 322 / 330, 338 / 346, 354 / 362, 370 / 378, 380 / 390, 392 / 400, 394 / 410, 396 / 420, 398 / 430, 398 / 440, 398 / 450, 400 / 460, 400 / 470, 400 / 480, 400 / 490, 410 / 420, 420 / 430, 430 / 440, 440 / 450, 450 / 460, 460 / 470, 470 / 480, 470 / 490, 480 / 490, 490 / 490, 500 / 500, 510 / 520, 520 / 530, 530 / 540, 540 / 550, 550 / 560, 560 / 570, 570 / 580, 580 / 590, 590 / 590, 600 / 600, 6 The present invention provides an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of 86 / 394, 402 / 410, 418 / 426, 434 / 442, 450 / 522, 458 / 522, 466 / 522, 474 / 522, 482 / 522, 490 / 522, 498 / 530, 506 / 530, 514 / 530, 538 / 546, and 554 / 562.

[0127] In certain embodiments, the invention provides an antibody or antigen-binding fragment thereof that is a human antibody, a humanized antibody, or a chimeric antibody. The antibody or antigen-binding fragment thereof can be, for example, an IgG1 antibody or an IgG4 antibody, such as, for example, a human IgG1 antibody or a human IgG4 antibody. The constant regions of these antibodies may correspond to wild-type constant regions or to constant regions into which mutations have been introduced.

[0128] In certain embodiments, the invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 577, 579, and 580. In certain embodiments, the invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 578, and 581. In certain embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof comprising a heavy / light chain amino acid sequence pair selected from the group consisting of SEQ ID NOs: 577 / 578, 579 / 578, and 580 / 581.

[0129] In one aspect, the invention provides a multispecific antigen-binding molecule comprising a first antigen-binding specificity that specifically binds to LAG3 and a second antigen-binding specificity that specifically binds to a second target epitope.

[0130] In one aspect, the invention provides a pharmaceutical composition comprising an anti-LAG3 antibody or antigen-binding fragment thereof of any of the above embodiments and a pharma- ceutically acceptable carrier or diluent.

[0131] In one aspect, the invention provides isolated polynucleotide molecules and vectors comprising the polynucleotide sequence of an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the invention provides isolated polynucleotide molecules and / or vectors comprising the polynucleotide sequence encoding the HCVR of an antibody set forth herein. In certain embodiments, the invention provides isolated polynucleotide molecules and / or vectors comprising the polynucleotide sequence encoding the LCVR of an antibody set forth herein.

[0132] In one aspect, the present invention provides a method of enhancing an immune response in a subject, comprising administering a pharmaceutical composition comprising an isolated anti-LAG3 antibody or antigen-binding fragment thereof as disclosed herein. In certain embodiments, the present invention provides a method of inhibiting regulatory T (Treg) cells in a subject, comprising administering a pharmaceutical composition comprising an isolated anti-LAG3 antibody or antigen-binding fragment thereof as disclosed herein. In certain embodiments, the present invention provides a method of enhancing T cell activation in a subject, comprising administering a pharmaceutical composition comprising an isolated anti-LAG3 antibody or antigen-binding fragment thereof as disclosed herein. In certain embodiments, the subject has a disease or disorder selected from the group consisting of blood cancer, brain cancer, renal cell carcinoma (e.g., renal clear cell carcinoma), ovarian cancer, bladder cancer, prostate cancer, breast cancer (e.g., triple-negative breast cancer), hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma), and melanoma. In certain embodiments, the subject has a chronic viral infection caused by a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), human papilloma virus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV). In certain embodiments, the anti-LAG3 antibody is administered to a subject in combination with a second therapeutic agent selected from the group consisting of a PD-1 inhibitor, a CTLA inhibitor, an antibody against a tumor-specific antigen, an antibody against a virally infected cell antigen, a PD-L1 inhibitor, a CD20 inhibitor, a bispecific antibody against CD20 and CD3, a dietary supplement such as an antioxidant, a VEGF antagonist, a chemotherapeutic agent, a cytotoxic agent, radiation, an NSAID, a corticosteroid, and any other treatment useful in ameliorating at least one symptom associated with a disease or disorder.

[0133] In one aspect, the invention provides a method of inhibiting tumor or tumor cell growth in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-LAG3 antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the tumor is primary or recurrent. In certain embodiments, the tumor is an established tumor. In certain embodiments, the subject has metastatic disease and / or has been treated with a prior therapy. In certain embodiments, the tumor is present in a subject with a disease or disorder selected from the group consisting of hematological cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, lymphoma, and melanoma. In certain embodiments, the anti-LAG3 antibody or antigen-binding fragment thereof is administered in one or more doses, with each dose administered 1-4 weeks after the immediately preceding dose. In certain embodiments, the anti-LAG3 antibody or antigen-binding fragment thereof is administered at a dose of about 0.1 mg to about 100 mg per kg of body weight of the subject. In certain embodiments, the anti-LAG3 antibody is administered to the subject in combination with a second therapeutic agent selected from the group consisting of PD-1 inhibitors, CTLA inhibitors, antibodies against tumor-specific antigens, PD-L1 inhibitors, CD20 inhibitors, bispecific antibodies against CD20 and CD3, dietary supplements such as antioxidants, VEGF antagonists, chemotherapeutic agents, cytotoxic agents, radiation, NSAIDs, corticosteroids, and any other treatment useful for ameliorating at least one symptom associated with a disease or disorder. In one embodiment, the second therapeutic agent is a PD-1 inhibitor, where the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PD-1. In one embodiment, the PD-1 inhibitor is REGN2810. In certain embodiments, the anti-LAG3 antibodies or antigen-binding fragments thereof are administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially.

[0134] In one aspect, the invention provides a method of rescuing LAG3-mediated inhibition of T cell activity, comprising contacting T cells with an anti-LAG3 antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the T cells are contacted with an anti-LAG3 antibody of the invention in combination with an anti-PD-1 antibody (e.g., REGN2810).

[0135] Antigen-binding fragment of an antibody Unless specifically indicated otherwise, the term "antibody" as used herein shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "full-length antibody molecules") as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to LAG3. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. In such embodiments, the term "antigen-binding fragment" includes, for example, an MHC class II molecule that specifically binds to LAG3. Antigen-binding fragments of antibodies can be derived from full-length antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering methods involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can also be sequenced and chemically engineered, and can be engineered using molecular biology techniques, for example, by placing one or more variable and / or constant domains into the appropriate configuration, or by introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0136] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides, consisting of amino acid residues that mimic the hypervariable regions of an antibody. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small molecule immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0137] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, the V H Domain and V L The domains can be in any suitable configuration relative to one another. For example, the variable region can be a dimer, with the V H -V H Dimer, V H -V L Dimer, or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V H Domain or V L It may contain domains.

[0138] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention can be composed of the configurations of variable and constant domains listed above, which can be linked to each other and / or to one or more monomeric V H Domain or V L The domains may comprise homodimers or heterodimers (or other multimers) in any of a number of configurations in which they are non-covalently associated (eg, by disulfide bonds).

[0139] As with full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain will be capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0140] Preparation of human antibodies Methods for producing human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to LAG3.

[0141] Using VELOCIMMUNE® technology (see, e.g., US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method for producing monoclonal antibodies, a high affinity chimeric antibody against LAG3 is first isolated, which has a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome that includes a human heavy chain variable region and a light chain variable region operably linked to an endogenous mouse constant region locus, such that the mouse produces an antibody that includes a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to the DNA encoding the human heavy chain constant region and the human light chain constant region. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0142] In general, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express the antibody. The lymphocytes are fused with a myeloma cell line to prepare an immortal hybridoma cell line, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and linked to constant regions of the desired isotype of heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be directly isolated from the antigen-specific lymphocytes.

[0143] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antibody can be characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody of the invention, e.g., wild-type IgG1 or wild-type IgG4 or modified IgG1 or modified IgG4. The constant region selected can vary depending on the specific use, but the high affinity antigen binding and target specificity characteristics reside within the variable region.

[0144] biological equivalent The anti-LAG3 antibodies and antibody fragments of the invention include proteins having amino acid sequences that vary from the amino acid sequences of the described antibodies but retain the ability to bind to LAG3. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions that exhibit essentially the same biological activity as the described antibodies when compared to the parent sequence. Similarly, DNA sequences encoding the antibodies of the invention include sequences that contain one or more nucleotide additions, deletions, or substitutions compared to the disclosed sequences, but that encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the invention.

[0145] Two antigen-binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or substitutes that do not show significant differences in their rate and extent of absorption when administered at the same molar dose under similar experimental conditions, e.g., in single or multiple doses. Some antibodies are equivalent in their extent of absorption but not in their rate of absorption, but are considered to be equivalents or pharmaceutical substitutes if such differences in absorption rates are considered to be bioequivalent because they are intentional, are not essential to achieving effective body drug concentrations, e.g., during chronic use, are reflected in the labeling, and are not considered medically significant for the particular drug product being studied.

[0146] In one embodiment, two antigen binding proteins are bioequivalent if they have no clinically meaningful differences in their safety, purity, or potency.

[0147] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched, one or more times, between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy, compared to continuing treatment without such a switch.

[0148] In one embodiment, two antigen binding proteins are bioequivalent if they both act through one or more common mechanisms of action for one or more conditions of use, to the extent such mechanisms are known.

[0149] Bioequivalence can be supported by in vivo and / or in vitro methods. Measures of bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) in vitro studies that correlate with and are reasonably predictive of in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals that measure the relevant short-term pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability, or bioequivalence, of an antibody.

[0150] Biologically equivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that ablate or eliminate glycosylation.

[0151] Anti-LAG3 antibodies containing Fc variants In accordance with certain embodiments of the present invention, anti-LAG3 antibodies are provided that include an Fc domain that includes one or more mutations that enhance or diminish binding of the antibody to the FcRn receptor at acidic pH, e.g., as compared to neutral pH. For example, the present invention provides an anti-LAG3 antibody that includes an Fc domain that includes one or more mutations that enhance or diminish binding of the antibody to the FcRn receptor at acidic pH, as compared to neutral pH. H Within 2 regions or C HThe present invention also includes anti-LAG3 antibodies that contain mutations in the FcRn-3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., within endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F, or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include a 428L modification (e.g., M428L) and a 434S modification (e.g., N434S); a 428L modification, a 259I modification (e.g., V259I), and a 308F modification (e.g., V308F); a 433K modification (e.g., H433K), and a modification at position 434 (e.g., 434Y); modifications at positions 252, 254, and 256 (e.g., 252Y, 254T, and 256E); a 250Q modification and a 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modifications include a 265A modification (e.g., D265A) and / or a 297A modification (e.g., N297A).

[0152] For example, the present invention provides peptides including 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the invention includes an anti-LAG3 antibody comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the foregoing Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated to be within the scope of the invention.

[0153] The present invention also relates to chimeric heavy chain constant (C H ) region, in which case the chimeric C H The region is a C region of more than one immunoglobulin isotype. H For example, the antibodies of the present invention may include segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H Part or all of the C2 domain derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule; H C, combined with some or all of the 3 domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the invention may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an amino acid sequence of an "upper hinge" (amino acid residues from positions 216-227 according to EU numbering) from the hinge region of human IgG1, human IgG2, or human IgG4, combined with a "lower hinge" (amino acid residues from positions 228-236 according to EU numbering) from the hinge region of human IgG1, human IgG2, or human IgG4. According to certain embodiments, the chimeric hinge region comprises amino acid residues from the upper hinge of human IgG1 or human IgG4 and amino acid residues from the lower hinge of human IgG2. In certain embodiments, the chimeric hinge region may comprise an amino acid sequence of a chimeric hinge as described herein, H Antibodies comprising the region exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Patent Publication No. 20140243504, the disclosure of which is incorporated herein by reference in its entirety). In certain embodiments, the Fc region comprises a sequence selected from the group consisting of SEQ ID NOs: 569, 570, 571, 572, and 573.

[0154] Antibody Biology In general, the antibodies of the invention function by binding to LAG3. The invention includes anti-LAG3 antibodies and antigen-binding fragments thereof that bind with high affinity to soluble monomeric or dimeric LAG3 molecules. For example, the invention includes antibodies that bind with high affinity to soluble monomeric or dimeric LAG3 molecules, and have a K of less than about 10 nM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein. D and antibodies and antigen-binding fragments thereof that bind to monomeric LAG3 (e.g., at 25° C. or 37° C.). In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.04 nM, as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 3 herein, or a substantially similar assay. D and binds to monomeric LAG3.

[0155] The present invention also provides a method for the preparation of a medicament having a K of less than about 1.1 nM, as measured by surface plasmon resonance, for example using the assay format defined in Example 3 herein. D Also included are antibodies and antigen-binding fragments thereof that bind to dimeric LAG3 (e.g., at 25° C. or 37° C.) at 100° C. In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 0.5 nM, less than about 0.25 nM, less than about 0.1 nM, less than about 0.05 nM, or less than about 0.01 nM, as measured by surface plasmon resonance, e.g., using the assay format set forth in Example 3 herein, or a substantially similar assay. D and binds to dimeric LAG3.

[0156] The present invention also relates to a method for producing a medicament having a dissociation half-life (t ) of greater than about 1.6 minutes, as measured by surface plasmon resonance, at 25° C. or 37° C., using, for example, the assay format defined in Example 3 herein, or a substantially similar assay. 1 / 2 In certain embodiments, the antibodies or antigen-binding fragments of the invention have a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1100 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using an assay format set forth in Example 3 herein (e.g., mAb capture or antigen capture format), or a substantially similar assay. 1 / 2 and binds to LAG3.

[0157] The present invention also provides a method for treating cancer with an EC of less than about 8 nM as measured by the flow cytometry assay defined in Example 5 herein, or a substantially similar assay. 50 and binds to human LAG3-expressing cells. In certain embodiments, the antibodies or antigen-binding fragments thereof have an EC of less than about 5 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by flow cytometry assay, e.g., using the assay format set forth in Example 5 herein, or a substantially similar assay. 50 and binds to hLAG3-expressing cells.

[0158] The present invention also provides a compound having an EC of less than about 2.5 nM as measured by the flow cytometry assay defined in Example 5 herein, or a substantially similar assay. 50 and binds to cynomolgus monkey LAG3 expressing cells. In certain embodiments, the antibody or antigen-binding fragment thereof has an EC of less than about 2 nM, or less than about 1 nM, as measured by a flow cytometry assay, e.g., using the assay format set forth in Example 5 herein, or a substantially similar assay. 50 and binds to mfLAG3-expressing cells.

[0159] The present invention also relates to a compound having an IC of less than about 32 nM, as determined, for example, using the cell adhesion assay set forth in Example 7, or a substantially similar assay. 50 and also include antibodies or antigen-binding fragments thereof that block the binding of LAG3 to MHC class II (human HLA-DR2). In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC of less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by a cell adhesion assay, e.g., using the assay format set forth in Example 7 herein, or a substantially similar assay. 50 and blocks the binding of LAG3 to human MHC class II.

[0160] The present invention also relates to compounds having an IC of less than about 30 nM, as determined, for example, using the cell adhesion assay set forth in Example 7, or a substantially similar assay. 50 and also include antibodies or antigen-binding fragments thereof that block the binding of LAG3 to MHC class II (mouse HLA-DR2). In certain embodiments, the antibodies or antigen-binding fragments thereof have an IC of less than about 25 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, as measured by a cell adhesion assay, e.g., using the assay format set forth in Example 7 herein, or a substantially similar assay. 50 blocks the binding of mouse LAG3 to human MHC class II.

[0161] The present invention also includes antibodies or antigen-binding fragments thereof that block the binding of LAG3 to human MHC class II or mouse MHC class II by more than 90%, as measured by the cell adhesion assay defined in Example 7 herein, or a substantially similar assay.

[0162] The present invention also provides a method for treating cancer with an EC of less than 9 nM as measured by the T cell / APC luciferase reporter assay defined in Example 8 herein, or a substantially similar assay. 50 and blocks LAG-induced T cell downregulation. In certain embodiments, the antibodies or antigen-binding fragments thereof have an EC of less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM, as measured by a T cell / APC luciferase reporter assay, e.g., using the assay format set forth in Example 8 herein, or a substantially similar assay. 50 and blocks LAG3-induced T cell downregulation.

[0163] The present invention also provides a compound having an EC of less than about 1.2 nM as measured by the fluorescent assay defined in Example 9 herein, or a substantially similar assay. 50and binds to cynomolgus monkey activated CD4+ and CD8+ T cells. In certain embodiments, the antibodies or antigen-binding fragments thereof have an EC of less than about 1.1 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.2 nM, or less than about 0.1 nM, as measured by a fluorescent assay, e.g., using the assay format set forth in Example 9 herein, or a substantially similar assay. 50 and binds to activated cynomolgus monkey CD4+ and CD8+ T cells.

[0164] In certain embodiments, the antibodies of the present invention may function by blocking or inhibiting MHC class II binding activity associated with LAG3 through binding to any other region or fragment of the full-length protein whose amino acid sequence is set forth in SEQ ID NO:582.

[0165] In certain embodiments, the antibodies of the present invention, when administered prophylactically to a subject in need thereof, are useful for inhibiting tumor growth or delaying the progression of cancer, and may prolong the survival of the subject. For example, administration of the antibodies of the present invention may result in the shrinkage of a primary tumor and may prevent the development of metastases or secondary tumors. In certain embodiments, the antibodies of the present invention, when administered therapeutically to a subject in need thereof, are useful for inhibiting tumor growth and may prolong the survival of the subject. For example, administration of a therapeutically effective amount of the antibodies of the present invention to a subject may result in the shrinkage and disappearance of an established tumor in the subject.

[0166] In one embodiment, the present invention relates to an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that binds to LAG3, comprising the following features: (i) a monoclonal antibody or antigen-binding fragment thereof selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402, 418, 434, 450, 458, 466, 474, 482, 490, 498, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 55 (ii) comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, (iii) comprising an LCVR having an amino acid sequence selected from the group consisting of: 346, 362, 378, 394, 410, 426, 442, 522, 530, 546, and 562, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; , 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 464, 472, 480, 488, 496, 504, 512, 520, 544, and 560, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;and an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 528, 536, 552, and 568, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; Nos. 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 460, 468, 476, 484, 492, 500, 508, 516, 540, and 556, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. R1 domain; an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 462, 470, 478, 486, 494, 502, 510, 518, 542, and 558, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 524, 532, 548, and 564, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto;and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​398, 414, 430, 446, 526, 534, 550, and 566, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) a binding dissociation equilibrium constant (K) of less than about 10 nM as measured in a surface plasmon resonance assay at 25° C.; D ) to monomeric human LAG3; (vi) a K of less than about 8 nM as measured in a surface plasmon resonance assay at 37° C. D (vii) binds to monomeric human LAG3 with a K of less than about 1.1 nM as measured in a surface plasmon resonance assay at 25° C. D (viii) binds to dimeric human LAG3 with a K of less than about 1 nM as measured in a surface plasmon resonance assay at 37° C. D (ix) an EC50 of less than about 8 nM as measured in a flow cytometry assay; 50 (x) binds to hLAG3-expressing cells with an EC of less than about 2.3 nM as measured in a flow cytometry assay; 50 (xi) binds to mfLAG3-expressing cells with an IC of less than about 32 nM as determined by a cell adhesion assay; 50 (xii) blocks the binding of hLAG3 to human MHC class II with an IC of less than about 30 nM as determined by a cell adhesion assay; 50 (xiii) blocks hLAG3 binding to mouse MHC class II by greater than 90%, as determined by a cell adhesion assay; (xiv) has an EC of less than about 9 nM, as measured in a luciferase reporter assay. 50(xv) rescues LAG3-mediated inhibition of T cell activity with an EC of less than about 1.2 nM as measured in a fluorescent assay; 50 (xvi) inhibiting tumor growth and prolonging survival in a subject with cancer; and (xvii) being fully human.

[0167] The antibodies of the invention may have one or more of the aforementioned biological characteristics, or any combination thereof. Other biological characteristics of the antibodies of the invention will be apparent to those of skill in the art from a review of this disclosure, including the working examples herein.

[0168] Species selectivity and species cross-reactivity According to certain embodiments of the present invention, the anti-LAG3 antibody binds to human LAG3 but not to LAG3 from other species. Alternatively, in certain embodiments, the anti-LAG3 antibody of the present invention binds to human LAG3 and LAG3 from one or more non-human species. For example, the anti-LAG3 antibody of the present invention may bind to human LAG3 but may or may not bind to one or more of mouse LAG3, rat LAG3, guinea pig LAG3, hamster LAG3, gerbil LAG3, pig LAG3, cat LAG3, dog LAG3, rabbit LAG3, goat LAG3, sheep LAG3, cow LAG3, horse LAG3, camel LAG3, cynomolgus monkey LAG3, marmoset LAG3, rhesus monkey LAG3, or chimpanzee LAG3, as the case may be. In certain embodiments, an anti-LAG3 antibody of the invention binds to human LAG3 and cynomolgus monkey LAG3 with the same or different affinity, but does not bind to rat and mouse LAG3.

[0169] Epitope Mapping and Related Techniques The present invention includes anti-LAG3 antibodies that interact with one or more amino acids found within one or more domains of the LAG3 molecule, including, for example, the extracellular, transmembrane, and intracellular domains D1-D4. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located in any of the aforementioned domains of the LAG3 molecule (e.g., a linear epitope within the domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) (e.g., a conformational epitope) located in one or both of the aforementioned domains of the LAG3 molecule.

[0170] A variety of techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include those described, for example, in "Antibodies," Harlow and Lane (Coldwell, 1999). Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004), Methods Mol. Biol., 248:443-63), peptide truncation analysis, crystal structure studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000), Prot. Sci., 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange involves labeling a protein of interest with deuterium, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, where exchangeable protons in amino acids protected by the antibody complex undergo deuterium to hydrogen back-exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface are able to retain deuterium and therefore exhibit a relatively large mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, which reveals deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999), Analytical Biochemistry, 267:252-259; Engen and Smith (2001), Anal. Chem., 73:256A-265A.

[0171] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically contain at least 3 amino acids, and usually at least 5 or 8-10 amino acids, in a unique spatial conformation.

[0172] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method (see US2004 / 0101920, specifically incorporated herein by reference in its entirety) of categorizing multiple monoclonal antibodies (mAbs) directed to the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces. Each categorization may reflect a unique epitope that is significantly different or partially overlapping with the epitope represented by another categorization. This technique allows rapid filtering of genetically identical antibodies so that characterization can be focused on genetically significantly different antibodies. When applied to hybridoma screening, MAP facilitates the identification of rare hybridoma clones that produce mAbs with desired characteristics. MAP can be used to sort the antibodies of the present invention into groups of antibodies that bind to different epitopes.

[0173] In certain embodiments, the anti-LAG3 antibodies or antigen-binding fragments thereof bind to an epitope within any one or more of the exemplified regions within LAG3, including the naturally occurring epitope exemplified by SEQ ID NO:582, or the recombinantly produced epitopes exemplified by SEQ ID NOs:574-576, or fragments thereof. In some embodiments, the antibodies of the invention bind to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 29-450 of LAG3. In some embodiments, the antibodies of the invention bind to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 1-533 of cynomolgus LAG3, exemplified by SEQ ID NO:576.

[0174] In certain embodiments, the present invention includes anti-LAG3 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the extracellular region of LAG3 (SEQ ID NO:588). An epitope may consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within the extracellular region of LAG3. Alternatively, an epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the extracellular region of LAG3. As shown in Example 18 herein, the epitope of LAG3 with which H4sH15482P, an exemplary antibody of the invention, interacts is defined by LRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 589), an amino acid sequence corresponding to amino acids 28-71 of SEQ ID NO: 588. Thus, the invention includes anti-LAG3 antibodies that interact with one or more amino acids contained within the region consisting of amino acids 28-71 of SEQ ID NO: 588 (i.e., the sequence LRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY [SEQ ID NO: 589]).

[0175] The present invention includes anti-LAG3 antibodies that bind to the same epitope, or a portion of an epitope, as any of the specific exemplary antibodies described in Table 1 herein, or an antibody having the CDR sequence of any of the exemplary antibodies described in Table 1. Similarly, the present invention also includes anti-LAG3 antibodies that compete for binding to LAG3 or a LAG3 fragment with any of the specific exemplary antibodies described in Table 1 herein, or an antibody having the CDR sequence of any of the exemplary antibodies described in Table 1. For example, the invention includes anti-LAG3 antibodies that cross-compete with one or more of the antibodies defined in Example 4 herein (e.g., H4sH15482P, H4sH15479P, H4sH14813N, H4H14813N, H4H15479P, H4H15482P, H4H15483P, H4sH15498P, H4H15498P, H4H17828P2, H4H17819P, and H4H17823P) for binding to LAG3.

[0176] By using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference anti-LAG3 antibody or competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as a reference anti-LAG3 antibody of the present invention, the reference antibody is bound to a LAG3 protein or LAG3 peptide under saturating conditions. The test antibody is then evaluated for its ability to bind to a LAG3 molecule. If the test antibody is able to bind to LAG3 after saturation binding with the reference anti-LAG3 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-LAG3 antibody. On the other hand, if the test antibody is unable to bind to the LAG3 protein after saturation binding with the reference anti-LAG3 antibody, the test antibody may bind to the same epitope as the reference anti-LAG3 antibody of the present invention.

[0177] To determine whether an antibody competes with a reference anti-LAG3 antibody for binding, the binding method described above is carried out in two directions. In the first direction, the reference antibody is allowed to bind to LAG3 protein under saturating conditions, followed by evaluation of the binding of the test antibody to the LAG3 molecule. In the second direction, the test antibody is allowed to bind to LAG3 molecules under saturating conditions, followed by evaluation of the binding of the reference antibody to the LAG3 molecule. If only the first (saturating) antibody is capable of binding to the LAG3 molecule in both directions, it is concluded that the test antibody and the reference antibody compete for binding to LAG3. As will be appreciated by those skilled in the art, an antibody that competes with a reference antibody for binding may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0178] Two antibodies bind to the same epitope or overlapping epitopes if each antibody competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits at least 50%, but preferably 75%, 90%, or even 99% of the binding of the other antibody as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res., 1990, vol. 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody.

[0179] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) contributes to the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0180] Immunoconjugates The present invention encompasses human anti-LAG3 monoclonal antibodies conjugated to a therapeutic moiety such as a cytotoxin or chemotherapeutic agent ("immunoconjugates") to treat cancer. The term "immunoconjugate" as used herein refers to an antibody that is chemically or biologically linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide or protein, or therapeutic agent. The antibody can be linked to a cytotoxin, radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, peptide, or therapeutic agent at any position along the molecule, so long as it is capable of binding to its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent can be a second, different antibody against LAG3. In certain embodiments, the antibody can be conjugated to an agent specific for tumor cells or virus-infected cells. The type of therapeutic moiety that can be conjugated to the anti-LAG3 antibody will take into consideration the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0181] multispecific antibody The antibody of the present invention may be monospecific, bispecific, or multispecific. A multispecific antibody may be specific for different epitopes of one target polypeptide, or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol., 147:60-69; Kufer et al., 2004, Trends Biotechnol., 22:238-244.

[0182] In one aspect, the invention includes multispecific antigen-binding molecules or antigen-binding fragments thereof, where one specificity of the immunoglobulin is specific for the extracellular domain of LAG3 or a fragment thereof and another specificity of the immunoglobulin is specific for binding outside the extracellular domain of LAG3 or for binding to a second therapeutic target or due to conjugation to a therapeutic moiety.

[0183] The multispecific antigen-binding molecules of the present invention, or any of their variants, can be constructed using standard molecular biology techniques known to those skilled in the art, such as recombinant DNA and protein expression techniques.

[0184] In some embodiments, LAG3-specific antibodies are produced in a bispecific format ("bispecific") in which variable regions that bind distinct domains of LAG3 are linked together to confer dual domain specificity in a single binding molecule. Properly designed bispecifics can enhance overall LAG3 inhibitory efficacy by increasing both specificity and binding avidity. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain), or variable regions that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to simultaneously bind to a distinct epitope, or to different regions within one domain. In one example of a bispecific, a heavy chain variable region (V) from a binder with specificity for one domain is combined with a heavy chain variable region (V H) with a light chain variable region (V) derived from a series of binders with specificity for a second domain. L ) and rearrange it into V H Without destroying the original singularity of H A non-cognate V that can be paired with L In this way, a single V L Segment (e.g., V L 1) Two different V H Domain (e.g., V H 1 and V H 2) to form two binding "arms" (V H 1-V L 1 and V H 2-V L 1) can be used to create a bispecific antibody composed of a single V L The use of segments reduces the complexity of the system, thereby simplifying and increasing the efficiency of the cloning, expression, and purification steps used to generate bispecific agents (see, e.g., USSN13 / 022759 and US2010 / 0331527).

[0185] Alternatively, using the techniques described herein or other techniques known to those skilled in the art, antibodies that bind to more than one domain and a second target, such as, but not limited to, a second, different anti-LAG3 antibody, can be prepared in a bispecific format. Antibody variable regions that bind to significantly different regions can be linked together with variable regions that bind to a site of interest, such as the extracellular domain of LAG3, to confer dual antigen specificity to a single binding molecule. Properly designed bispecifics of this nature perform dual functions. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for outside the extracellular domain, and paired on a structural scaffold that allows each variable region to bind a separate antigen.

[0186] An exemplary bispecific antibody format that may be used in the context of the present invention is H3 domain and second Ig C H 3 domains, in this case the first Ig C H 3 domain and second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H The 3 domain binds to Protein A and the second Ig C H The C3 domain contains mutations that reduce or abolish binding to Protein A, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (by IMGT; Y436F by EU). H Further modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I in the case of IgG1 antibodies (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); N44S, K52N, and V82I in the case of IgG2 antibodies (N384S, K392N, and V422I by IMGT; EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I in the case of IgG4 antibodies (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variations on the bispecific antibody format described above are contemplated as being within the scope of the present invention.

[0187] Other exemplary bispecific formats that may be used in the context of the present invention include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, KIH (knobs-into-holes), common light chains (such as common light chains with KIH (knobs-into-holes)), crossed Mabs, crossed Fabs, (SEED)bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mabs. 2 Bispecific formats include (for a review of the aforementioned formats, see, e.g., Klein et al., 2012, mAbs, vol. 4:6, pp. 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates that self-assemble into multimeric complexes with defined composition, valency, and shape (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0188] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-LAG3 antibodies of the present invention or antigen-binding fragments thereof. The therapeutic compositions according to the present invention will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to improve entry, delivery, and tolerance. A large number of suitable formulations can be found in a formulary known to all medicinal chemists: "Remington's Pharmaceutical Sciences", Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA, (1998), J Pharm Sci Technol 52:238-311.

[0189] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, the condition, the route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is advantageous to administer the antibody of the present invention in a single dose, usually about 0.1 to about 60 mg per kg of body weight, more preferably about 5 to about 60, about 20 to about 50, about 10 to about 50, about 1 to about 10, or about 0.8 to about 11 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment of the present invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 10 to about 100 mg, or about 10 to about 50 mg. In certain embodiments, an initial dose can be followed by a second dose, or multiple subsequent doses of the antibody or antigen-binding fragment thereof, in an amount that can be about the same as or less than the amount of the initial dose, where the subsequent doses are separated by at least 1 to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0190] A variety of delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present invention (see, for example, Wu et al. (1987), J. Biol. Chem., vol. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered via any convenient route, such as infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), and can be administered in conjunction with other bioactive agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see, eg, Langer (1990) Science 249:1527-1533).

[0191] The use of nanoparticles to deliver the antibodies of the present invention is also envisioned herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and preparation and use methods are described in detail in Arruebo, M. et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications", J. Nanomat., Vol. 2009, Literature ID: 439389, p. 24, doi:10.1155 / 2009 / 439389), which are incorporated herein by reference. Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target tumor cells or autoimmune tissue cells or virus-infected cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995, each of which is incorporated herein in its entirety.

[0192] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0193] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intracranial injection, intraperitoneal injection, intramuscular injection, drip infusion, and the like. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by dissolving, suspending, or emulsifying, for example, the above-described antibody or its salt in a sterile aqueous medium or a sterile oily medium that is conventionally used for injection. The aqueous medium for injection includes, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents that can be used in combination with suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. The oily medium includes, for example, sesame oil, soybean oil, and the like that can be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. The injection thus prepared is preferably filled into an appropriate ampoule.

[0194] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously by a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is also easily applied to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. In this way, the pen delivery device can be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir in the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0195] A number of reusable pen and autoinjector delivery devices have application for the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical), to name a few. Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices having application in subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name a few.

[0196] The above-described pharmaceutical compositions for oral or parenteral use are advantageously prepared into a dosage form of a unit dose suitable for the dose of the active ingredient. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and in particular, in the form of an injection, the antibody is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0197] Therapeutic Uses of Antibodies The antibodies of the present invention are useful for treating, preventing, and / or ameliorating, inter alia, any disease or disorder associated with or mediated by LAG3 expression, signaling, or activity, or treatable by blocking the interaction of LAG3 with MHC class II, the ligand of LAG3, or by otherwise inhibiting LAG3 activity and / or signaling. For example, the present invention provides methods for treating cancer (inhibiting tumor growth) and / or treating viral infections by administering the anti-LAG3 antibodies (or pharmaceutical compositions comprising anti-LAG3 antibodies) described herein to patients in need of such treatment, as well as anti-LAG3 antibodies (or pharmaceutical compositions comprising anti-LAG3 antibodies) for use in treating cancer (inhibiting tumor growth) and / or treating viral infections. The antibodies of the present invention are useful for treating, preventing, and / or ameliorating a disease or disorder or condition, such as cancer or viral infection, and / or ameliorating at least one symptom associated with such a disease, disorder, or condition. In the context of the treatment methods described herein, the anti-LAG3 antibody can be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0198] In some embodiments of the invention, the antibodies described herein are useful for treating subjects suffering from primary or recurrent cancers, including, but not limited to, blood cancers, brain cancer (e.g., glioblastoma multiforme), renal cell carcinoma (e.g., clear cell renal carcinoma), ovarian cancer, bladder cancer, prostate cancer, breast cancer (e.g., triple-negative breast cancer), hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, and melanoma.

[0199] The term "hematological cancer" as used herein includes hematological malignancies affecting blood, bone marrow, lymph, or lymphatic system. As such, the term includes malignancies of cells derived from lymphoid and myeloid lineages. Myeloid lineages usually produce granulocytes, erythrocytes, platelets, macrophages, mast cells, while lymphoid lineages produce B cells, T cells, NK cells, plasma cells. Thus, the term includes the above-mentioned cellular malignancies, i.e., lymphoma, myeloma, lymphocytic leukemia, and myeloid leukemia. Examples include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma), and myeloma (including multiple myeloma).

[0200] The antibodies can be used to treat early or late stage symptoms of cancer. In one embodiment, the antibodies or fragments thereof of the present invention can be used to treat advanced or metastatic cancer. The antibodies are useful for reducing or inhibiting or shrinking the growth of tumors, both solid and blood cancers. In certain embodiments, treatment with the antibodies or antigen-binding fragments thereof of the present invention results in greater than 40% regression, greater than 50% regression, greater than 60% regression, greater than 70% regression, greater than 80% regression, or greater than 90% regression of tumors in a subject. In certain embodiments, the antibodies can be used to prevent tumor recurrence. In certain embodiments, the antibodies are useful for extending progression-free or overall survival in subjects with cancer. In some embodiments, the antibodies are useful for reducing toxicity resulting from chemotherapy or radiation therapy while maintaining long-term survival in patients with cancer.

[0201] In certain embodiments, the subject is a patient suffering from cancer, Patients who have not previously been treated with anti-PD-1 / PD-L1 therapy but are suitable candidates for anti-PD-1-based therapy; and / or Patients who have previously received anti-PD-1 / PD-L1-based therapy and have had an objective response (CR or PR) or SD for at least 3 months on anti-PD-1 / PD-L1 therapy, but have subsequently progressed on that treatment, or have had SD or PR as their best response followed by a stable response for 6 months; and / or Patients who are not candidates for standard of care or who have no available treatments predicted to provide clinical benefit and who are suitable for mAb1 monotherapy; and / or Patients with stage IIIB or IV NSCLC who have not undergone anti-PD-1 / PD-L1 therapy and who have no prior treatment for metastatic disease or who have disease progression / recurrence after one platinum-containing regimen; and / or Patients with stage IIIB or IV NSCLC who have received anti-PD-1 / PD-L1 therapy and have not received more than two prior therapies for metastatic disease; and / or Patients with advanced or metastatic ccRCC with a clear cell component who have not undergone anti-PD-1 / PD-L1 therapy and who have received one or two prior regimens of anti-angiogenic therapy; and / or - Anti-PD-1 / PD-L1 * Patients with advanced or metastatic ccRCC with a clear cell component who have received one or two prior regimens of antiangiogenic therapy; and / or Patients with metastatic TNBC (estrogen, progesterone, and human epidermal growth factor receptor 2 negative) who have not undergone anti-PD-1 / PD-L1 and have received five or fewer prior lines of therapy; and / or Patients with advanced or metastatic melanoma who have not undergone anti-PD-1 / PD-L1 therapy and who have received no more than two prior regimens for metastatic disease; and / or Patients with advanced or metastatic melanoma who have received anti-PD-1 / PD-L1 and have received no more than two prior regimens for metastatic disease; and / or Patients with relapsed / refractory DLBCL who have not undergone anti-PD-1 / PD-L1 and who have progressed after or are not candidates for autologous stem cell transplantation; and / or - Anti-PD-1 / PD-L1 * Patients with relapsed / refractory DLBCL who have progressed after or are not candidates for autologous stem cell transplantation It is.

[0202] In certain embodiments, the antibodies of the present invention are useful for treating subjects suffering from chronic viral infections. In some embodiments, the antibodies of the present invention are useful for reducing viral titers in a host and / or rescuing exhausted T cells. In certain embodiments, the antibodies of the present invention or fragments thereof can be used to treat chronic viral infections with lymphocytic choriomeningitis virus (LCMV). In some embodiments, the antibodies of the present invention or antigen-binding fragments thereof can be administered in therapeutic doses to patients infected with human immunodeficiency virus (HIV) or human papillomavirus (HPV) or hepatitis B virus / hepatitis C virus (HBV / HCV). In related embodiments, the antibodies of the present invention or antigen-binding fragments thereof can be used to treat infections with simian immunodeficiency virus (SIV) in monkey subjects, such as cynomolgus monkeys.

[0203] In certain embodiments, the blocking antibodies of the invention can be administered in a therapeutically effective amount to a subject suffering from cancer or a viral infection.

[0204] One or more antibodies of the invention can be administered to alleviate, prevent, or reduce the severity of one or more symptoms or conditions of a disease or disorder.

[0205] Also contemplated herein is the prophylactic use of one or more antibodies of the invention in patients at risk of developing a disease or disorder, such as cancer and viral infections.

[0206] In a further embodiment of the invention, the antibody is used to prepare a pharmaceutical composition for treating a patient suffering from cancer or a viral infection. In another embodiment of the invention, the antibody is used as an adjunct therapy with any other drug or any other therapy known to those skilled in the art to be useful in treating cancer or a viral infection.

[0207] Combination Therapies and Combination Preparations The combination therapy may include an anti-LAG3 antibody of the invention and any additional therapeutic agent that may be advantageously combined with the antibody of the invention, or a biologically active fragment of the antibody of the invention.

[0208] The antibody of the present invention can be synergistically combined with one or more anti-cancer drugs or anti-cancer therapies used to treat or inhibit cancer, including, for example, blood cancer, brain cancer (e.g., glioblastoma multiforme), renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, colorectal cancer, mesothelioma, and melanoma. It is envisioned herein that the anti-LAG3 antibody of the present invention can be used in combination with immune stimulatory and / or immune supportive therapy to inhibit tumor growth and / or prolong the survival of cancer patients. Immune stimulatory therapy includes direct immune stimulatory therapy that enhances immune cell activity by "releasing the brakes" on suppressed immune cells or "stepping on the gas" to activate immune responses. Examples include targeting other checkpoint receptors, vaccination, and adjuvants. Immune supportive modalities may increase tumor antigenicity by promoting immunogenic cell death, inflammation, or have other indirect effects that promote anti-tumor immune responses. Examples include radiation, chemotherapy, anti-angiogenic agents, and surgery.

[0209] In various embodiments, one or more of the antibodies of the invention are administered in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody such as nivolumab, pembrolizumab, pidilizumab, BGB-A317, or REGN2810), a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody such as avelumab, atezolizumab, durvalumab, MDX-1105, or REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, another T cell co-inhibitor, or an antagonist of a T cell ligand. Antibodies against CD28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept, "VEGF-Trap," or other VEGF inhibitory fusion proteins as specified in US 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, , sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate-specific antigen (PSA), mucin 1, MART-1, and CA19-9], vaccines (e.g., Calmette-Gaesthesia oryzae vaccines, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3×CD20 bispecific antibody, or PSMA×CD3 bispecific antibody), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy,It can be used in combination with IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADCs, and anti-DS6-DM4 ADCs), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or any other therapeutic care to treat cancer. In certain embodiments, the anti-LAG3 antibodies of the present invention can be used in combination with cancer vaccines, including dendritic cell vaccines, oncolytic viruses, tumor cell vaccines, and the like, to enhance anti-tumor responses. Examples of cancer vaccines that can be used in combination with the anti-LAG3 antibodies of the present invention include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., Rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers). ,

[0210] In certain embodiments, the anti-LAG3 antibody of the present invention can be administered in combination with radiation therapy in a manner that generates long-term and durable anti-tumor responses and / or enhances the survival of patients with cancer. In some embodiments, the anti-LAG3 antibody of the present invention can be administered before, concomitantly with, or after radiation therapy is administered to cancer patients. For example, radiation therapy can be administered to tumor lesions in one or more doses, followed by administration of one or more doses of the anti-LAG3 antibody of the present invention. In some embodiments, radiation therapy can be administered locally to tumor lesions to enhance the local immunogenicity of the patient's tumor (adjuvinating radiation) and / or to kill tumor cells (ablative radiation), followed by systemic administration of the anti-LAG3 antibody of the present invention. For example, intracranial radiation can be administered to patients with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of the anti-LAG3 antibody of the present invention. In certain embodiments, the anti-LAG3 antibodies of the invention may be administered in combination with radiation therapy and a chemotherapeutic agent (eg, temozolomide) or a VEGF antagonist (eg, aflibercept).

[0211] In certain embodiments, the anti-LAG3 antibody of the present invention can be administered in combination with one or more antiviral drugs to treat chronic viral infections caused by LCMV, HIV, HPV, HBV, or HCV. Examples of antiviral drugs include, but are not limited to, zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine, and corticosteroids.

[0212] The additional therapeutically active agent / component may be administered prior to, concurrently with, or subsequent to the anti-LAG3 antibody of the present invention. For purposes of this disclosure, such administration regimes are considered administration of the anti-LAG3 antibody "in combination with" the second therapeutically active ingredient.

[0213] The additional therapeutic active ingredient can be administered to the subject before the administration of the anti-LAG3 antibody of the present invention.For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before the administration of the second component, the first component can be considered to be administered "before" the second component.In other embodiments, the additional therapeutic active ingredient can be administered to the subject after the administration of the anti-LAG3 antibody of the present invention. For example, a first component can be considered to be administered "after" a second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours after administration of the second component. In yet other embodiments, a further therapeutically active component can be administered to a subject contemporaneously with administration of an anti-LAG3 antibody of the invention. "Contemporaneous" administration for purposes of the present invention includes, for example, administration of an anti-LAG3 antibody and a further therapeutically active component to a subject in a single dosage form (e.g., a co-formulated dosage form) or in separate dosage forms that are administered to a subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-LAG3 antibody and the additional therapeutically active ingredient may be administered intravenously, subcutaneously, etc.), or alternatively, each dosage form may be administered via a different route (e.g., the anti-LAG3 antibody may be administered intravenously and the additional therapeutically active ingredient may be administered subcutaneously). In any case, whether the ingredients are administered in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes, all are considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-LAG3 antibody "before," "concurrent with," or "after" administration of an additional therapeutically active ingredient (as these terms are defined herein above) is considered administration of the anti-LAG3 antibody "in combination with" the additional therapeutically active ingredient.

[0214] The present invention includes pharmaceutical compositions in which the anti-LAG3 antibodies of the present invention are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein, using various dosage combinations.

[0215] In exemplary embodiments in which an anti-LAG3 antibody of the invention is administered in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody disclosed in US2015 / 0203579, which is incorporated herein by reference in its entirety), including administration of a co-formulation comprising an anti-LAG3 antibody and a PD-1 inhibitor, the individual components can be administered to a subject and / or can be co-formulated using various dosage combinations. Thus, the invention includes combinations of (i) an anti-LAG3 antibody of the invention and (ii) a PD-1 inhibitor (e.g., an anti-PD-1 antibody disclosed in US2015 / 0203579, which is incorporated herein by reference in its entirety) for simultaneous, separate, and / or sequential use in the treatment of cancer or viral infection. For example, each of the anti-LAG3 antibody and the PD-1 inhibitor (e.g., an anti-PD-1 antibody) can be administered to a subject and / or included in a co-formulation in an amount selected from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg. The combination / co-formulation can be administered to a subject according to any of the dosing regimens disclosed elsewhere herein, including, for example, twice weekly, once weekly, once every other week, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, once every six months, etc. The anti-LAG3 antibodies of the invention could be administered, for example, at a dose of about 0.8 to about 11, about 1 to about 10, about 3 to about 10, about 1, about 3, or about 10 mg / kg, co-administered with a PD-1 inhibitor (e.g., an anti-PD-1 antibody disclosed in US2015 / 0203579) at a dose of about 3 to 5, or about 3.0 mg / kg. Co-administration could occur, for example, every 14 days, every 21 days, or every 28 days.

[0216] In exemplary embodiments in which an anti-LAG3 antibody of the present invention is administered in combination with a VEGF antagonist (e.g., a VEGF trap such as aflibercept), including administration of a co-formulation comprising an anti-LAG3 antibody and a VEGF antagonist, the individual components can be administered to a subject and / or can be co-formulated using various dosage combinations. For example, an anti-LAG3 antibody may be administered to a subject and / or included in a co-formulation in an amount selected from the group consisting of 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg, and 10.0 mg, and a VEGF antagonist (e.g., A VEGF trap such as aflibercept) may be administered to a subject and / or included in a co-formulation in an amount selected from the group consisting of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, and 3.0 mg. The combination / co-formulations can be administered to a subject according to any of the dosing regimens disclosed elsewhere herein, including, for example, twice weekly, once weekly, once every other week, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, once every six months, etc.

[0217] Dosing regimen According to certain embodiments of the invention, multiple doses of anti-LAG3 antibody (or pharmaceutical composition comprising a combination of anti-LAG3 antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a defined time course. The method according to this aspect of the invention comprises sequentially administering multiple doses of the anti-LAG3 antibody of the invention to the subject. As used herein, "sequentially administering" means that each dose of anti-LAG3 antibody is administered to the subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient a single initial dose of anti-LAG3 antibody, followed by one or more secondary doses of anti-LAG3 antibody, optionally followed by one or more tertiary doses of anti-LAG3 antibody. The anti-LAG3 antibody can be administered at a dose between 0.1 mg and 100 mg per kg of the subject's body weight.

[0218] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the anti-LAG3 antibody of the present invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-LAG3 antibody, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-LAG3 antibody contained in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., adjusted up or down as necessary). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5 doses) are administered at the beginning of a treatment regimen as a "loading dose", followed by subsequent doses (e.g., "maintenance doses") at a lower frequency.

[0219] In certain embodiments, the amount of anti-LAG3 antibody contained in the initial dose, secondary dose, and / or tertiary dose may be suboptimal or subtherapeutic. As used herein, the term "subtherapeutic" or "suboptimal" refers to a dose of antibody administered at a level that is too low to produce a therapeutic effect or below the level required to treat a disease such as cancer.

[0220] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 weeks or more) after the immediately preceding administration. As used herein, the phrase "the immediately preceding dose" refers to a dose of an anti-LAG3 antibody in a multiple administration sequence that is administered to a patient prior to the immediately subsequent dose in a sequence in which there are no intervening doses.

[0221] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of anti-LAG3 antibody to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0222] In embodiments involving multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose can be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the invention, the frequency with which the secondary and / or tertiary doses are administered to the patient can vary over the course of the treatment regimen. The frequency of administration can also be adjusted by the physician during the course of treatment according to the needs of the individual patient following clinical testing.

[0223] Diagnostic Uses of Antibodies The anti-LAG3 antibodies of the present invention can be used to detect and / or measure LAG3 in a sample, for example, for diagnostic purposes. Some embodiments contemplate the use of one or more of the antibodies of the present invention in an assay for detecting a disease or disorder, such as cancer, an autoimmune disease, or a viral infection. An exemplary diagnostic assay for LAG3 can include, for example, contacting a sample obtained from a subject (e.g., a patient) with an anti-LAG3 antibody of the present invention, where the anti-LAG3 antibody is labeled with a detectable label or reporter molecule, or is used as a capture ligand to selectively isolate LAG3 from the subject sample. Alternatively, an unlabeled anti-LAG3 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125The LAG3 may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that may be used to detect or measure LAG3 in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence-activated cell sorting (FACS).

[0224] The sample that can be used in the LAG3 diagnostic assay according to the present invention includes any tissue or body fluid sample obtained from a subject, which contains a detectable amount of LAG3 protein or its fragment under normal or pathological conditions.Generally, the LAG3 level in a particular sample obtained from a healthy patient (e.g., a patient not suffering from cancer or autoimmune disease) is measured to first establish a baseline LAG3 level or standard LAG3 level.This baseline LAG3 level can then be compared with the LAG3 level measured in a sample obtained from an individual suspected of having a cancer-related condition or a symptom associated with such a condition.

[0225] The antibody specific for LAG3 may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it is bound. For example, when the surface is coated with avidin, a peptide containing an N-terminal biotin is oriented so that the C-terminal portion of the peptide is distal to the surface.

[0226] Aspects of the invention relate to the use of the disclosed antibodies as markers for predicting the prognosis of cancer or viral infection in a patient. The antibodies of the invention can be used in diagnostic assays to assess the prognosis and predict survival of cancer in a patient. EXAMPLES

[0227] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0228] (Example 1: Production of human antibodies against LAG3) Human antibodies against LAG3 were generated using a fragment of LAG3 from approximately amino acids 29-450 of GenBank Accession No. NP_002277.4 (SEQ ID NO:582) coupled to a mouse Fc region. The immunogen was administered directly, together with an adjuvant to stimulate an immune response, to VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the heavy and kappa light chain variable regions of human immunoglobulins) described in US8502018B2 or humanized Universal Light Chain (ULC) VelocImmune® mice described in WO2013022782. Antibody immune responses were monitored by LAG3-specific immunoassays. Once the desired immune response was achieved, spleen cells were harvested, their viability preserved, and fused with mouse myeloma cells to form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing LAG3-specific antibodies. Using this technique and the immunogens described above, several anti-LAG3 chimeric antibodies (i.e., antibodies possessing a human variable domain and a mouse constant domain) were obtained; thus, exemplary antibodies generated from VELOCIMMUNE® mice were named H1M14985N, H1M14987N, H2M14811N, H2M14885N, H2M14926N, H2M14927N, H2M14931N, H2M18336N, H2M18337N, and H4H14813N.

[0229] Anti-LAG3 antibodies were also isolated directly from antigen-positive B cells (from any of the immunized mice) without fusion to myeloma cells, as described in U.S. Patent No. 7,582,298, which is specifically incorporated herein by reference in its entirety. Using this method, several fully human anti-LAG3 antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained, and exemplary antibodies generated in this manner are as follows: H4H15477P, H4H15483P, H4H15484P, H4H15491P, H4H17823P, H4H17826P2, H4H17828P2, H4sH15460P, H4sH15462P, H4sH15463P, H4sH15464P, H4sH15466P, H4sH15468P, H4sH15469P, H4sH15476P, H4sH15478P, H4sH15479 ... The antibodies were named as follows: H4sH15467P, H4sH15470P, H4sH15475P, H4sH15479P, H4sH15480P, H4sH15482P, H4sH15488P, H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, H4sH15567P2, and H4H17819P.

[0230] Exemplary antibodies H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, and H4sH15567P2 were generated from B cells derived from ULC Velocimmune® mice.

[0231] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0232] Example 2: Amino acid and nucleotide sequences of heavy and light chain variable regions Table 1 sets forth amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-LAG3 antibodies of the invention. Corresponding nucleic acid sequence identifiers are set forth in Table 2.

Table 1-1

Table 1-2

Table 2-1

Table 2-2

[0233] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H1M", "H4sH", "H4H", etc.), followed by a numerical identifier (e.g., "14813", "17828", etc., as shown in Table 1), followed by a "P", "P2", or "N" suffix. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H4sH14813N", "H4H17819P", "H4H17828P2", etc. The H4sH and H4H prefixes in the designation of antibodies used herein indicate the isotype of the particular Fc region of the antibody. For example, an "H4sH" antibody has a human IgG4 Fc with two or more amino acid changes as disclosed in US20140243504 (incorporated herein in its entirety), an "H4H" antibody has a human IgG4 Fc with a serine to proline mutation (S108P) in the hinge region, an "H1M" antibody has a murine IgG1 Fc, and an "H2M" antibody has a murine IgG2 Fc (all variable regions are fully human, as indicated by the first "H" in the antibody designation). As will be appreciated by those skilled in the art, an antibody with a particular Fc isotype can be converted into an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted into an antibody with a human IgG4, etc.), but in any case, the variable domains (including the CDRs) (shown by the numerical identifiers in Table 1) will remain the same, and the antigen binding characteristics are expected to be the same or substantially similar, regardless of the nature of the Fc domain.

[0234] In certain embodiments, the selected antibodies with mouse IgG1 Fc were converted to antibodies with human IgG4 Fc. In certain embodiments, the antibodies comprise human IgG4 Fc with two or more amino acid changes as disclosed in US20100331527 (incorporated herein in its entirety). In one embodiment, the IgG4 Fc domain comprises a serine to proline mutation (S108P) in the hinge region to promote dimer stabilization. In certain embodiments, the Fc region of the antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 569, 570, 571, 572, or 573. Table 3 sets forth the amino acid sequence identifiers of the heavy and light chain sequences of selected anti-LAG3 antibodies with human IgG4 Fc. [Table 3]

[0235] Each heavy chain sequence in Table 3 is H or HCVR; including HCDR1, HCDR2, and HCDR3) and a constant region (C H 1 domain, C H 2 domain, and C H Each light chain sequence in Table 3 contained a variable region (V L or LCVR; containing LCDR1, LCDR2, and LCDR3) and a constant region (C L ). SEQ ID NO:577 contained an HCVR comprising amino acids 1 to 123 and a constant region comprising amino acids 124 to 449. SEQ ID NO:578 contained an LCVR comprising amino acids 1 to 107 and a constant region comprising amino acids 108 to 214. SEQ ID NO:579 contained an HCVR comprising amino acids 1 to 123 and a constant region comprising amino acids 124 to 450. SEQ ID NO:580 contained an HCVR comprising amino acids 1 to 119 and a constant region comprising amino acids 120 to 445. SEQ ID NO:581 contained an LCVR comprising amino acids 1 to 107 and a constant region comprising amino acids 108 to 214.

[0236] Control constructs used in the following examples For comparison purposes, the following experiments include two control constructs (anti-LAG3 antibodies): "Comparator 1": V of antibody "25F7" according to WO2010 / 019570 H / V L and "Control 2": a human monoclonal antibody against LAG3 having the sequence VIII of antibody "v3.5" according to US2014 / 0093511. H / V L A human monoclonal antibody against LAG3 having the sequence

[0237] Example 3: Binding of antibodies to LAG3 as determined by surface plasmon resonance The association and dissociation rate constants (k a and k d ), equilibrium dissociation constant and dissociation half-life (K D and t 1 / 2) was determined using a real-time surface plasmon resonance biosensor assay on a BiaCore 4000 instrument or a BiaCore T200 instrument. The BiaCore sensor surface was derivatized with rabbit anti-mouse polyclonal antibody (GE; model no. BR-1008-38) or mouse anti-human Fc monoclonal antibody (GE; model no. BR-1008-39) to capture approximately 50-85 RU of anti-LAG3 monoclonal antibody expressed with mouse or human Fc, respectively. LAG3 reagents tested for binding to anti-LAG3 antibodies included recombinant human LAG3 expressed with a C-terminal myc-myc-hexahistidine tag (hLAG3-mmh; SEQ ID NO: 574) and recombinant human LAG3 expressed with a C-terminal mouse IgG2a mFc tag (hLAG3-mFc; SEQ ID NO: 575). Different concentrations (50, 25, 12.5, and 6.25 nM) of LAG3 reagent were injected over the anti-LAG3 monoclonal antibody captured surface at a flow rate of 50 μL / min on a BiaCore T200. Binding of LAG3 reagent to the captured monoclonal antibodies was monitored over 4 min and dissociation from the antibodies was monitored over 8 min in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20). Experiments were performed at 25° C. and 37° C. The kinetic association rate constant (k a ) and the dissociation rate constant (k d The binding dissociation equilibrium constant (K) was then determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t 1 / 2 ) to K D (M)=k d / k a and t 1 / 2 (min)=[ln2 / (60 * k d ) was calculated from the kinetic rate constant.

[0238] The binding kinetic parameters for binding of different anti-LAG3 monoclonal antibodies to monomeric (mmH tagged) or dimeric (mFc tagged) LAG3 reagents at 25°C and 37°C are shown in Tables 4-7. [Table 4]

[0239] Table 4 shows that all anti-LAG3 antibodies of the present invention have a K in the range of 49 pM to 10 nM at 25°C. D values ​​of 0.58 nM and 0.8 nM, whereas the comparator drugs bound to hLAG3-mmh D Indicates that the join was made by value. [Table 5]

[0240] Table 5 shows that all anti-LAG3 antibodies of the present invention have a K in the range of 32 pM to 7.66 nM at 37°C. D values, whereas the comparator drugs bound to hLAG3-mmh with K values ​​of 2.1 nM and 3.0 nM. D Indicates that the join was made by value.

[0241] The data in Tables 4 and 5 indicate that all anti-LAG3 antibodies bind to monomeric hLAG3-mmh with very similar affinities at both 25°C and 37°C. [Table 6]

[0242] Table 6 shows that all anti-LAG3 antibodies of the present invention have a K in the range of 4.3 pM to 1.0 nM at 25°C. D values, whereas the comparator drugs bound to the hLAG3 dimeric protein with K values ​​of 97 pM and 0.16 nM. D Indicates that the join was made by value. [Table 7]

[0243] Table 7 shows that all anti-LAG3 antibodies of the present invention have a K in the range of 4.0 pM to 0.9 nM at 37°C. D values, whereas the comparator drugs bound to the hLAG3 dimeric protein with K values ​​of 0.26 nM and 0.45 nM. D Indicates that the join was made by value.

[0244] The data in Tables 6 and 7 indicate that all anti-LAG3 antibodies bind with similar affinity to the hLAG3 dimer reagent at both 25°C and 37°C.

[0245] Example 4: Cross-competition between anti-LAG3 antibodies by Octet To assess whether two antibodies compete with each other for binding to monomeric human LAG3 (hLAG3.mmh), binding competition between anti-LAG3 monoclonal antibodies was determined using a real-time label-free biolayer interference assay on an Octet RED384 biosensor (Pall ForteBio Corp.). Cross-competition experiments were performed at 25° C. in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant Tween-20, 0.1 mg / mL BSA (Octet HBS-P buffer) with plate shaking at a speed of 1000 rpm. All tested anti-LAG3 antibodies and tested hLAG3 solutions were formulated in Octet HBS-P buffer. To assess whether the two antibodies could compete with each other for binding to hLAG3, about 0.6-0.85 nM hLAG3.mmh was first captured from wells containing 5 μg / mL hLAG3 onto an anti-His coated Octet biosensor chip for 75 seconds. The Octet biosensor chip with captured hLAG3 was saturated by immersing it in a well containing 50 ug / ml of a first anti-LAG3 monoclonal antibody (referred to as mAb-1 in this example) for 5 minutes, followed by immersion in a well containing a second anti-LAG3 monoclonal antibody (referred to as mAb-2 in this example). Between each step, the Octet biosensor chip was washed in HBS-P buffer for 30 seconds.

[0246] Real-time binding responses were monitored during the course of the experiment and the binding responses at the end of every step were recorded. The binding responses of hLAG3 to mAb-1 and then to the blocking mAb were corrected for background binding and compared to determine the competitive / non-competitive behavior of the different anti-Lag monoclonal antibodies.

[0247] Table 8 clearly defines the relevance of competing antibodies in either orientation, independent of the order of binding. [Table 8]

[0248] Example 5: Binding of antibodies to cells overexpressing LAG3 as determined by flow cytometry HEK293 cells (HEK293; ATCC number: CRL-1573), a stable cell line expressing recombinant hLAG3 (NCBI accession number: NP_002277.4) or monkey LAG3 (mfLAG3) [based on the rhesus monkey (Macaca mulata) sequence: NCBI accession number: XP_001108923.1, with further modification of the cynomolgus monkey sequence: NCBI accession number: XP_005570011.1 to replace the "X" at amino acid position 74 with a proline] (SEQ ID NO: 576) on the cell surface, were developed and used to determine the binding specificity of the anti-LAG3 monoclonal antibodies of the present invention by flow cytometry analysis.

[0249] Anti-LAG3 antibody binding was assessed as follows: stable HEK293 cells expressing hLAG3 or mfLAG3 were washed once with D-PBS (Irvine Scientific product no. 9240), trypsinized (Specialty Media product no. SM-2004-C), and blocked with HEK293 cell culture medium (DME + 10% FBS + P / S / G + non-essential amino acids). After centrifugation, cells were cultured at 2.0 x 10 cells per mL in staining buffer (D-PBS + 2% FBS). 6Anti-LAG3 antibodies and isotype controls, including a no antibody and buffer only control, were serially diluted in staining buffer at doses ranging from 5 pM to 100 nM. Serially diluted antibodies were added to the cell suspension and incubated on ice for 15-30 minutes. Cells were centrifuged and the pellet washed with staining buffer to remove unbound antibody. Cells were then incubated with allophycocyanin-conjugated secondary F(ab')2 that recognizes human Fc (Jackson ImmunoResearch; product no. 109-136-170) for 15-30 minutes on ice. Cells were centrifuged, the pellet washed with staining buffer to remove unbound secondary F(ab')2, and fixed overnight in a 1:1 dilution of Cytofix (BD Biosciences; product no. 554655) and staining buffer. The following day, the fixed cells were centrifuged and the pellet washed with staining buffer, resuspended, and filtered. Fluorescence measurements were collected on a HyperCyt® cytometer and analyzed by ForeCyt™ (IntelliCyt; Albuquerque, NM) to determine mean fluorescence intensity (MFI). 50 Values ​​were calculated from a four-parameter logistic equation for 11-point response curves using GraphPad Prism. EC 50 was defined as the antibody concentration at which 50% of the maximum binding signal was detected. The MFI ratio for each antibody was calculated by dividing the MFI at an antibody concentration of 100 nM by the MFI at an antibody concentration of 0 nM. [Table 9]

[0250] As shown in Table 9, the comparators and isotype controls did not demonstrate any binding to wild-type parental HEK293 cells as measured by FACS analysis. The calculated MFI ratios ranged between 1.0-1.6x. Few anti-LAG3 antibodies showed large ratios on wild-type cells, suggesting non-specific binding, with calculated MFI ratios ranging between 0.9-8.2x. Under these staining conditions, the EC 50 The value was not determined. [Table 10]

[0251] As shown in Table 10, the isotype control did not demonstrate any measurable binding to HEK293 / hLag3 cells. All 15 anti-LAG3 antibodies of the invention had EC values ​​ranging from 300 pM to 7.9 nM. 50 The calculated MFI ratios ranged from 62 to 414-fold. 50 Values ​​were 0.65 nM and 0.75 nM in the binding assay, and MFI ratios were less than 200-fold for either antibody. [Table 11]

[0252] As shown in Table 11, the isotype control did not demonstrate any measurable binding to HEK293 / mfLag3 cells. A total of 13 of the 15 anti-LAG3 antibodies of the invention had EC values ​​ranging from 900 pM to over 10 nM. 50 The calculated MFI ratios ranged from 8.8-fold to 21.5-fold. 50 Values ​​were above 10 nM with calculated MFI ratios of 6.7-fold and 8.6-fold.

[0253] Example 6: Binding of antibodies to cells overexpressing LAG3 as determined by electrochemiluminescence immunoassay. To examine the ability of a panel of anti-LAG3 monoclonal antibodies to bind to LAG3 expressed on the cell surface, we developed an in vitro binding assay utilizing cell lines expressing human and monkey LAG3 within an electrochemiluminescence-based detection platform [Meso Scale Diagnostics, Rockville, MD; MSD].

[0254] Stable HEK293 (ATCC number: CRL-1573) cell lines were generated by lentiviral transduction expressing recombinant hLAG3 (NCBI accession number: NP_002277.4) or monkey LAG3 (mfLAG3) (SEQ ID NO: 576) [based on the rhesus monkey (Macaca mulata) sequence: NCBI accession number: XP_001108923.1, modified from the cynomolgus monkey sequence Macaca fascicularis: NCBI accession number: XP_005570011.1 to replace the "X" at amino acid position 74 with a proline]. A parental HEK293 cell line that does not result in detectable expression of LAG3 by fluorescence-activated cell sorting (FACS) was included in the experiment as a background binding control. Unrelated human IgG4 and hIgG4 were included as isotype control antibodies.

[0255] The experiment was carried out according to the following procedure. Cells from the three cell lines described above were cultured in a Ca 2+ / Mg 2+ The cells were rinsed once in 1x PBS buffer without CaCl, followed by incubation with Enzyme Free Cell Dissociation Solution for 10 min at 37°C. 2+ / Mg 2+The cells were washed once with 1x PBS with 1x PBS and counted with a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience). Approximately 10,000 cells per well were seeded into a 96-well carbon electrode plate (MULTI-ARRAY high bind plate; MSD) in cell wash buffer and incubated at 37°C for 1 hour to allow cell attachment. Nonspecific binding sites were blocked with 2% BSA (w / v) in PBS for 1 hour at room temperature. Solutions of anti-LAG3 antibodies ranging from 1.7 pM to 100 nM in serial dilutions and no antibody were added in duplicate to the plate-bound cells, and the plate was incubated at room temperature for 1 hour. The plate was then washed to remove unbound antibody using an AquaMax2000 plate washer (MDS Analytical Technologies). Plate-bound antibodies were detected with SULFO-TAG™ conjugated anti-human IgG antibody (MSD) for 1 hour at room temperature. After washing, plates were developed with Read Buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded with a SECTOR Imager 6000 (MSD) instrument. Direct binding signals (in relative light units: RLU) were analyzed as a function of antibody concentration, and data were fitted with a sigmoidal dose-response model (four-parameter logistic model) using GraphPad Prism™ software. The potency of each antibody was calculated as EC 50 The EC for binding to HEK293-hLAG3 and HEK293-mfLAG3 cells was determined by calculating the EC 50 was defined as the antibody concentration at which 50% of the maximum binding signal was detected. The ratio of the signal detected by binding of 100 nM of antibody to HEK293-hLAG3 or HEK293-mfLAG3 cells compared to the signal detected by binding to HEK293 parental cells was recorded as an indication of the specificity of LAG3 binding. If the ratio was less than 2-fold at the highest antibody concentration (100 nM), the antibody was concluded as nonspecific (NS).

[0256] The binding results are summarized in Table 12. [Table 12]

[0257] As shown in Table 12, the potency of the antibodies was determined by the EC 50 Values ​​ranged from 690 pM to 52 nM, and all of the antibodies provided specific binding with >2-fold greater binding to cells expressing human LAG3 compared to parental HEK293 cells at 100 nM antibody binding. Only seven of the antibodies had EC values ​​ranging from 7.9 nM to 74 nM. 50 The binding potency of the comparative drugs on HEK293-hLag3 cells was approximately 4.0 nM and 7.0 nM. The comparative drugs did not show specific binding to HEK293-mfLAG3 cells. Binding of the non-participating controls to HEK293-hLAG3 and HEK293-mfLAG3 cells was non-specific as predicted by H4H and H4sH isotype control binding, with ratios ranging from 0.7-1.1 fold over parental HEK293 cells.

[0258] Example 7: Blocking LAG3 binding to MHC class II in a cell adhesion assay A cell adhesion assay was used to measure the ability of anti-LAG3 monoclonal antibodies to block adhesion of cells expressing human MHCII or mouse MHCII (RAJI and A20 cells, respectively) to hLAG3-expressing HEK293 cells attached on microtiter plates (based on the assay described in Huard et al., Eur J Immunol. 1995, 25:2718-21).

[0259] Stable HEK293 (ATCC number: CRL-1573) cell lines were generated by lentiviral transduction expressing recombinant hLAG3 (NCBI accession number: NP_002277.4) or mouse LAG3 (mLAG3) (GenBank accession number: NP_032505.1). The parental HEK293 cell line did not yield detectable expression of human LAG3 by fluorescence-activated cell sorting (FACS) techniques and was used to confirm the requirement of LAG3 for adhesion of MHCII-expressing RAJI and A20 cells.

[0260] The experiment was carried out as follows: Subconfluent HEK293-hLAG3 expressing cells were washed once with 1x PBS, trypsinized, and centrifuged at 1,200 rpm for 5 min. The cell pellet was resuspended in 1x PBS and then counted to determine cell number. An appropriate number of cells was isolated, and each well contained 1.2 x 10 cells in 100 μL. 4 The cells were resuspended in complete medium (DME + 10% FBS + penicillin / streptomycin / glutamine) to contain 1000 cells / well. The cells were then plated in a sterile Nunclon™ Delta 96-Well MicroWell™ Black The cells were seeded onto Optical Bottom Plates (Thermo Scientific). To avoid edge effects, wells at the edge of the plate were not included in the assay design. 100 μL of 1× PBS was added to the edge of the wells instead of cells. The seeded HEK293-hLAG3 cells were incubated overnight at 37° C., 5% CO2. After incubation, the cells were treated with anti-LAG3 antibodies and isotype controls.

[0261] On the day of treatment, all antibodies were serially diluted (1:3) in RPMI medium (no FBS or supplements added). Nine dilutions were made ranging between 45 pM and 300 nM, with the last dilution point containing no mAb. To test for H4sH antibodies in adhesion assays using RAJI cells, nine dilutions were made ranging between 23 pM and 150 nM, with the last dilution point containing no mAb. Anti-LAG3 antibodies or isotype controls were added to HEK293-hLAG3 cells in a final volume of 50 μL and incubated at 37° C., 5% CO2 for 1 hour. While the seeded HEK293-LAG3 cells were incubated with the test or control antibodies, non-adherent B cells (RAJI or A20 cells) were labeled with Calcein AM using the following procedure. RAJI and A20 suspension cells were harvested and centrifuged at 1,200 rpm for 5 minutes. The cell pellet was resuspended in 1× PBS and counted. Cells were washed with RPMI and resuspended at 10 cells / ml in RPMI. 6 Cells were labeled by adding 5 μL of Calcein AM per ml of cell suspension and incubated at 37° C., 5% CO2 for 30 min. Labeled cells were washed once with RPMI, centrifuged, washed with 1× PBS, and resuspended at a concentration of 5×10 cells per ml in PBS. 6 Calcein AM stained, Fc blocked RAJI or A20 cells were resuspended at a concentration of 3 x 10 cells per ml in RPMI. Then, 10 μL of BD Pharmingen Fc Block (0.5 mg / mL) was added per ml of Calcein AM labeled cells, which was incubated at RT for 10 min. Calcein AM stained, Fc blocked RAJI or A20 cells were resuspended at a concentration of 3 x 10 cells per ml in RPMI. 6 The cells were resuspended to a final concentration of 100 mM MgCl and then used in the adhesion assay.

[0262] At the end of treatment of HEK293-LAG3 cells with LAG3 antibody for 1 hour, 50 μl of labeled RAJI or A20 cells were added to each well at a concentration of 1.5×10 cells per well. 5Labeled suspension cells were incubated with the HEK293 monolayer for 1 hour at 37°C, 5% CO2. Wells were gently washed 4 times with RPMI and after each wash non-adherent cells were washed off by wiping the plate on a paper towel followed by two washes with PBS. A final volume of 200 μl of PBS was added to each well and Calcein AM fluorescence was read on a VICTOR™ X5 Multilabel Plate Reader with excitation / emission wavelengths of 485 nm / 535 nm. IC 50 Values ​​were calculated using a four-parameter logistic equation for nine-point response curves using GraphPad Prism. IC 50 was defined as the antibody concentration at which 50% blocking of adhesion of HEK cells to RAJI or A20 cells was observed.

[0263] A cell-based adhesion assay was used to evaluate the ability of anti-LAG3 antibodies to block LAG3 binding to both human and mouse MHCII. The assay utilized a format of binding of cells that endogenously express fluorescently labeled MHCII [human MHCII (RAJI) or mouse MHCII (A20)] to cells engineered to express human LAG3. The ability of LAG3 antibodies to block this interaction was evaluated and the results are shown in Table 13. [Table 13]

[0264] As shown in Table 13, the isotype controls did not demonstrate any measurable blocking of RAJI or A20 cell binding to HEK293-hLAG3 cells. All LAG3 antibodies of the invention blocked 92-98% of RAJI or A20 cell adhesion to HEK293-hLAG3 cells at the highest antibody concentrations, with IC50 values ​​ranging from 2.5 nM to 31 nM for RAJI cell adhesion and 3.6 nM to 30 nM for A20 cell adhesion to HEK293-hLAG3 cells. The IC50 values ​​of the comparators were 3.4 nM and 7.2 nM for RAJI cell adhesion and 5.1 nM and 9.6 nM for A20 cell adhesion to HEK293-Lag3 cells.

[0265] Example 8: Blockade of LAG3-induced T cell downregulation in a T cell / APC luciferase reporter assay T cell activation is achieved by stimulating the T cell receptor (TCR), which recognizes specific peptides presented by major histocompatibility complex class I or II (MHCI or MHCII) proteins on antigen-presenting cells (APCs) (Goldrath et al., 1999, Nature, vol. 402, pp. 255-262). The activated TCR in turn triggers a cascade of signaling events that can be monitored by reporter genes driven by transcription factors such as activator protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). T cell responses are elaborated through the engagement of co-receptors, which are constitutively or inducibly expressed on T cells. One such receptor is LAG3, a negative regulator of T cell activity. LAG3 interacts with its ligand, MHCII, expressed on target cells, including APCs or cancer cells, and inhibits T cell activation by disrupting positive signals induced by the TCR signalosome (Workman et al., 2002, J Immunol 169:5392-5395).

[0266] To characterize the ability of anti-LAG3 antibodies to antagonize LAG3-mediated signaling in T cells, a LAG3 T cell / APC reporter assay was developed to measure T cell signaling induced by APC interaction with T cells by utilizing mixed cultures derived from two mammalian cell lines: Jurkat-derived T cells (describe below, clone JRT3.T3.5) and engineered APCs (described below) in a HEK293 background (Figure 1).

[0267] For the first component, a T cell line for the LAG3 T cell / APC reporter bioassay was developed as follows: A Jurkat-derived T cell clone, JRT3.T3.5 (ATCC number: TIB-153), was first transduced with Cignal Lenti AP-1 Luc reporter (Qiagen (Sabiosciences); model number CLS-011L) according to the manufacturer's instructions. The lentivirus encodes a firefly luciferase gene under the control of a minimal CMV promoter, tandem repeats of a TPA-inducible transcription response element (TRE), and a puromycin resistance gene. After antibiotic selection, puromycin-resistant pools of reporter cells were further engineered by transduction with human CD28 (NP_006130.1), the alpha and beta subunits of Ob2F3 TCR (alpha: AGA92550.1, beta: AAA61026.1), a human LAG3 chimera (comprising the extracellular domain of human LAG3 (NP_002277.4) and the transmembrane / cytoplasmic domain of human CD300a (amino acids 181-299; accession no. NP009192.2)), and human PD-1 (accession no. NP_005009.2). Protein expression was confirmed by FACS analysis after each round of transduction. A single clone, JRT3.T3 / AP1-Luc / CD28 / hLAG3-CD300a chimera / hPD-1 clone 2D2, was then generated and used in the T cell / APC reporter bioassay. The cell line was maintained in RPMI+10% FBS+Penicillin / Streptomycin / Glutamine (P / S / G) supplemented with 100ug / mL Hygromycin+500ug / mL G418+1ug / mL Puromycin, referred to as selection medium in this example. These cells are referred to herein as reporter T cells.

[0268] In the second component, APC cell lines for LAG3T cell / APC reporter bioassay were generated as follows: A stable HEK293 cell line (ATCC number: CRL-1573) expressing human CD20 (number: NP_068769.2) was transduced with human MHCII subunits HLA-DR2A (NP_061984.2) and HLA-DR2B (NP_002115). HLA-DR2 positive cells were isolated by FACS, and the resulting cell line, HEK293 / CD20 / HLA-DR2, was maintained in DME+10%+P / S / G+non-essential amino acids (supplemented with 100ug / mL hygromycin+500ug / mL G418), referred to as selection medium in this example. These cells are referred to as APC cells herein.

[0269] Two approaches were used to engage T cells with APCs to induce signaling through the TCR: (1) a bispecific approach; and (2) a peptide pulsing approach. In this bioassay, anti-LAG3 antibodies rescued T cell activity by blocking the LAG3 / MHCII interaction and abolishing the inhibitory signaling delivered by the cytosolic tail of the CD300a molecule, followed by increased AP1-Luc signaling. T cell activation was monitored by luciferase readout.

[0270] Bispecific approach: This approach utilized a bispecific antibody (CD3xCD20 bispecific antibody; e.g., as disclosed in US20140088295) composed of one Fab arm that binds to CD3 on T cells and a second Fab that binds to CD20 on HEK293 cells. The presence of the bispecific molecule results in the formation of an immunological synapse and activation of the TCR complex due to the clustering of CD3 molecules on the engineered T cells.

[0271] The day before screening, engineered reporter T cells were plated at 5 x 10 cells per mL in selection medium. 5 The EC of anti-LAG3 antibody was cultured in 100 ml of 10 ... 50Values ​​were determined in the presence of a fixed concentration of CD3xCD20 bispecific antibody (100 pM). Reagents were added in the following order: Anti-Lag3 antibody and isotype control were serially diluted in RPMI1640 (assay medium) supplemented with 10% FBS and penicillin / streptomycin / glutamine. Ten dilutions were made ranging between 15 pM and 100 nM, with the last dilution point containing no mAb (buffer only control). Serially diluted antibodies were added to corresponding wells in a 96-well white flat-bottom plate containing a fixed concentration (100 pM) of bispecific antibody. Overnight cultured reporter T cells were diluted at 2 x 10 per mL in assay medium. 6 The reporter T cells were resuspended in 100 ml of PBS and added to the plate containing the bispecific anti-LAG3 antibody and incubated for 30 min at 37° C. / 5% CO2. The final concentration of reporter T cells was 5×10 per well. 4 There were 100 pieces.

[0272] APC cells were then washed once with D-PBS (Irvine Scientific; model no. 9240), trypsinized (Specialty Media; model no. SM-2004-C), and blocked with assay medium. After centrifugation, cells were resuspended at 4 × 10 per mL in assay medium. 5 The APC cells were resuspended at 1 × 10 per well and added to the plate containing the reporter T cells and incubated with anti-LAG3 antibody plus bispecific antibody. The final concentration of APC cells was 1 × 10 per well. 4 Plates were incubated at 37°C, 5% CO2 for 4-6 hours. AP1-Luc activity was detected after adding ONE-Glo™ (Promega; model no. E6051) reagent and relative light units (RLU) were captured on a Victor luminometer. All samples were tested in duplicate.

[0273] Peptide pulsing using MBP85-99: TCR recognizes specific MHC / peptide complexes, resulting in T cell activation. Ob2F3 used in this assay The TCR heterodimer has been reported to interact with HLA-DR2AB, an MHCII protein, complexed with the MBP85-99 peptide (myelin basic protein: NP_001020272; SEQ ID NO: 583).

[0274] The day before screening, engineered reporter T cells were prepared as described above for the bispecific format. APC cells were incubated at 37° C. / 5% CO2 in the corresponding antibiotic-containing cell culture medium with 0.2 μM MBP85-99 peptide (Celtek HLA-DR2 The cells were pulsed overnight with MBP85-99 (in DMSO); ER-15; Lot number: 140411; Molecular weight: 1797.1 g / mol). 50 The value is the pulsed APC1×10 4 The mAb concentration was determined in the presence of 100 μg / mL of mAb. Reagents were added in the following order: Anti-LAG3 antibodies and controls were serially diluted in assay medium. 10-point serial dilutions of anti-LAG3 antibodies ranged between 15 pM and 100 nM, with the last dilution point containing no mAb. Antibodies were added to corresponding wells in a 96-well white flat-bottom plate. Overnight cultured reporter T cells were diluted at 2 × 10 per mL in assay medium. 6 The reporter T cells were resuspended in 100 ml of PBS and added to the plate and incubated at 37° C., 5% CO for 30 min. The final concentration of reporter T cells was 5×10 T cells per well. 4 The MBP85-99 peptide-pulsed APC cells were then washed once with D-PBS (Irvine Scientific; model no. 9240), trypsinized (Specialty Media; model no. SM-2004-C), and blocked with assay medium. After centrifugation, the cells were diluted to 4 × 10 per mL in assay medium. 5 The APC cells were resuspended in 100 ml of PBS and added to the plate containing anti-LAG3 plus peptide-pulsed reporter T cells. The final concentration of APC cells was 1 × 10 per well. 4The assay plates were incubated at 37°C / 5% CO2 for 4-6 hours. AP1-Luc activity was detected after addition of ONE-Glo™ (Promega; model no. E6051) reagent and relative light units (RLU) were captured on a Victor luminometer. All samples were tested in duplicate.

[0275] EC 50 Values ​​were determined from a four-parameter logistic equation for a 10-point response curve using GraphPad Prism. The RLU values ​​for each antibody screened were normalized by setting the control, the last serial dilution containing no mAb, as 100%, which should theoretically represent the maximum AP1-Luc response elicited by the bispecific molecule (in the bispecific format) or APC pulsed with MBP85-99 peptide. The results are summarized in Table 14. [Table 14]

[0276] As shown in Table 14, all anti-Lag3 mAbs of the present invention tested in the T cell / APC bioassay inhibited LAG3 / HLA-DR2 with ECs ranging from 135 pM to 8.83 nM in the peptide format of the assay. 50 Rescued at low EC values, the bispecific format of the assay showed EC values ​​ranging from 87 pM to over 100 nM. 50 Rescue was achieved at EC values ​​of 280 pM and 350 pM for the comparator drugs. 50 Values ​​are presented. The isotype control tested did not show significant rescue activity in the bioassay. In further experiments, the addition of an anti-PD-1 antibody (REGN2810; described in Example 12 herein) did not enhance rescue of T cell activation.

[0277] In a second experiment, HEK293 / CD20 / HLA-DR2 cell line was transduced with human PD-L1 gene (amino acids 1-290 of accession number NP_054862.1) cloned into lentiviral (pLVX) vector system. Jurkat-derived T cells expressing hLAG-3 and AP-1-driven luciferase reporter were incubated with PD-L1 positive antigen-presenting cells expressing human MHCII / peptide complexes as described above. In this assay, anti-LAG-3 antibody rescued T cell activation only in the presence of anti-PD-1 antibody (REGN2810; described in Example 12 herein), but not in the absence of anti-PD-1 antibody. In summary, anti-LAG-3 antibody rescued T cell activation by blocking LAG-3 / MHCII inhibitory signaling in this T cell assay, and acted synergistically with anti-PD-1 antibody.

[0278] Example 9: Anti-LAG3 antibodies exhibit binding to stimulated cynomolgus monkey CD4+ and CD8+ T cells. In this example, selected anti-LAG3 antibodies were assessed for their ability to bind to LAG3-expressing cynomolgus monkey CD4+ and / or CD8+ T cells via FACS.

[0279] First, T cells were isolated from cynomolgus monkey whole blood (Bioreclamation, Westbury, NY). Briefly, whole blood from two cynomolgus monkey donors was diluted 1:1 in PBS and layered on 85% Ficoll-Paque solution. Red blood cells were separated from mononuclear cells according to standard procedures, and T cells were isolated using a pan-T cell isolation kit (Miltenyi Biotech).

[0280] Isolated T cells were activated using a T-cell Activation / Expansion kit for non-human primate (Miltenyi Biotech). The cell-bead mixture was diluted to 1 × 10 cells per mL in complete medium. 6After 72 hours of incubation, the beads were removed and the activated T cells were expanded in complete medium for 96 hours.

[0281] Stimulated cynomolgus T cells from each donor as described above were then stained with LIVE / DEAD dye (Molecular Probes) to distinguish between live and dead cells via FACS, after which the cells were co-incubated with PE-conjugated anti-CD8 and FITC-conjugated anti-CD4 for 15 minutes on ice to allow detection of CD4 and CD8 positive and negative cell populations.

[0282] To assess the binding of anti-LAG3 antibodies to previously isolated cynomolgus CD4+ and CD8+ T cells, anti-LAG3 antibodies and isotype controls were serially diluted in blocking buffer and seeded into 96-well V-bottom plates with final doses ranging from 100 nM to 50 pM. The serially diluted antibodies were then directly labeled with Zenon Alexa-Fluor 647 (Molecular Probes) and incubated with cynomolgus CD4+ / CD8+ T cell suspensions for 15-30 min on ice. Cells were centrifuged and the pellets were washed with staining buffer to remove unbound antibodies and fixed with a 1:1 dilution of Cytofix (BD Biosciences) and staining buffer for 12 h. After the incubation period, the fixation buffer was removed and the pellets were resuspended in staining buffer, filtered, and used for fluorescence measurements.

[0283] Fluorescence measurements were collected on a Fortessa (Becton Dickinson) and data were analyzed by FlowJo to determine mean fluorescence intensity (MFI). 50Values ​​were calculated from a four-parameter logistic equation for 11-point response curves using GraphPad Prism. The MFI ratio for each antibody was calculated by dividing the MFI at 11.1 nM antibody concentration by the MFI at 0 nM antibody concentration (negative control). [Table 15]

[0284] As previously shown, the anti-LAG3 antibodies, H4sH15482P and H4sH14813N, exhibited binding to engineered cynomolgus LAG3-HEK293 cell lines. In this example, H4sH15482P and H4sH14813N exhibited specific binding to CD4+ LAG3-expressing cynomolgus T cells and CD8+ LAG3-expressing cynomolgus T cells with EC values ​​ranging from 1.1 nM to 85 pM. 50 In summary, the selected anti-LAG3 antibodies described in this example show cross-reactivity with LAG3 expressed on activated cynomolgus monkey CD4+ and CD8+ T cells. In contrast, comparator 1 only demonstrated binding to cynomolgus monkey CD8+ T cells.

[0285] (Example 10: Efficacy of anti-LAG3 antibody against Colon 26 tumor in vivo) The in vivo efficacy of anti-mouse LAG3 antibody alone and in combination with anti-mouse PD-1 antibody was investigated in the Colon 26 preclinical synergy tumor model.

[0286] BALB / c mice were purchased from Taconic Biosciences. Colon 26, a mouse adenocarcinoma cell line derived from the BALB / c mouse strain, was purchased from the American Type Culture Collection (ATCC). On day 0, 1 × 10 Colon 26 cells (a mouse adenocarcinoma cell line derived from the BALB / c strain) were subcutaneously injected into 50 BALB / c mice. 6On day 8, the average tumor volume was reduced to 50 mm 3 Forty mice were selected and randomized into four treatment groups (N=10 per group). On days 14, 17, 21, 24, and 28, mice received the following antibodies: Group 1: rat IgG2a isotype control antibody (clone 2A3, BioXCell; model no. BE0089) and mIgG1 isotype control antibody (clone MOPC-21, BioXCell; model no. BE0083); Group 2: anti-mouse PD-1 antibody (rat IgG2a anti-mouse PD-1 antibody, clone RPMI-14, BioXCell; model no. BE0089) and mIgG1 isotype control antibody; Group 3: anti-mouse LAG3 antibody (rat IgG1 anti-mouse LAG3 antibody, clone C9B7W, BioXCell; model no. BE0174) and rat IgG2a control antibody; Group 4: anti-mouse PD-1 antibody and anti-mouse LAG3 antibody. All antibodies were administered at 10 mg / kg by intraperitoneal injection. Tumor volumes were monitored by caliper measurements twice weekly for the duration of the experiment (42 days).

[0287] A significant reduction in tumor growth was observed in mice treated with anti-mouse PD-1 antibody (Figures 2A-2B), with 5 of 10 mice being tumor-free by day 45, the end of the experiment. A reduction in tumor growth was also observed in a subset of mice treated with anti-mouse LAG3 antibody, with 3 of 10 mice being tumor-free by day 45. In contrast, combined treatment with anti-LAG3 and anti-PD-1 antibodies was superior to either monotherapy, with 8 of 10 mice being tumor-free at the end of the experiment, suggesting a strong additive effect of treatment with anti-LAG3 and anti-PD-1. No tumor-free mice were found in the control group at the end of the experiment. A statistically significant reduction in tumor volume (p<0.05, Figure 2B) was observed by day 35 in mice treated with combination therapy, but not in mice treated with monotherapy. Despite effective tumor elimination, no evidence of weight loss was observed (not shown). Thus, in an established Colon 26 tumor model, the combination regimen of anti-PD-1 and anti-LAG3 antibodies was significantly more effective than the corresponding monotherapies.

[0288] The activity of anti-mouse PD-1 and anti-mouse LAG3 antibodies in vivo was also studied in preclinical synergistic tumor models with 4T1, RENCA, and A20. Combination treatment with both antibodies resulted in at least additive antitumor effects compared to single antibody treatment (data not shown).

[0289] Example 11: Efficacy of anti-mouse LAG3 antibody against MC38 tumor in vivo We investigated the effect of dual blockade of PD-1 and LAG3 on established MC38 tumors in C57BL / 6 mice using anti-mouse PD-1 and anti-mouse LAG3 blocking antibodies.

[0290] C57BL / 6 mice were purchased from Taconic Biosciences. MC38, a mouse adenocarcinoma cell line derived from the C57BL / 6 mouse strain, was obtained from a deposit at the National Institutes of Health. Anti-LAG3 and anti-PD-1 antibodies and controls were obtained as described in Example 10.

[0291] On day 0, 50 C57BL / 6 mice were subcutaneously injected with 3 × 10 MC38 cells in the flank. 5 On day 8, the average tumor volume was 45 mm 3 Forty mice were selected and randomized into four treatment groups (N=10 per group). On days 8, 12, 15, 19, and 23, mice were administered the following antibodies: Group 1: rat IgG2a isotype control antibody and mIgG1 isotype control antibody; Group 2: anti-mouse PD-1 antibody and mIgG1 antibody; Group 3: anti-mouse LAG3 antibody and rat IgG2a antibody; Group 4: anti-mouse PD-1 antibody and anti-mouse LAG3 antibody. All antibodies were administered at 10 mg / kg by intraperitoneal injection. Tumor volumes were monitored by caliper measurements twice weekly for the duration of the experiment (28 days).

[0292] Anti-PD-1 antibody therapy significantly reduced tumor growth in a subset of mice (p<0.0001; day 23) (Figures 3A-3B), with 2 of 10 mice being tumor-free at the end of the experiment. While anti-LAG3 antibody monotherapy did not show a significant anti-tumor effect (0 of 10 mice were tumor-free at the end of the experiment), the combination of anti-LAG3 antibody and anti-PD-1 antibody was superior to anti-PD-1 therapy alone, indicating a synergistic effect.

[0293] To investigate the immunomodulatory properties of the combined anti-PD-1 and anti-LAG3 antibodies, T cell markers were examined in the spleens and draining lymph nodes (DLN) of treated mice (Figure 4). Spleens and draining lymph nodes of tumor-bearing mice were removed by dissection and placed in 15 mL of RPMI1640 Glutamax I medium (Invitrogen). Total RNA was isolated and RT-PCR Taqman analysis was performed using a primer / probe mix specific for mouse CD8β [probe: AGCAGCTCTGCCCTCAT (SEQ ID NO: 584), forward primer: GCTCTGGCTGGTCTTCAGTATG (SEQ ID NO: 585), reverse primer: TTGCCGTATGGTTGGTTTGAAC (SEQ ID NO: 586)] and mouse IFNγ (Mm01168134_m1, Applied Biosystems). Samples were normalized to mouse cyclophilin B transcript expression levels. TaqMan analysis confirmed the expansion of CD8+ T cells in both the DLN and spleens of mice in the combination treatment group, as well as increased production of the T cell effector molecule IFNγ in both combination and single anti-PD-1 treated animals, indicating that blockade of PD-1 and LAG3 increases T cell proliferation and effector function.

[0294] Example 12: Efficacy of anti-human LAG3 antibody against MC38 tumor in vivo In this experiment, the efficacy of exemplary anti-human LAG3 antibodies of the present invention against MC38 tumors was studied. Anti-human LAG3 mAbs do not bind to mouse LAG3. Therefore, to study the anti-tumor properties of these antibodies in vivo in mice, mice humanized to express human LAG3 protein were used. For the experiments herein, VelociGene® technology (Valenzuela et al., 2003, Nat. Biotechnol. 21:652-659) was used to generate double humanized mice expressing the extracellular portions of human PD-1 and human LAG3, as well as the transmembrane and intracellular portions of the mouse forms of these proteins.

[0295] Using VelociGene® technology, double humanized LAG3 / PD-1 mice were engineered to replace the extracellular domains of mouse Pdcd1 and Lag3 genes with the corresponding regions of human PD-1 and LAG3 genes (U.S. Patent Application No. 62 / 258,181, filed November 20, 2015). Expression of humanized PD-1 and humanized LAG3 was confirmed by examining the expression of PD-1 and LAG3 proteins on humanized mouse T cells after stimulation with anti-CD3 / anti-CD28 antibodies. Interaction of human PD-1 and LAG3 proteins with their corresponding mouse ligands was verified by examining the binding of human LAG3 to mouse MHCII in a cell adhesion assay and by examining the binding of human PD-1 to mouse PD-L1 in an SPR-Biacore binding study.

[0296] The exemplary anti-LAG3 antibody used in this study is a fully human antibody that specifically binds to human LAG3 and comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of SEQ ID NOs: 420-422-424-428-430-432, and HCVR / LCVR of SEQ ID NOs: 418 / 426 (also known as H4sH15482P, hereinafter referred to as "mAb1").

[0297] Anti-human PD-1 antibodies are disclosed in US Patent Application Publication No. US20150203579, which is incorporated herein by reference in its entirety. The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810. More specifically, H4H7798N comprises a heavy chain of SEQ ID NO: 330 and a light chain of SEQ ID NO: 331, as disclosed in US20150203579). REGN2810 (anti-hPD-1) binds to human PD-1 with high affinity and blocks the interaction of PD-1 with PD-L1 and PD-L2.

[0298] To increase tumor immunogenicity, mouse colon adenocarcinoma cells were engineered to express chicken ovalbumin (MC38.Ova).

[0299] Day 0, LAG3 hum / hum PD-1 hum / hum Mice were subcutaneously injected with 1.5 × 10 MC38.Ova cells. 6 Mice were implanted and randomized into three treatment groups (N=7 per group). Mice were administered mAb1 (anti-hLAG3) (25 mg / kg), REGN2810 (anti-hPD-1) (10 mg / kg), or isotype control antibody (25 mg / kg) by intraperitoneal injection on days 3, 6, 10, 14, and 17. Tumor volumes were monitored by caliper measurements twice weekly for the duration of the experiment (24 days).

[0300] Day 0, LAG3 hum / hum PD-1 hum / humDoubly humanized mice were inoculated with MC38.Ova cells and administered mAb1 (anti-hLAG3) (25 mg / kg), REGN2810 (anti-hPD-1) (10 mg / kg), or isotype control (25 mg / kg) via the intraperitoneal route on days 3, 6, 10, 14, and 17. REGN2810 (anti-hPD-1) showed partial tumor growth inhibition (Figure 5), with tumor regression observed in 2 of 7 mice, whereas none of the mice in the isotype control group were tumor-free. Monotherapy with mAb1 (anti-hLAG3) at 25 mg / kg had no significant effect on tumor growth, with only 1 of 7 mice being tumor-free by the end of the experiment. Mouse body weight was not affected by mAb1 (anti-hLAG3), REGN2810 (anti-hPD-1), or isotype control treatment.

[0301] Example 13: Efficacy of a combination of anti-human LAG3 antibody and anti-human PD-1 antibody against MC38 tumors in vivo In this experiment, the double humanized LAG3 hum / hum PD-1 hum / hum The efficacy of mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) against MC38.Ova tumors in mice was studied. The double humanized mice are described in Example 12 herein.

[0302] The exemplary anti-LAG3 antibody used in this study is a fully human antibody that specifically binds to human LAG3 and comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of SEQ ID NOs: 420-422-424-428-430-432, and HCVR / LCVR of SEQ ID NOs: 418 / 426 (also known as H4sH15482P, hereinafter referred to as "mAb1").

[0303] The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1) binds to human PD-1 with high affinity and blocks the interaction of PD-1 with PD-L1 and PD-L2.

[0304] On day 0, mice were subcutaneously injected with 1 × 10 MC38.Ova cells. 6 Mice were implanted and randomized into four treatment groups (N=7 for control group, N=12 for mAb1 (anti-hLAG3), REGN2810 (anti-hPD-1), and mAb1 (anti-hLAG3) + REGN2810 (anti-hPD-1) combination treatment groups, respectively. On days 3, 7, 10, 14, and 17, mice were administered mAb1 (anti-hLAG3) (25 mg / kg), REGN2810 (anti-hPD-1) (10 mg / kg), a combination of mAb1 (anti-hLAG3) (25 mg / kg) and REGN2810 (anti-hPD-1) (10 mg / kg), or isotype control (25 mg / kg) by intraperitoneal injection. Tumor volumes were monitored by caliper measurements twice weekly for the duration of the experiment (32 days) and tumor-free animals were monitored for the absence of tumor recurrence for up to 80 days.

[0305] REGN2810 (anti-hPD-1) monotherapy resulted in tumor growth inhibition (FIGS. 6A-6B), with tumor regression in 2 of 12 animals (17%), whereas mAb1 (anti-hLAG3) was less effective, confirming the findings of previous experiments. Tumor regression was observed in only 1 of 12 mice treated with mAb1 (anti-hLAG3). No tumor-free mice were observed in the isotype control group, even at day 32. In contrast, the combination of mAb1 (anti-hLAG3) and REGN2810 (anti-hPD-1) demonstrated robust inhibition of MC38.Ova tumor growth, resulting in 5 of 12 (42%) tumor-free mice by the end of the experiment. None of the tumor-free mice showed tumor recurrence over 80 days after implantation, indicating a long-lasting effect of the combined immunotherapy. One-way ANOVA with post-Tukey's test for multiple comparisons revealed significant differences in tumor volume in the mAb1 (anti-hLAG3) and REGN2810 (anti-hPD-1) combination treatment group compared to mAb1 (anti-hLAG3) monotherapy (p<0.05) or isotype control (p<0.01) at day 22 (Figure 6B). REGN2810 (anti-hPD-1) monotherapy also showed a significant reduction in tumor growth compared to the isotype control (p<0.05). Combination treatment with mAb1 (anti-hLAG3) and REGN2810 (anti-hPD-1) also resulted in a significant improvement in animal survival (p<0.0001) as analyzed by the log-rank (Mantel-Cox) test (Figure 6C). Despite the improved efficacy of the combination therapy, no evidence of weight loss was observed (data not shown). In summary, the combination of mAb1 (anti-hLAG3) and REGN2810 (anti-hPD-1) resulted in improved efficacy including reduced tumor growth, improved tumor elimination, and improved survival compared to REGN2810 (anti-hPD-1) or mAb1 (anti-hLAG3) monotherapy.

[0306] Example 14: Dose-finding study of anti-LAG3 and anti-PD-1 antibody combinations against MC38 tumors. This example shows the in vivo double humanized LAG3 hum / hum PD-1 hum / hum A set of dose-ranging experiments is described to measure the efficacy of a combination of anti-human PD-1 and anti-human LAG3 antibodies against MC38.Ova tumors in mice. The bi-humanized mice are described in Example 12 herein.

[0307] The exemplary anti-LAG3 antibody used in this study is a fully human antibody that specifically binds to human LAG3 and comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of SEQ ID NOs: 420-422-424-428-430-432, and HCVR / LCVR of SEQ ID NOs: 418 / 426 (also known as H4sH15482P, hereinafter referred to as "mAb1").

[0308] The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1) binds to human PD-1 with high affinity and blocks the interaction of PD-1 with PD-L1 and PD-L2.

[0309] In the first experiment, REGN2810 (anti-hPD-1) was titrated and examined at dose levels of 10 mg / kg and 1 mg / kg, both dose groups as single agents and in combination with mAb1 (anti-hLAG3) (25 mg / kg). Results of both combination treatment groups were compared to monotherapy with REGN2810 (1 mg / kg or 10 mg / kg) or mAb1 (25 mg / kg) to determine whether additive / synergistic antitumor effects were observed. Treatment with an isotype control antibody (25 mg / kg) was also examined. On day 0, LAG-3 hum / hum PD-1 hum / hum mice were implanted with MC38.Ova cells and randomized into six treatment groups (N=6 for isotype control group, N=12 for mAb1, REGN2810, or REGN2810+mAb1 treatment groups). Mice were administered mAb1 (25 mg / kg); REGN2810 (10 mg / kg); REGN2810 (1 mg / kg); mAb1 (25 mg / kg) + REGN2810 (10 mg / kg); mAb1 (25 mg / kg) + REGN2810 (1 mg / kg); or isotype control (25 mg / kg) by intraperitoneal injection on days 3, 7, 10, 14, and 17. Tumor volumes were monitored for 36 days and tumor-free animals were monitored for up to 80 days.

[0310] Table 16 shows the mean tumor volumes at various time points during the study, and the number of tumor-free mice at day 36 is shown in Table 17. [Table 16] [Table 17]

[0311] REGN2810 monotherapy at 1 mg / kg showed minimal tumor regression over 36 days, similar to the results for the isotype control treatment group (Figures 7A-7B). At day 36, 2 of 12 mice (approximately 16%) and 0 of 6 mice (0%) were tumor-free in the REGN2810 (1 mg / kg) and isotype control groups, respectively. In contrast, the mAb1 (25 mg / kg), REGN2810 (10 mg / kg), and REGN2810 (1 mg / kg) + mAb1 (25 mg / kg) treatment groups demonstrated similar reductions in tumor growth over 22 days (Figure 7A). At day 36, mAb1 (25 mg / kg) treatment resulted in 1 of 12 (approximately 8%) tumor-free mice, whereas 4 of 12 (approximately 33%) and 3 of 12 (25%) mice were tumor-free in the REGN2810 (10 mg / kg) and REGN2810 (1 mg / kg) + mAb1 (25 mg / kg) groups, respectively. Overall, mAb1 (25 mg / kg) + REGN2810 (10 mg / kg) demonstrated the most robust reduction in MC38.Ova tumor growth, with 6 of 12 (50%) mice being tumor-free by day 36. None of the tumor-free mice showed tumor recurrence over 80 days post-implantation despite cessation of treatment, indicating a long-lasting effect of the combined immunotherapy. mAb1 (25 mg / kg) + REGN2810 (10 mg / kg) treatment also resulted in a significant improvement in animal survival (p=0.0197) as analyzed by the log-rank (Mantel-Cox) test (Figure 7C). No evidence of weight loss was observed.

[0312] In the second experiment, mAb1 (anti-hLAG3) was titrated and examined at dose levels of 25 mg / kg and 5 mg / kg, both dose groups as single agents and in combination with REGN2810 (anti-hPD-1) (10 mg / kg). Results of both combination treatment groups were compared to monotherapy with mAb1 (5 mg / kg or 25 mg / kg), or REGN2810 (10 mg / kg) to determine whether additive / synergistic antitumor effects were observed. Treatment with an isotype control antibody (25 mg / kg) was also examined. On day 0, LAG-3 hum / hum PD-1 hum / hum mice were implanted with MC38.Ova cells and randomized into six treatment groups (N=6 for isotype control group, N=12 for mAb1, REGN2810, or REGN2810+mAb1 treatment groups). Mice were administered mAb1 (5 mg / kg); mAb1 (25 mg / kg); REGN2810 (10 mg / kg); mAb1 (5 mg / kg) + REGN2810 (10 mg / kg); mAb1 (25 mg / kg) + REGN2810 (10 mg / kg); or isotype control (25 mg / kg) by intraperitoneal injection on days 3, 7, 10, 14, and 17. Tumor volumes were monitored over a 31-day period.

[0313] mAb1 monotherapy at 5 mg / kg showed only minimal tumor regression, similar to the isotype control treatment group. The combination of mAb1 (25 mg / kg) + REGN2810 (10 mg / kg) showed the most robust tumor reduction (Figure 8A). The strong combination effect was also supported by the mAb1 (5 mg / kg) + REGN2810 (10 mg / kg) treatment group. The tumor volumes measured on day 23 are shown in Figure 8B. The combination of anti-LAG-3 antibody with REGN2810 also showed a significant improvement in animal survival (p<0.001; analyzed by the Mantel-Cox log-rank test) (Figure 8C). The results indicate that the strong combination effect is still seen with low doses of anti-LAG3 antibody. Based on the results shown herein, it may not be necessary to use high doses of anti-LAG3 antibody in combination with anti-PD-1 antibody in the treatment regimen against tumors.

[0314] In summary, the combination of REGN2810 (anti-hPD-1) and mAb1 (anti-hLAG3) in the MC38.ova tumor model with double humanized LAG3 / PD-1 mice, allowing testing of clinical antibodies that do not cross-react with mouse receptors, demonstrated improved efficacy in a dose-dependent manner, including reduced tumor growth and improved survival, compared to REGN2810 (anti-hPD-1) and mAb1 (anti-hLAG3) monotherapy. The robust anti-tumor efficacy of the combination of REGN2810 (anti-hPD-1) and mAb1 (anti-hLAG3) in the preclinical setting supports their clinical development as a combination cancer immunotherapy.

[0315] Example 15: Efficacy of a combination of anti-human LAG3 and anti-human PD-1 antibodies against established MC38 tumors in vivo In this experiment, the double humanized LAG3 hum / hum PD-1 hum / hum The efficacy of mAb1 (anti-hLAG3) in combination with REGN2810 (anti-hPD-1) against established MC38.Ova tumors in mice was investigated. The double humanized mice are described in Example 12 herein.

[0316] The exemplary anti-LAG3 antibody used in this study is a fully human antibody that specifically binds to human LAG3 and comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of SEQ ID NOs: 420-422-424-428-430-432, and HCVR / LCVR of SEQ ID NOs: 418 / 426 (also known as H4sH15482P, hereinafter referred to as "mAb1").

[0317] The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1) binds to human PD-1 with high affinity and blocks the interaction of PD-1 with PD-L1 and PD-L2.

[0318] Day 0, LAG-3 hum / hum PD-1 hum / hum Mice were inoculated subcutaneously with MC38.Ova cells. On day 10, the average tumor volume was 100 mm 3 Mice were selected and randomized into four treatment groups. Mice received mAb1 (25 mg / kg; N=9), REGN2810 (10 mg / kg; N=10), mAb1 (25 mg / kg) + REGN2810 (10 mg / kg) combination (N=11), or isotype control antibody (25 mg / kg; N=7) by IP injection on days 10, 14, 17, and 22. Tumor volumes were monitored for 28 days after tumor implantation.

[0319] Treatment of humanized mice bearing MC38.ova tumors with a combination of anti-hPD-1 and anti-hLAG-3 antibodies elicited activation of intratumoral and peripheral T cells. Whereas REGN2810 monotherapy resulted in partial tumor growth inhibition, mAb1 monotherapy showed no reduction in mean tumor volume compared to mice treated with isotype control (Figure 9). In contrast, the combination of mAb1 (25 mg / kg) and REGN2810 (10 mg / kg) demonstrated robust inhibition of MC38.Ova tumor growth. One-way analysis of variance (ANOVA) with Bonferroni post-test multiple comparisons revealed significant differences in mean tumor volume in the mAb1 and REGN2810 combination group compared to mAb1 monotherapy (p<0.01) or isotype control (p<0.05) (Figure 10). When the combination therapy was compared with REGN2810 monotherapy, the overall mean tumor volume over the course of the experiment was smaller in the combination therapy, but this difference did not reach statistical significance. The combination of anti-LAG-3 antibody and REGN2810 also resulted in a significant increase in the survival rate of animals and a significant prolongation of the duration of survival (p<0.01; analyzed by the Mantel-Cox log-rank test). At day 25, 50% of the mice treated with the combination therapy were alive compared with the control and monotherapy (Figure 11). Based on this result, the combination treatment exhibited an additive and dose-dependent antitumor effect compared with each monotherapy.

[0320] Example 16: Pharmacokinetics of anti-LAG-3 antibodies in cynomolgus monkeys The pharmacokinetics (PK) of anti-LAG3 antibody mAb1 was characterized following a single intravenous (IV) dose of 1.0, 5.0, or 15.0 mg / kg to female cynomolgus monkeys (5 animals per dose group). Blood samples for determining functional mAb1 concentrations were collected from all animals at various time points. Serum functional mAb1 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA). Anti-mAb1 antibodies in serum were analyzed using an electrochemiluminescence-based bridging immunoassay. PK parameters were estimated using noncompartmental analysis (NCA). Mean concentration-time profiles are characterized by a short initial distribution phase followed by a linear beta elimination phase (Figure 12). No target-mediated clearance phase was observed over the 8-week study duration. Mean AUC inf The AUCs were comparable across the dose levels tested. inf Consistent with this finding is the fact that mean total body clearance (CL) values ​​were similar for the three dose groups. In addition, the beta-phase half-lives (t 1 / 2 ) were comparable over a range of 10.8–11.5 days. Based on the results, a no-observed-adverse-effect level (NOAEL) of up to 50 mg / kg could be established. These results indicated that mAb1 demonstrates linear kinetics under the conditions of this study.

[0321] Example 17: Toxicological evaluation of anti-LAG3 antibodies in cynomolgus monkeys The in vivo toxicology and toxicokinetic profile of mAb1 was evaluated during a 4-week, repeated-dose, GLP-compliant toxicology study in male and female cynomolgus monkeys. Monkeys (5 animals per sex per group) received 0, 2, 10, or 50 mg / kg mAb1 weekly by IV infusion. Toxicity assessment was based on mortality, moribundity, clinical observations, body weight, food consumption, ophthalmologic examination, ECG evaluation, respiratory rate, pulse oximetry, temperature, and blood pressure evaluation, and neurological examination. Clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis) and immunophenotyping were also evaluated. Gross necropsy examination, organ weight measurements, and histopathological evaluation were also performed. Complete necropsies were performed at the end of the dosing period or at the end of the 8-week recovery period. Selected organs were weighed and tissues were examined grossly and microscopically.

[0322] mAb1 was well tolerated at all dose levels evaluated when administered via weekly IV infusion to cynomolgus monkeys. Because mAb1 was well tolerated and there were no mAb1-related changes in any of the safety parameters evaluated, the no-observed-adverse-effect level (NOAEL) for the study is considered to be 50 mg / kg per dose, the highest dose administered in this study.

[0323] Example 18: Hydrogen / Deuterium Exchange (H / D)-Based Epitope Mapping of Anti-LAG3 Antibody H4sH15482P Binding to the Extracellular Domain of hLAG3 Experiments were performed to determine the amino acid residues of the human LAG3 extracellular domain (amino acids Leu23-Leu450 of human LAG3; UniProt accession number: P18627; generated with a human IgG1 tag at the C-terminus ("hLag3.Fc") (R&D Systems, Minneapolis, MN) (SEQ ID NO: 587) with which the anti-LAG3 antibody H4sH15482P interacts. For this purpose, hydrogen / deuterium (H / D) exchange epitope mapping with mass spectrometry (HDX-MS) was utilized. A general description of the HDX method is set forth in Ehring (1999), Analytical Biochemistry, 267(2):252-259; and Engen and Smith (2001), Anal. Chem., 73:256A-265A.

[0324] Experimental procedure HDX-MS experiments were performed on an integrated Waters HDX / MS platform (Waters Corporation, Milford, MA) consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradients, and a Synapt G2-Si mass spectrometer for mass measurement of digested peptides.

[0325] Labeling solutions were prepared in 10 mM PBS buffer at pD 7.0 under D2O. For deuterium labeling, 3.8 μL of hLAG3.Fc (5 picomoles per μL) or hLAG3.Fc premixed with anti-Lag3 antibody H4sH15482P at a 1:1 molar ratio was incubated with 56.2 μL of D2O labeling solution for various time points (e.g., non-deuterated control=0 seconds, deuterium labeling: 1 minute and 20 minutes). Deuteration was quenched by transferring 50 μL of sample to 50 μL of pre-chilled quench buffer (0.2 M TCEP, 6 M guanidine hydrochloride in 100 mM phosphate buffer, pH 2.5). The mixture was incubated at 1.0° C. for 4 minutes. The quenched samples were then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were captured on an ACQUITY UPLC BEH C18 1.7 μm, 2.1×5 mm VanGuard pre-column (Waters Corporation) at 0° C. and eluted onto an analytical column ACQUITY UPLC BEH C18 1.7 μm, 1.0×50 mm for gradient separation over 9 min with 5%-40% B phase (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set with a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50-1700 Thomson units (Th).

[0326] To identify the peptide residues of human Lag3 that interact with H4sH15482P, LC-MS analysis was performed using non-deuterated samples. EThe data was processed and searched against a database containing the sequence of human LAG3, and the sequences were digested with pepsin and randomized using Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum products per amino acid = 0.2, and 2) duplicate file threshold = 3. The DynamX software then automatically determined the deuterium incorporation by each peptide based on retention time and high mass accuracy (<10 ppm) at each time point, in triplicate, and across multiple time points.

[0327] result MS E Using an online pepsin / protease XIII column coupled to data collection, a total of 123 peptides derived from human LAG3 were reproducibly identified in the absence or presence of antibodies, displaying 78.3% sequence coverage. Four Lag-3 peptides exhibited significantly reduced deuterium incorporation upon binding to H4sH15482P (centroid delta values ​​>0.8 Daltons, as p-values ​​<0.05). Reported peptide masses were calculated using the centroid MH-H4sH15482P ... + These peptides, corresponding to amino acids 34-77 of hLAG3.Fc (SEQ ID NO:587), reduced the deuteration rate upon binding to H4sH15482P. These identified residues also correspond to residues 28-71 of human LAG3, defined by Uniprot Accession Number: P18627 (LAG3_HUMAN; SEQ ID NO:588) and also exemplified in Table 18. [Table 18]

[0328] Example 19: Clinical Trial of Anti-LAG3 Antibodies in Patients with Advanced Malignant Disease This example describes a clinical trial evaluating the safety, tolerability, and anti-tumor activity of an anti-LAG3 antibody as monotherapy and in combination with an anti-PD-1 antibody in patients with advanced malignant disease.

[0329] The exemplary anti-LAG3 antibody used in this study is a fully human antibody that specifically binds to human LAG3 and comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 of SEQ ID NOs: 420-422-424-428-430-432, and HCVR / LCVR of SEQ ID NOs: 418 / 426 (also known as H4sH15482P, hereinafter referred to as "mAb1").

[0330] The anti-PD-1 antibody used in this example is H4H7798N (disclosed in US20150203579; also known as REGN2810). REGN2810 (anti-hPD-1) binds to human PD-1 with high affinity and blocks the interaction of PD-1 with PD-L1 and PD-L2.

[0331] Research Objective The main objectives of the study are as follows:

[0332] In the dose escalation phase: To evaluate safety and pharmacokinetics (PK) of mAb1 as monotherapy and in combination with REGN2810 in patients with advanced malignancies, including lymphoma, to determine dose for Phase 2. Endpoints include dose-limiting toxicity (DLT) rates, adverse events (AEs) (including immune-related AEs), serious adverse events (SAEs), deaths, and laboratory abnormalities (Grade 3 or greater per Common Terminology Criteria for Adverse Events [CTCAE]), and pharmacokinetics.

[0333] In the dose expansion phase: To evaluate the preliminary antitumor activity of mAb1 alone and in combination with REGN2810 (separately for each cohort), as measured by ORR. The endpoint is overall response rate (ORR) based on Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 (Eisenhauer et al., 2009, Eur. J. Cancer 45:228-247) (solid tumors), and the Lugano criteria (Cheson et al., 2014, J. Clin. Oncol. 32:3059-3068) (lymphomas).

[0334] Secondary objectives were (a) ORR based on immune-related Response Evaluation Criteria in Solid Tumors (irRECIST) (Wolchok et al., 2009, Clin. Cancer Res. 15:7412-7420; Nishino et al., 2013, Clin. Cancer Res. 19:3936-3943), best overall response (BOR), duration of response (DOR), disease control rate, and PFS based on RECIST, irRECIST, and Lugano criteria (Cheson et al., 2014, J. Clin. (b) evaluate the preliminary anti-tumor activity of mAb1 alone and in combination with REGN2810 (separately for each cohort); (b) characterize the safety profile within each expansion cohort as determined during the dose escalation phase; (c) characterize the PK of mAb1 as monotherapy and of mAb1 and REGN2810 when administered in combination; and (d) evaluate immunogenicity as measured by anti-drug antibodies (ADA) against mAb1 and REGN2810.

[0335] The exploratory objectives of the study are (a) to assess tumor volume; (b) to explore the pharmacodynamic effects of REGN2810 in serum, plasma, peripheral blood mononuclear cells (PBMCs), and tumor tissue samples (including archived tumor tissue) obtained from patients treated with mAb1 as monotherapy or mAb1 in combination with REGN2810 at baseline, during treatment, and at progression; and (c) to assess (i) circulating tumor nucleic acid; (ii) distribution of PBMC subsets and expression of immune checkpoint molecules and other biomarkers of interest; (iii) expression of tumor RNA; (iv) tumor-infiltrating lymphocytes (CD8+ T cells, CD4+ Biomarkers of interest, which may include, but are not limited to, numbers and distribution of T cells, regulatory T cells, and other tissue-penetrant subtypes such as B cells, bone marrow-derived cells, NK cells; (v) expression levels (messenger RNA and / or protein) of PD-1, PD-L1, LAG3, and possibly other checkpoint modulators; (vi) mutations in known oncogenes and potential tumor neo-antigens; and (vii) tumor mutational burden, are assessed for predictive potential and correlation with clinical response.

[0336] Study design This is a Phase 1, first-in-human (FIH), open-label, multicenter, dose-escalation study of the safety, tolerability, activity, and PK of mAb1 administered as monotherapy or in combination with REGN2810 in patients with progressive malignant disease. Three dose levels (1.0, 3.0, and 10 mg / kg) of mAb1 as monotherapy and in combination with REGN2810 at a dose of 3.0 mg / kg will be examined.

[0337] After a screening period of up to 28 days, patients received up to 17 21-day treatment cycles (maximum total treatment of 51 weeks), followed by a follow-up period of 24 weeks. Each patient received mAb1 (±REGN2810) every 21 days.

[0338] Treatment will continue until the 51-week treatment period is completed or disease progression, unacceptable toxicity, withdrawal of consent, or study discontinuation criteria are met. Response assessments will be every 6 weeks for the first 24 weeks, regardless of delays in administration of study drug, and then every 9 weeks for the following 27 weeks. Patients who achieve a confirmed complete response (CR) after a minimum of 24 weeks of treatment (minimum of 8 treatment cycles) may be selected to discontinue treatment and continue with all relevant study evaluations. Similarly, patients who have been treated for a minimum of 24 weeks, after physician consultation, and who maintain stable disease (SD) or partial response (PR) for three consecutive tumor evaluations may also be selected to discontinue treatment but continue with all relevant study evaluations. Patients who experience a response but then progress will be required to have a tumor biopsy at the time of progression. Patients who tolerate 4 doses of mAb1 monotherapy and have an initial tumor assessment of at least SD, but subsequently demonstrate progressive disease (PD), will have the option to add 3.0 mg / kg REGN2810 to the highest dose (if known) of mAb1 safely administered to that point in an attempt to "rescue" the response with combined lymphocyte activation gene 2 (LAG-3) blockade and programmed cell death 1 (PD-1) blockade. Safety assessments will be performed at each study drug administration visit.

[0339] Dose escalation Three dose-escalating monotherapy and combination therapy cohorts are planned to be enrolled (mAb1 at 1.0, 3.0, and 10 mg / kg with or without REGN2810 at 3 mg / kg). The first cohort enrolled will receive mAb1 monotherapy at 1.0 mg / kg. Enrollment of each additional cohort thereafter may be limited by the number of dose-limiting toxicities (DLTs) observed in the preceding cohort. If necessary, a dose-decreasing cohort (mAb1 at 0.3 mg / kg with or without REGN2810 at 3 mg / kg) will be enrolled.

[0340] Dose Titration Rules A modification of the traditional 3+3 design ("4+3") will be used to evaluate all dose levels in both the mAb1 monotherapy cohort and the mAb1+REGN2810 cohort. A minimum of three patients at each dose level are required to be evaluable for DLT, but to maximize the efficiency of dose escalation in Phase 1 while maintaining patient safety in the event that a patient discontinues before being evaluable for DLT, four patients will be enrolled at each dose level. The DLT evaluation period will be 28 days. Dose level tolerability will be achieved only if all potential DLT-evaluable patients complete the 28-day DLT period without DLT (0 of 3 or 0 of 4). If three patients complete the DLT period without a DLT, but a fourth patient is within the DLT evaluation period, dose level tolerability will be achieved only if the fourth patient completes the DLT evaluation period or discontinues treatment before being evaluable for DLT. If one of 3 or 4 DLT-evaluable patients experiences a DLT, then 4 or 3 additional patients (respectively) will be enrolled for a total of 7 patients. Similarly, 1 of 6 or 1 of 7 DLTs would be considered tolerable. A DLT in 2 of 2-7 evaluable patients would be above the maximum tolerated dose (MTD). At the highest dose level tolerated, 3-4 additional patients will be enrolled for a total of 6-10 DLT-evaluable patients for further evaluation for safety. A DLT in 0-1 of 6-8 or 2 of 9-10 would be considered tolerable. At the sponsor's discretion (investigator consultation), an additional 3 patients may be enrolled at any dose level for further evaluation for safety.

[0341] If 1.0 mg / kg mAb1 (dose level 1; DL1) is deemed safe (after 3–7 patients), enrollment will begin with 3.0 mg / kg mAb1 (DL2). After enrollment of 4 patients in DL2, enrollment in the first combination cohort (1.0 mg / kg mAb1 + 3.0 mg / kg REGN2810; DL3) can begin. Once 3.0 mg / kg mAb1 (DL2) is deemed safe, dose escalation to 10 mg / kg mAb1 (DL4) can begin. The second combination cohort (3.0 mg / kg mAb1 + 3.0 mg / kg REGN2810; DL5) will be enrolled only after both DL2 and DL3 are deemed safe. The third combination cohort (10 mg / kg mAb1 + 3.0 mg / kg REGN2810; DL6) will be enrolled only after both DL4 and DL5 are deemed safe. If multiple cohorts are opened simultaneously, prioritize lower numbered cohorts (i.e., DL2 over DL3 or DL3 over DL4).

[0342] Dose-limiting toxicity To initiate a dose escalation or a new combination therapy, the DLT observation period to determine safety is defined as a 28-day period beginning on cycle 1, day 1, intended to monitor the safety and tolerability of the first two doses of study drug (mAb1 with or without REGN2810, as appropriate). To be evaluable for DLT, patients must receive at least the first two doses of study drug (i.e., days 1 and 22) before completing the DLT period, be monitored for at least 28 days after the first dose, and be monitored for at least 7 days after the second dose or experience a DLT (defined below). A delay in administration of the second dose of study drug beyond day 35 and / or interruption of study drug is considered a DLT if it is study drug related. Thus, for patients whose second dose is delayed and for patients who experience an AE, the duration of the DLT observation period will be longer, and the duration must also be evaluated to determine whether the event was a DLT.

[0343] In addition to not being able to administer Dose #2 within the window (due to toxicity of the study drug), a DLT is generally defined as any of the following toxicities related to the study drug:

[0344] Non-hematological toxicity: Grade ≥2 uveitis Any non-hematologic toxicity of grade ≥ 3 (excluding clinically insignificant laboratory abnormalities such as asymptomatic elevations of amylase or lipase)

[0345] Hematological toxicity: Grade 4 neutropenia lasting >7 days Grade 4 thrombocytopenia Grade 3 thrombocytopenia with bleeding Febrile neutropenia of grade ≥3 or neutropenia of grade ≥3 with documented infection

[0346] Any irAE or non-irAE that meets the definition of a DLT will be considered a DLT.

[0347] maximum capacity The MTD is defined as the dose level immediately below which dosing is stopped due to DLT in 2 or more of 6-7 evaluable patients and is determined for monotherapy and combination therapy on an individual basis. However, due to the known occurrence of AEs due to monotherapy with REGN2810 and other PD-1 / PD-L1 antibodies, the intensity, frequency, and novelty of combination toxicity may be considered when determining the MTD for combination cohorts and deciding to add additional patients at the dose level. If dose escalation is not stopped due to DLT, the MTD is considered not determined. Each of the monotherapy and combination cohorts deemed tolerable at the highest dose level will enroll 3 more patients (i.e., 6-10 patients in each of these cohorts). If dose escalation for mAb1 monotherapy or combination therapy with REGN2810 is stopped at 1.0 mg / kg due to DLT, the cohort will enroll at the 0.3 mg / kg dose. If dose escalation for mAb1 monotherapy or combination therapy is stopped at the 3.0 or 10 mg / kg dose level due to DLT, newly enrolled patients (in the monotherapy or combination therapy cohort, respectively) will have the dose of mAb1 reduced to the dose level already examined. No patient will be allowed to start combination therapy at a dose of mAb1 that they were unable to tolerate as monotherapy.

[0348] Recommended Phase 2 Dosage The RP2D for the expansion cohorts will not be higher than the MTD or the highest dose tested and may differ for the monotherapy and combination therapy cohorts. The determination of the RP2D will be based on safety and PK data.

[0349] Patients in the monotherapy cohort who tolerate four doses of mAb1 and have an initial tumor assessment of at least SD, but subsequently demonstrate PD, have the option to add 3.0 mg / kg REGN2810 to the highest dose (if known) of mAb1 safely administered to that point in an attempt to "rescue" the response from the combination of LAG-3 and PD-1 blockade.

[0350] Expansion cohort Once tolerability of mAb1 has been established, alone and in combination with REGN2810, multiple expansion cohorts using monotherapy or combination therapy in select indications (non-small cell lung cancer (NSCLC), clear cell renal carcinoma (ccRCC), triple-negative breast cancer (TNBC), melanoma, and diffuse large B-cell lymphoma (DLBCL)) will be enrolled to further evaluate safety and obtain preliminary evidence of antitumor activity in tumors known to express or overexpress LAG3; where antitumor activity of mAb1 alone or in combination with REGN2810 may occur. Enrollment in expansion cohorts will begin after dose escalation is complete. After confirmation of the RP2D, an additional 10 expansion cohorts (Table 19) utilizing a two-stage design according to Simon (1989, Controlled Clinical Trials, 10:1-10) will be enrolled. Patients will be assigned to specific treatment cohorts based on the patient's tumor type, the presence or absence of prior anti-PD-1 / anti-programmed cell death ligand 1 (PD-L1) therapy, the investigator's assessment of the suitability of the treatment regimen for the patient, and the availability of patient slots in the assigned treatment cohort. Enrollment may be stopped if a safety issue arises in an individual expansion cohort during the first stage of the Simon two-stage design. After discussion between the investigator and Regeneron, enrollment may be resumed at the same or lower dose of mAb1. Safety issues triggering discontinuation may include early or late safety events. Patients will be treated with mAb1 (at a dose determined by dose escalation findings) monotherapy or with mAb1 in combination with REGN2810 at 3 mg / kg Q3W for up to 51 weeks. Enrollment in the second stage for each expansion cohort will occur only if a minimum number of tumor responses are observed during the first stage. [Table 19]

[0351] Study population Study population in the dose escalation phase: Patients with progressive malignancies who have not been previously treated with anti-LAG3 and / or PD-1 / PD-L1 inhibitors and who are not candidates for standard of care or for which no viable treatment is predicted to provide clinical benefit, and patients with malignancies that are incurable, have not responded to standard of care, or have shown tumor progression despite standard therapy.

[0352] Study population in dose expansion cohort: Patients with select malignancies who had not received prior treatment with anti-LAG3 agents. · Patients who have not previously been treated with anti-PD-1 / PD-L1 but are suitable candidates for anti-PD-1-based therapy (cohorts 1, 3, 5, 6, and 9); or Patients who had previously received anti-PD-1 / PD-L1-based therapy and had a confirmed objective response (CR or PR) or SD for at least 3 months on anti-PD-1 / PD-L1 therapy, but subsequently progressed on that treatment, or had SD or PR as their best response followed by a stable response for 6 months (Cohorts 2, 4, 7, and 10); or Patients who are not candidates for standard of care or for whom there is no available treatment predicted to provide clinical benefit and who are suitable for mAb1 monotherapy (Cohort 8).

[0353] Inclusion Criteria: Patients must meet the following criteria to be eligible for inclusion in the study: 1. Male or female aged ≥ 18 years; 2. Dose escalation cohort: Patients with a histologically or cytologically confirmed diagnosis of malignant disease (including lymphoma) with documented tumor progression, for which there is no alternative therapeutic option with the standard of care or for which there is no alternative standard of care with the potential for cure (i.e., have failed to respond or have developed tumor progression despite standard therapy), and who have not been previously treated with a PD-1 / PD-L1 inhibitor. These patients do not require measurable disease according to RECIST 1.1 or Lugano criteria. 3. Dose expansion cohort: Patients with a histologically or cytologically confirmed diagnosis of one of the following tumors with measurable disease according to RECIST 1.1 or Lugano criteria and who meet the following criteria: i. Patients with stage IIIB or IV NSCLC who have not undergone anti-PD-1 / PD-L1 and who have no prior treatment for metastatic disease or who have disease progression / recurrence after one platinum-containing regimen (Cohort 1); ii. Anti-PD-1 / PD-L1 * , patients with stage IIIB or IV NSCLC with no more than two prior therapies for metastatic disease (cohort 2); iii. Patients with advanced or metastatic ccRCC with a clear cell component who have not undergone anti-PD-1 / PD-L1 and who have received one or two prior regimens of antiangiogenic therapy (Cohort 3); iv. Anti-PD-1 / PD-L1 * , patients with advanced or metastatic ccRCC with a clear cell component who had received one to two prior regimens of antiangiogenic therapy (cohort 4); v. Patients with metastatic TNBC (estrogen, progesterone, and human epidermal growth factor receptor 2 negative) who have not undergone anti-PD-1 / PD-L1 and have received five or fewer prior lines of therapy (Cohort 5); vi. Patients with advanced or metastatic melanoma who have not undergone anti-PD-1 / PD-L1 and have received no more than two prior regimens for metastatic disease (Cohort 6); vii. Anti-PD-1 / PD-L1 * , patients with advanced or metastatic melanoma who had received no more than two prior regimens for metastatic disease (cohort 7); viii. Patients with relapsed / refractory DLBCL who have not undergone anti-PD-1 / PD-L1 and who have progressed after or are not candidates for autologous stem cell transplant (Cohorts 8 and 9) ix. Anti-PD-1 / PD-L1 * , patients with relapsed / refractory DLBCL who have progressed after or are not candidates for autologous stem cell transplantation (cohort 10); Note: * Patients who underwent anti-PD-1 / PD-L1 therapy were defined as having tolerated or being tolerating anti-PD-1 / PD-L1 therapy (i.e., not discontinued due to toxicity); CR / PR confirmed by repeat imaging or SD for at least 12 weeks from the first dose prior to disease progression. Disease progression must have occurred during anti-PD-1 / PD-L1 treatment or within 12 weeks of the last dose. b. Demonstration of disease control during anti-PD-1 / PD-L1 therapy, defined as either SD or PR as the best response, with a stable response (no more than 70% decrease from baseline) for the following 6 months. 4.Eastern Cooperative Oncology Group performance status of 0 or 1; 5. Life expectancy is at least 3 months; 6. Adequate organ and bone marrow function as follows: (a) hemoglobin ≥ 9.0 g / dL; (b) absolute neutrophil count ≥ 1.5 x 10 per L 9(c) platelet count per liter ≥ 75 x 10 9 (d) Serum creatinine ≤ 1.5 × upper limit of normal (ULN) or 1.73 m 2 (e) estimated glomerular filtration rate >50 mL per minute; (f) total bilirubin ≤1.5×ULN; (g) aspartate aminotransferase and alanine aminotransferase (ALT) ≤3×ULN, or in the case of liver metastases, aspartate aminotransferase and alanine aminotransferase (ALT) ≤5×ULN; and / or (h) alkaline phosphatase ≤2.5×ULN (or in the case of liver or bone metastases, alkaline phosphatase ≤5.0×ULN); 7. Willing and able to comply with clinic visits and study related procedures; 8. Provide signed informed consent must be satisfied.

[0354] Exclusion Criteria: The following criteria were met: 1. Currently receiving treatment in another study, or have participated in a study with an investigational drug and received treatment or used an investigational device within 4 weeks of the first dose of study treatment, or have received treatment with an approved systemic therapy within 3 weeks of the first dose of study treatment, or have received any prior systemic therapy within 5 half-lives of the first dose of study treatment (whichever is longer). For expansion cohorts 2, 4, 7, and 10 (via anti-PD-1 / PD-L1) only, prior anti-PD-1 / PD-L1 therapy, regardless of drug half-life or approval status, must not have been administered within 3 weeks of the first dose of study treatment; 2. Prior treatment with any biologic or small molecule targeting LAG-3; 3. Radiation therapy within 2 weeks prior to randomization and no recovery to baseline from any radiation-related AEs; 4. Expansion cohort only: Progressing or requiring active treatment, another malignancy excluding non-melanomatous skin cancer or in situ cervical cancer that has been treated with potentially curative therapy, or any other tumor that has been deemed effectively treated with definitive local control for at least 2 years prior to enrollment; 5. Untreated or active central nervous system metastases. Patients with previously treated CNS metastases may also be enrolled, provided they are stable (i.e., no evidence of progression by imaging for at least 6 weeks prior to the first dose of study treatment and any neurological symptoms have returned to baseline), have no evidence of new or spreading CNS metastases, and have not required systemic corticosteroids for management of CNS metastases within 4 weeks prior to the first dose of REGN2810; 6. Encephalitis, meningitis, or uncontrolled seizures in the year prior to informed consent; 7. Ongoing or recent (within 5 years) evidence of significant autoimmune disease that has required treatment with systemic immunosuppressive measures that may indicate risk of irAEs.The following do not require exclusion: vitiligo, resolved childhood asthma, hypothyroidism requiring only hormone replacement therapy, type 1 diabetes mellitus or psoriasis not requiring systemic treatment;8. Corticosteroid treatment (>10 mg prednisone per day or equivalent) within 1 week prior to first dose of study medication. Patients requiring a short course of steroids will not be excluded;9. Known history or any evidence (within the past 5 years) of interstitial lung disease or active noninfectious pneumonia;10. Uncontrolled human immunodeficiency virus infection, hepatitis B infection, or hepatitis C infection; or diagnosis of immunodeficiency;11. Active infection requiring systemic therapy;12. Receipt of live vaccine within 30 days prior to planned initiation of study medication;13. Major surgery, open biopsy, or significant traumatic injury within 4 weeks prior to screening;14. Study treatment Patients who meet any of the following criteria will be excluded from the study: 1. Myocardial infarction within 9 months prior to the first dose of study drug; 2. Previous allogeneic stem cell transplant; 3. Any medical condition that, in the opinion of the investigator, would not be in the patient's best interest to participate in the study; 4. Documented allergic or acute hypersensitivity reaction attributable to antibody treatment; 5. Known allergy to doxycycline or other tetracycline antibiotics; 6. Known psychiatric or substance abuse disorder that would prevent participation in the study according to its requirements; 7. Sexually active men or women of childbearing potential who are unwilling to use contraception during the study. Women who are pregnant, breastfeeding, or expect to become pregnant, or men who intend to father a child, within the planned duration of the study (screening visit to 180 days after the last dose of study drug); 8. Sexually active men or women of childbearing potential who are unwilling to use adequate contraception prior to the start of the first treatment, throughout the study, and for at least 6 months after administration of the last dose of study drug.

[0355] Study Procedures Monot...

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[Claim 1] The invention as depicted in the drawings.

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