human antibodies with high affinity for human angiopoietin-2, their use and pharmaceutical compositi

专利摘要:
HUMAN ANTIBODIES WITH HIGH AFFINITY FOR HUMAN ANGIOPOIETIN-2. The present invention provides antibodies that bind to agiopoeitin-2 (Ang-2) and methods of using them. According to certain embodiments of the invention, antibodies are fully human antibodies that bind to human Ang-2. The antibodies of the invention are useful, inter alia, for the treatment of diseases and disorders associated with one or more biological activities of Ang-2 including angiogenesis.
公开号:BR112012001984B1
申请号:R112012001984-8
申请日:2010-07-27
公开日:2020-10-27
发明作者:Gavin Thurston；Christopher Daly
申请人:Regeneron Pharmaceuticals, Inc；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The present invention relates to antibodies, and antigen-binding fragments thereof, which are specific for angiopoietin-2 (Ang-2). BACKGROUND
[0002] Angiogenesis is the biological process through which new blood vessels are formed. Aberrant angiogenesis is associated with a number of disease conditions including, for example, prolerative retinopathies, rheumatoid arthritis and psoriasis. Still, it is well established that angiogenesis is critical for tumor growth and maintenance. Angiopoietin-2 (Ang-2) is a ligand for the Tie-2 receptor (Tie-2) and has been shown to play a role in angiogenesis. Ang-2 is also referred to as a Tie-2 linker. (U.S. 5,643,755; Yancopoulos et al., 2000, Nature407: 242-248).
[0003] Antibodies and other peptide inhibitors that bind to Ang-2 are mentioned in U.S. 6,166,185; 7,521,053; 7,205,275; 2006/0018909 and 2006/0246071. There is a need in the art for new Ang-2 modulating agents, including antibodies to Ang-2, that can be used to treat diseases and conditions caused by or exacerbated by angiogenesis. BRIEF SUMMARY OF THE INVENTION
[0004] The present invention provides human antibodies that bind to human Ang-2. The present inventors, in view of various lines of evidence and research, have recognized the need for Ang-2 inhibitors that do not bind to or antagonize the related Ang-1 molecule. For example, previous studies have demonstrated or suggested a beneficial role for Ang-1 in hemostasis (see, for example, Li et al., 2001, Thrombosis and Haemostasis, 85: 191-374) and in the production of the adult vasculature against plasma leakage ( see, for example, Thurston et al., 2000, Nature Medicine 6: 460-463; Thurston et al., 1999, Science 286: 2511-2514). In this way, the present inventors recognized that, in certain antiangiogenic therapeutic situations, it may be beneficial to preserve the activity of Ang-1. In this way, the present invention provides antibodies that specifically bind to Ang-2, but do not substantially bind to Ang-1. The present invention also includes antibodies that block the interaction between Ang-2 and its Tie-2 receptor, but do not block the interaction between Ang-1 and Tie-2. The antibodies of the invention are useful, inter alia, for inhibiting Ang-2 angiogenesis-promoting activities and for treating diseases and disorders caused by or related to the angiogenesis process.
[0005] The antibodies of the invention can be full-length (for example, an IgG1 or IgG4 antibody) or can comprise only an antigen-binding portion (for example, a Fab, F (ab ') 2 or scFv fragment) and can be modified to affect functionality, for example, to eliminate residual effector functions.
[0006] In one embodiment, the invention comprises an antibody or antigen binding fragment of an antibody comprising a heavy chain variable region (HCRV) having an amino acid sequence selected from the group consisting of SEQ ID NO: 2.18, 22 , 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162,166, 170, 186, 190, 194, 210, 214, 218, 234 , 238, 242, 258, 262, 266,282, 286, 290, 306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382,386, 402, 406, 410, 426, 430, 434, 450 , 454, 458, 474, 478, 482, 498,502, 506, 514 and 516 or a sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or antigen-binding portion of an antibody comprises an HCRV having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, 42, 66, 162, 210, 266 and 434.
[0007] In one embodiment, the invention comprises an antibody or antigen binding fragment of an antibody comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 20, 24 , 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154,164, 168, 178, 188, 192, 202, 212, 216, 226, 236 , 240, 250, 260, 264,274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380,384, 394, 404, 408, 418, 428, 432, 442, 452 , 456, 466, 476, 480, 490,500 and 504 or a sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or antigen-binding portion of an antibody comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 44, 68, 164, 212, 274 and 442.
[0008] In specific embodiments, the antibody or antigen binding fragment thereof comprises a pair of HCVR or LCVR amino acid sequence (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118 / 120,122 / 130, 138/140, 142/144, 146/154, 162/164, 166/168, 170 / 178,186 / 188, 190/192, 194/202, 210/212, 214/216, 218/226, 234 / 236,238 / 240, 242/250, 258/260, 262/264, 266/274, 282/284, 286 / 288,290 / 298, 306/308, 310/312, 314/322, 330/332, 334 / 336, 338 / 346,354 / 356, 358/360, 362/370, 378/380, 382/384, 386/394, 402 / 404,406 / 408, 410/418, 426/428, 430/432, 434/442, 450/452, 454 / 456,458 / 466, 474/476, 478/480, 482/490, 498/500 and 502/504. In a modality, the antibody or fragment thereof comprises an HCVR or LCVR selected from the amino acid sequence pairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212, 266 / 274 and 434/442.
[0009] In a following aspect, the invention provides an antibody or antigen-binding fragment of an antibody comprising a heavy chain CDR3 domain (HCDR3) having an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 32 , 56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320, 344, 368, 392, 416, 440, 464, 488 and 512 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) selected from the group consisting of SEQ ID NO: 16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448, 472 and 496 or a sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00010] In certain embodiments, the antibody or antigen binding portion of an antibody comprises a pair of amino acid sequence HCDR3 // LCDR3 selected from the group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 80/88, 104/112, 128/136, 152/160, 176/184, 200/208, 224/232, 248/256, 272/280, 296/304, 320/328, 344/352, 368 / 376, 392/400, 416/424, 440/448, 464/472 and 488/496. In one embodiment, the antibody or antigen-binding portion of an antibody comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of 8/16, 32/40, 56/64, 152/160, 200/208, 272/280 and 440/448. Non-limiting examples of anti-Angi-2 antibody having such HCDR3 / LCDR3 pairs are antibodies called H1H685, H1H690, H1H691, H1H696, H1H706, H1M724 and H2M744, respectively.
[00011] In a further embodiment, the invention comprises an antibody or fragment thereof further comprising an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 28, 52, 76, 100, 124, 148 , 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436, 460, 484 and 508 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 domain (HCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 30, 54, 78, 102, 126, 150, 174, 198, 222, 246, 270, 294, 318, 342, 366, 390, 414, 438, 462, 486 and 510 or a sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276, 300, 324, 348, 372, 396 , 420, 444, 468 and 492 or a sequence substantially similar to it having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 38, 62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326, 350, 374 , 398, 422, 446, 470 and 494 or a sequence substantially similar to the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00012] Certain exemplary, non-limiting antibodies and antigen-binding fragments of the invention comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 domains, respectively, selected from the group consisting of: (i) SEQ ID NO: 4, 6 , 8, 12, 14 and 16 (for example, H1H685); (ii) SEQ ID NO: 28, 30, 32, 36, 38 and 40 (for example, H1H690); (iii) SEQ ID NO: 52, 54, 56, 60, 62 and 64 (for example, H1H691); (iv) SEQ ID NO: 148, 150, 152, 156, 158 and 160 (for example, H1H696); (v) SEQ ID NO: 196, 198, 200, 204, 206 and 208 (for example, H1H706); (vi) SEQ ID NO: 268, 270, 272, 276, 278 and 280 (for example, H1M724); and (vii) SEQ ID NO: 436, 438, 440, 444, 446 and 448 (for example, H2M744).
[00013] In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to Ang-2, wherein the antibody or fragment comprises the heavy and light chain CDR domains (i.e. , CDR1, CDR2 and CDR3) contained within the heavy and light chain variable domain sequences selected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46 / 48, 50/58, 66/68, 70/72, 74 / 82.90 / 92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142 / 144,146 / 154, 162/164, 166/168, 170/178, 186/188, 190/192, 194 / 202,210 / 212, 214/216, 218/226, 234/236, 238/240, 242/250, 258 / 260,262 / 264, 266/274, 282/284, 286/288, 290/298, 306/308, 310 / 312,314 / 322, 330/332, 334/336, 338/346, 354/356, 358/360, 362 / 370,378 / 380, 382/384, 386/394, 402/404, 406/408, 410/418, 426 / 428,430 / 432, 434/442, 450/452, 454/456, 458/466, 474 / 476, 478 / 480,482 / 490, 498/500 and 502/504. In one embodiment, the antibody or fragment thereof comprises the CDR sequences contained within HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164 , 210/212, 266/274 and 434/442.
[00014] In another aspect, the invention provides nucleic acid molecules encoding anti-Ang-2 antibodies or fragments thereof. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also understood by the invention, as well as the methods of producing antibodies by culturing the host cells under conditions that allow the production of antibodies and recovery of antibodies produced.
[00015] In one embodiment, the invention provides an antibody or fragment thereof comprising an HCRV encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 17, 21.25, 41.45, 49, 65 , 69, 73, 89, 93, 97, 113, 117, 121, 137,141, 145, 161, 165, 169, 185, 189, 193, 209, 213, 217, 233, 237, 241,257, 261, 265, 281 , 285, 289, 305, 309, 313, 329, 333, 337, 353, 357,361, 377, 381, 385, 401, 405, 409, 425, 429, 433, 449, 453, 457, 473,477, 481,497, 501,505 , 513 and 515 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with it. In one embodiment, the antibody or fragment thereof comprises an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, 41.65, 161, 209, 265 and 433.
[00016] In one embodiment, the invention provides an antibody or fragment thereof comprising an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, 19, 23, 33,43,47, 57, 67 , 71,81,91,95, 105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187, 191, 201, 211, 215, 225, 235, 239, 249,259, 263, 273 , 283, 287, 297, 307, 311, 321, 331, 335, 345, 355, 359,369, 379, 383, 393, 403, 407, 417, 427, 431, 441, 451, 455, 465, 475,479, 489 , 499 and 503 or a sequence substantially identical to the same having at least 90%, at least 95%, at least 98% or at least 99% identity with the same. In one embodiment, the antibody or fragment thereof comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19, 43, 67, 163, 211,273 and 441.
[00017] In one embodiment, the invention provides an antibody or antigen binding fragment of an antibody comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, 31.55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343, 367, 391,415, 439, 463, 487 and 511 or a substantially identical sequence having at least 90%, at least 95%, at least 98 % or at least 99% identity with the same; and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303, 327, 351,375, 399, 423, 447, 471 and 495 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with it. In one embodiment, the antibody or fragment thereof comprises HCDR3 and LCDR3 sequences encoded by the nucleic acid sequence pairs selected from the group consisting of SEQ ID NO: 7/15, 31/39, 55/63, 151/159, 199 / 207, 271/279 and 439/447.
[00018] In an additional embodiment, the antibody or fragment thereof further comprises: an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 27, 51, 75, 99, 123, 147, 171, 195, 219, 243, 267, 291,315, 339, 363, 387, 411,435, 459, 483 and 507 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with it; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, 29, 53, 77, 101, 125, 149, 173, 197, 221, 245, 269, 293, 317, 341, 365 , 389, 413, 437, 461, 485 and 509 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with the same; an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 11.35, 59, 83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347 , 371, 395, 419, 443, 467 and 491 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with the same; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181, 205, 229, 253, 277, 301, 325, 349, 373, 397, 421,445, 469 and 493 or a substantially identical sequence having at least 90%, at least 95%, at least 98% or at least 99% identity with the same.
[00019] In one embodiment, the antibody or fragment thereof comprises the heavy and light chain CDR sequences encoded by the nucleic acid sequences of SEQ ID NO: 17 and 19; SEQ ID NO: 41 e43; SEQ ID NO: 65e67; SEQ ID NO: 161 and 163; SEQ ID NO: 209 and 211; SEQ ID NO: 265 and 273; or SEQ ID NO: 433 and 441.
[00020] The invention comprises anti-Ang-2 antibodies having a modified glycosylation pattern. In some applications, modification to remove unwanted glycosylation sites may be useful. For example, the present invention comprises modified versions of any antibody shown here in which the modified version does not have a fucose portion present in the oligosaccharide chain, for example, to increase the function of antibody dependent cell cytotoxicity (ADCC) (Antibody Dependent Cellular Cytotoxicity) (see Shield et al. (2002) JBC 277: 26733). In other applications, modification of galactosylation can be done in order to modify Complement Dependent Cytotoxicity (CDC).
[00021] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to Ang-2 and a pharmaceutically acceptable carrier or diluent. In a related aspect, the invention relates to a composition that is a combination of an Ang-2 inhibitor and a second therapeutic agent. In one embodiment, the Ang-2 inhibitor is an antibody or fragment thereof. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an Ang-2 inhibitor. Exemplary agents that can be advantageously combined with an Ang-2 inhibitor include, without limitation, any agent that inhibits or reduces angiogenesis, other therapeutic agents for cancer, anti-inflammatory agents, cytokine inhibitors, factor inhibitors growth, anti-hematopoietic factors, non-steroidal anti-inflammatory drugs (NSAIDs) (Non-steroidal Anti-inflammatory Drugs), antiviral agents and antibiotics.
[00022] In yet another aspect, the invention provides methods for inhibiting Ang-2 activity using the anti-Ang-2 antibody or antigen binding portion of the antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen binding fragment of an antibody of the invention. The disorder to be treated is any disease or condition that is improved, improved, inhibited or prevented by removing, inhibiting or reducing Ang-2 activity. Preferably, the angi-Ang-2 antibody or antibody fragment of the invention is useful for treating any disease or condition caused by, associated with or perpetuated by the process of angiogenesis. In certain embodiments of the invention, anti-Ang-2 antibodies or antigen binding portions thereof are useful for the treatment of cancer. In the context of cancer therapies, the anti-Ang-2 antibodies of the invention or their antigen binding portions can be administered alone or in combination with other therapeutic anti-cancer antibodies, chemotherapeutic agents and / or radiation therapy. In other embodiments of the present invention, anti-Ang-2 antibodies or antigen-binding fragments thereof are useful for the treatment of one or more eye disorders, for example, age-related macular degeneration, diabetic retinopathy, etc., and / or one or more inflammatory or infectious diseases.
[00023] Other modalities will become apparent from a review of the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS
[00024] Figure 1 is an alignment of the at least 88 C-terminal amino acids of human Ang-2 (residues 409 to 496 of SEQ ID NO: 518) with the corresponding amino acid sequence of human Ang-1 (SEQ ID NO: 531). Residues that differ between hAng-1 and hAng-2 are indicated by white text and black shadow. Asterisks (*) indicate the amino acids of hAng-2 that have been shown to interact with Tie-2 through crystal structure analysis. See Barton et al., Nat. Struct. Mol. Biol. 73: 524-532 (2006). Triangles (A) indicate the amino acid positions interacting with Tie-2 that differ between hAng-2 and hAng-1.
[00025] Figure 2 (Panels A-C) shows the results of Western blots that illustrate the degree to which Ang-2 binding molecules inhibit, or fail to inhibit, Ang-1 induced Tie-2 phosphorylation.
[00026] Figure 3 is a summary of the Ang-2F-mFc point mutant binding experiment in Example 13 showing the amino acid changes that resulted in a greater than five-fold reduction in dissociation Ti / 2 (shown by solid circles • ) with respect to the wild type for the various antibodies and peptibodies tested. DETAILED DESCRIPTION
[00027] Before the present invention is described, it should be understood that the present invention is not limited to the particular experimental methods and conditions described, since such methods and conditions may vary. It should also be understood that the terminology used here is for the purpose of describing particular modalities only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[00028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which the present invention belongs. As used herein, the term "about", when used with reference to a particular mentioned numerical value, means that the value may vary from the mentioned value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (for example, 99.1, 99.2, 99.3, 99.4, etc.).
[00029] While any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Definitions
[00030] As used herein, the term "angiopoietin-2" or "Ang-2", unless specified as being of a non-human species (for example, "Mouse Ang-2", "Ang-2 of monkey ", etc.), refers to a human Ang-2 or a biologically active fragment thereof (for example, a fragment of the Ang-2 protein that is capable of inducing angiogenesis in vitro or in vivo). Human Ang-2 is encoded by the nucleic acid sequence shown in SEQ ID NO: 517 and has the amino acid sequence of SEQ ID NO: 518. The amino acid sequences of mouse and monkey Ang-2 proteins are available from the NCBI protein sequence database under Nos. Access Code NPJD31452 and BAE89705.1, respectively.
[00031] The term "angiopoietin-1" or "Ang-1", unless specified as being of a non-human species (e.g., "mouse Ang-1", "monkey Ang-1, etc. ), refers to a human Ang-1 or a biologically active fragment thereof. Human Ang-1 has the amino acid sequence as shown in the NCBI protein sequence database under Accession No. AAB50557. The term " Tie-2 "(also referred to in the art as" TEK ") unless specified as being of a non-human species (for example," Mouse Tie-2 "," Monkey Tie-2 ", etc.), to a human Tie-2 or a biologically active fragment thereof Human tie-2 has the amino acid sequence as shown in the NCBI protein sequence database under Accession No. AAA61130.
[00032] The term "antibody" as used herein is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as their multimers. (for example, IgM). Each heavy chain comprises a heavy chain variable region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated here as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain (Ci_1). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determination regions (CDRs) (Complementarity Determining Regions), interspersed with regions that are more conserved, called structure regions (FR) (Framework Regions). VH and VL is composed of three CDRs and four FRs arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-Ang-2 antibody (or its antigen binding portion) can be identical to human germline sequences or can be naturally or artificially modified. A consensus amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs.
[00033] The term "antibody", as used herein, also includes antigen-binding fragments of entire antibody molecules. The terms "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that binds specifically to an antigen to form a complex. Antigen binding fragments of an antibody can be derived, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding variable and optionally constant domains of antibody. Such DNA is known and / or is readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries) or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
[00034] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab ') 2j fragments (iii) Fd fragments; (iv) Fv fragments; (v) single-stranded Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (for example, an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, tribodies, tetribodies and minibodies, are also included within the term "antigen binding fragment", as used here.
[00035] An antigen binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or in structure with one or more main structure sequences. In antigen binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be located with respect to each other in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL OR VL-VL dimers. Alternatively, the antigen binding fragment of an antibody may contain a monomeric VH OR VL domain.
[00036] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Exemplary, non-limiting configurations of variable and constant domains that can be found within an antigen binding fragment of an antibody of the present invention include: (i) VH-CH1; (Ü) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to each other or can be linked by an integral or partial hinder region or ligand. An impairment region can consist of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible link between variable domains and / or adjacent constants in a polypeptide molecule only. In addition, an antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or more VH OR Vimonomer domain (for example, by disulfide bond (s)).
[00037] As with integral antibody molecules, antigen binding fragments can be monospecific or multispecific (for example, bispecific). A multi-specific antigen binding fragment of an antibody will typically comprise at least two different variable domains, each variable domain capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine techniques available in the field.
[00038] An antibody constant region is important in an antibody's ability to fix complement and mediate cell-dependent cytotoxicity. In this way, the isotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.
[00039] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced through random or site specific mutagenesis in vitro or via somatic mutation in vivo), for example, in CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been inserted into human structure sequences.
[00040] The term "recombinant antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected in a host cell (described below), antibodies isolated from a recombinant, combinatorial human antibody library (described below), antibodies isolated from an animal (for example, a mouse) that is transgenic to human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res.20: 6287-6295) or antibodies prepared, expressed, raised or isolated by other means that involve binding human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, somatic mutagenesis in vivo) and then the amino acid sequences of the VH and VL regions of the Recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[00041] Human antibodies can exist in two forms that are associated with fold heterogeneity. In one form, an immunoglobulin molecule comprises a construct of four stable chains of approximately 150-160 kDa in which the dimers are held together by an inter-chain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of coupled light and heavy chains (half antibody). These forms have been extremely difficult to separate, even after affinity purification.
[00042] The frequency of appearance of the second form in various IgG isotypes is due to, but not limited to, structural differences associated with the isotype of the antibody fold region. A single amino acid substitution in the fold region of the human IgG4 fold can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology30: 105) to levels typically seen using a human IgG1 fold. The present invention comprises antibodies having one or more mutations in the fold, CHh or CH3 region that may be desirable, for example, in production, to improve the yield of the desired antibody form.
[00043] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to human Ang-2 or a fragment of Human Ang-2 is substantially free of antibodies that specifically bind to antigens other than human Ang-2). The term "specifically binds", or similar, means that an antibody or antigen binding fragment likewise forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by a KD of about 1x10'8 M or less. Methods for determining whether two molecules specifically bind are well known in the field and include, for example, equilibrium dialysis, surface plasmon resonance and the like. An isolated antibody that specifically binds to human Ang-2, however, is cross-reactive with other antigens, such as Ang-2 molecules from other species. In addition, an isolated antibody can be substantially free of other cellular material and / or chemical agents.
[00044] An "neutralizing" or "blocking" antibody, as used herein, is intended to refer to an antibody whose binding to Ang-2 blocks the interaction between Ang-2 and its receptor (Tie-2) and / or results in inhibition of at least one biological function of Ang-2. The inhibition caused by an Ang-2 neutralizing or blocking antibody need not be complete as long as it is detectable using an appropriate assay. Exemplary assays for detecting Ang-2 inhibition are described elsewhere here.
[00045] The fully human anti-Ang-2 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the CDR structure and / or regions of the heavy and light chain variable domains compared to the line sequences corresponding germline. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed here with germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and the antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, in which one or more amino acids within one or more of the main structure and / or regions of CDR are reassembled for the corresponding germline residue (s) or for a conservative amino acid substitution (natural or unnatural) for the corresponding germline residue (s) (such sequence changes are referred to here such as "germline retromutations"). A person skilled in the art, starting with the variable region sequences of heavy and light chains disclosed here, can easily produce several antibodies and antigen binding fragments that comprise one or more individual germline retromutations or combinations thereof. In certain embodiments, all of the main structure and / or CDR residues within the VH and / or VL domains are reassigned to the germline sequence. In other embodiments, only certain residues are reassigned to the germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In addition, the antibodies of the present invention may contain any combination of two or more germline retromutations within the main structure and / or CDR regions, that is, in which certain individual residues are retromuted into the germline sequence while certain other residues that differ from the germline sequence are maintained. Once obtained, antibodies and antigen-binding fragments that contain one or more germline retromutations can be easily tested for one or more desired properties such as binding specificity, increased binding affinity, improved or increased antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen binding fragments obtained in this general manner are understood in the present invention.
[00046] The present invention also includes anti-Ang-2 antibodies comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-Ang-2 antibodies having HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., substitutions of conservative amino acids with respect to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed here. In one embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO: 18 with 8 or less conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO: 18 with 6 or less conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO: 18 with 4 or less conservative amino acid substitutions. In another embodiment, the antibody comprises an HCVR having the amino acid sequence of SEQ ID NO: 18 with 2 or less conservative amino acid substitutions. In one embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO: 20 with 8 or less conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO: 20 with 6 or less conservative amino acid substitutions. In another embodiment, the antibody comprises an LCVR having the amino acid sequence of SEQ ID NO: 29 with 4 or less conservative amino acid substitutions. In another embodiment, the antibody comprising an LCVR having the amino acid sequence of SEQ ID NO: 20 with 2 or less conservative amino acid substitutions.
[00047] The term "surface plasmon resonance", as used here, refers to an optical phenomenon that allows the analysis of interactions in real time by detecting changes in protein concentrations with a biosensor matrix, for example , using the BIAcore® system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
[00048] The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
[00049] 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 can have more than one epitope. In this way, different antibodies can bind to different areas in an antigen and can have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include portions of saccharides, phosphoryl groups, or sulfonyl groups in the antigen.
[00050] The term "substantial identity" or "substantially identical", when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand) , there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99%, of the nucleotide bases, as measured by any well sequence identity algorithm known, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule cell having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar sequence as the polypeptide encoded by the reference nucleic acid molecule.
[00051] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as through the GAP or BESTFIT program using default gap weights, share at least 95% of sequence identity, more preferably still at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (group R) with similar chemical properties (for example, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percentage sequence identity or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic hydroxyl side chains: serine or 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: aspartate and glutamate; and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the log PAM250 probability matrix disclosed in Gonnet and others (1992) Science 256: 1443-1445. A "moderately conservative" substitution is any change having a non-negative value in the log PAM250 probability matrix.
[00052] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software compares similar sequences using measures of similarity given 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 or a mutein from it. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using recommended or default parameters, a program in GCG Version 6.1. FASTA (for example, FASTA2 and FASTA3) provides alignments and percent identity of the regions of the best overlap between the query and search sequences (Pearson (2000), supra). Another preferred algorithm when comparing a sequence of the invention with a database comprising a large number of sequences of different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) , J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402. Preparation of Human Antibodies
[00053] Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to human Ang-2 and that have one or more antigen-binding and / or functional characteristics of any of the anti-Ang-2 antibodies copies revealed here.
[00054] Using VELOCIMMUNE® technology or any other method for generating monoclonal antibodies, chimeric antibodies of high affinity to Ang-2 are initially isolated having a human variable region and a constant mouse region. As in the experimental section below, antibodies are characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example, wild-type or modified IgG1 or IgG4. Although the selected constant region may vary according to specific use, antigen binding characteristics with high affinity and target specificity reside in the variable region. Bioequivalents
[00055] The anti-Ang-2 antibodies and antibody fragments of the present invention comprise proteins having amino acid sequences that vary from those of the described antibodies, but which retain the ability to bind to human Ang-2. Such variant antibodies and antibody fragments comprise one or more amino acid additions, deletions or substitutions when compared to the original sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the anti-Ang-2 antibody encoding DNA sequences of the present invention comprise sequences that comprise one or more nucleotide additions, deletions or substitutions when compared to the disclosed sequence, but which encode an anti-Ang- 2 or antibody fragment that is essentially bioequivalent to an anti-Ang-2 antibody or antibody fragment of the invention. Examples of such amino acid and variant DNA sequences are discussed above.
[00056] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and degree of absorption do not show a significant difference when administered in the same molar dose under similar experimental conditions, or single dose or multiple dose. Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in the degree of their absorption, but not in their absorption rate and can still be considered bioequivalent because such differences in the absorption rate are intentional and are reflected in the label, are not essential for obtaining effective drug concentrations in the body in, for example, chronic use, and are considered medically insignificant for the particular drug product studied.
[00057] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.
[00058] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can change one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity , or less efficacy, compared to continued therapy without such a change.
[00059] In one embodiment, two antigen-binding proteins are bioequivalent if they both act through a mechanism or mechanisms of action common to the condition or conditions of use, to the extent that such mechanisms are known.
[00060] Bioequivalence can be demonstrated using in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) an in vivo test in humans and other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been related to and is reasonably predictive of bioavailability data in vivo; (c) an in vivo test on humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical test that establishes the safety, effectiveness or bioavailability or bioequivalence of an antibody.
[00061] Bioequivalent variants of anti-Ang-2 antibodies of the invention can be constructed by, for example, carrying out various substitutions of residues or sequences or deletion of terminal or internal residues or sequences not necessary for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds when renatured. Biological and Therapeutic Characteristics of Antibodies
[00062] In general, the antibodies of the present invention bind to human Ang-2 with a KD of less than 100 pM, typically with a KD of less than 50 pM and, in certain embodiments, with a Kode less than 40 pM, when measured by binding to antigen or immobilized in solid phase or in solution phase.
[00063] In addition, certain exemplary anti-Ang-2 antibodies of the invention may exhibit one or more of the following characteristics: (1) ability to bind to human Ang-2, but not to mouse Ang-2; (2) ability to bind to human Ang-2 and mouse Ang-2; (3) ability to bind to human Ang-2, but not to human Ang-1, -3 or -4; (4) ability to bind to human Ang-2, but not to mouse Ang-1, -3 or -4; (5) ability to bind to human Ang-2 and human Ang-1, -3 or -4; (6) ability to bind to human Ang-2 and mouse Ang-2, -3 or -4; (7) availability to block binding of human Ang-2 to human Tie-2; (8) ability to block binding of human Ang-2 to human Tie-2; (9) ability to block binding of human Ang-2 to human Tie-2; (10) ability to block binding of mouse Ang-2 to mouse Tie-2; (11) ability to block binding of human Ang-1 to human Tie-2; (12) ability to block binding of human Ang-1 to mouse Tie-2; (13) ability to block mouse Ang-1 binding to human Tie-2: (14) ability to block mouse Ang-1 binding to mouse Tie-2; (15) ability to inhibit phosphorylation induced by human Ang-2 of human Tie-2; (16) ability to inhibit mouse Tie-2-induced human Ang-2 phosphorylation; (17) ability to inhibit Ang-2-induced phosphorylation of human Tie-2 mice; (18) ability to inhibit tie-2 mouse Ang-2 induced phosphorylation; (19) ability to inhibit phosphorylation induced by human Ang-1 of human Tie-2; (20) ability to inhibit phosphorylation induced by human mouse Tie-2 Ang-1; (21) ability to inhibit phosphorylation induced by human Ang-1 of human Tie-2; (22) ability to inhibit mouse Tie-2-induced Ang-1 phosphorylation; (23) ability to inhibit angiogenesis in vivo in an experimental model (eg, angiogenesis induced by a Matrigel buffer containing MCF-7 cells subcutaneously implanted in nude mice); and / or (24) ability to inhibit or decrease tumor volume in a mouse xenograft model.
[00064] The present invention also includes antibodies that bind with high affinity to a construct comprising the Ang-2 fibronectin-like domain, but without the N-terminal coiled-coil domain of Ang-2 ( such constructs are referred to here as "Ang-2FD"). Exemplary Ang-2FD constructs include human Ang-2FD (SEQ ID NO: 519), mouse Ang-2FD (SEQ ID NO: 520) and monkey Ang-2FD (SEQ ID NO: 521). The human, mouse and monkey Ang-2FD constructs can be monomeric or dimeric. Ang-2FD constructs can also include other non-Ang-2 amino acid sequences such as a human or mouse Fc domain linked to Ang-2FD molecules. Another exemplary Ang-2FD construct is referred to here as "hBA2" (or human "bow-Ang2") which is a tetramer of fibrinogen-like domains of human Ang-2 associated with one another via a human Fc domain or mouse to form a "bow-tie" configuration. Typically, hBA2 consists of two Ang-2 dimers, where each Ang-2 dimer contains two Ang-2 fibronectin-like domains connected to each other via an Fc domain. Exemplary hBA2 components include polypeptides called hBA2-hlgG1 (SEQ ID NO: 52) and hBA2-mlgG2a (SEQ ID NO: 523). Unexpectedly, certain anti-Ang-2 antibodies of the present invention have been found to bind to Ang-2FD constructs with much greater affinities than a known Ang-2 control antibody (see Examples shown here).
[00065] High affinity binding, in the context of anti-Ang-2 antibody binding to a human or mouse dimeric Ang-2FD construct, means that the anti-Ang-2 antibody binds to Ang-2FD di- human or mouse serum with a KD of less than 300 pM. For example, anti-Ang-2 antibodies that bind with high affinity to human or mouse dimeric Ang-2FD include antibodies that bind to human or mouse dimeric Ang-2-FD with a KD of less than 300 pM, less than 250 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, less than 160 pM, less than 150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM or less than 50 pM , as measured at 25 ° C in a surface Plasmon resonance assay.
[00066] High affinity binding, in the context of anti-Ang-2 antibody binding to a monkey dimeric Ang-2FD construct, means that the anti-Ang-2 antibody binds to mouse dimeric Ang-2FD with a KD less than 500 pM. For example, anti-Ang-2 antibodies that bind with high affinity to mouse dimeric Ang-2FD include antibodies that bind to mouse Ang-2-FD with a KD of less than 500 pM, less than 450 pM , less than 400 pM, less than 350 pM, less than 300 pM, less than 250 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, less than 160 pm, less than 150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM, less than 90 pM or less than 80 pM, as measured at 25 ° C in a surface Plasmon resonance assay.
[00067] High affinity binding, in the context of binding anti-Ang-2 antibody to a human monomeric Ang-2FD construct, means that the anti-Ang-2 antibody binds to human monomeric Ang-2FD with a KD of less than 40 nM. For example, anti-Ang-2 antibodies that bind with high affinity to human monomeric Ang-2FD include antibodies that bind to monomeric Ang-2FD with a KD of less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM , less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM or less than 0.6 nM as measured at 25 ° C in a surface Plasmon resonance assay.
[00068] High affinity binding, in the context of anti-Ang-2 antibody binding to an hBA2 construct, means that the anti-Ang-2 antibody binds to hBA2 with a KD of less than 80 pM. For example, anti-Ang-2 antibodies that bind with high affinity to hBA2 include antibodies that bind to hBA2 with a KD of less than 80 pM, less than 75 pM, less than 70 pM, less than 65 pM, less than 60 pM, less than 55 pM, less than 50 pM, less than 45 pM, less than 40 pM, less than 35 pM, less than 30 pM, less than 25 pM, less than 20 pM, less than 18 pM, less than 16 pM, less than 14 pM or less than 12 pM, as measured at 25 ° C in a surface Plasmon resonance assay.
[00069] The present invention includes antibodies that bind to Ang-2, but do not substantially bind to Ang-1. As used herein, an antibody "does not substantially bind to Ang-1" if the antibody, when tested for binding to Ang-1 in a surface Plasmon resonance assay in which the antibody is captured in a human Ang-1 wild-type and full-length at a concentration of about 25 nM, is injected into the surface of the captured antibody at a flow rate of about 60 pl / min for about 3 minutes at 25 ° C, exhibits a higher KD than about 1 nM, for example, a KD greater than about 5 nM, greater than about 10 nM, greater than about 50 nM, greater than about 100 nM, greater than about 150 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 350 nM, greater than about 400 nM, greater than about 450 nM, greater than about 500 nM or more (see, for example, Example 4). In addition, an antibody "does not substantially bind to Ang-1" if the antibody fails to exhibit any binding to Ang-1 when tested in such an assay or equivalent thereof.
[00070] The present invention also includes antibodies that block the binding of Ang-2 to Tie-2, but do not substantially block the binding of Ang-1 to Tie-2. As used herein, an antibody "does not substantially block the binding of Ang-1 to Tie-2" if, when the antibody is premixed with Ang-1 antigen in a ratio of about 100: 1 (antibody: antigen) and allowed to incubate at 25 ° C for about 60 minutes and then the balanced mixture is tested for binding to Tie-2 through surface Plasmon resonance on a surface coated with Tie-2 (5 pl / min for 5 min at 25 ° C), the amount of Ang-1 bound to Tie-2 is at least 50% the amount of Ang-1 bound to Tie-2 in the presence of an irrelevant control molecule. (See, for example, Example 6). For example, if the amount of Ang-1 bound to Tie-2 following preincubation with an antibody is at least about 50%, at least about 55%, at least about 60%, at least about 65% at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% the amount of Ang-1 that binds to Tie-2 following pre-incubation with an irrelevant control molecule under the experimental conditions mentioned above, so the antibody is considered "not to substantially block the binding of Ang-1 to Tie-2".
[00071] In addition, the present invention includes antibodies that block or substantially attenuate biological activity of Ang-2 (e.g., Ang-2-mediated Tie-2 phosphorylation; Ang-2 induces angiogenesis, etc.), but does not block or substantially attenuate the corresponding biological activity of Ang-1 (e.g., Ang-1-mediated Tie-2 phosphorylation; Ang-1-induced angiogenesis, etc.). Assays and tests useful for determining whether an antibody meets one or more of the characteristics listed above will be readily known and easily practiced by persons of ordinary skill in the art and / or can be fully determined from the present invention. For example, the experimental procedures detailed below can be used to determine whether a given antibody binds to Ang-2 and / or Ang-2 or not; blocks or does not block binding of Ang-2 and / or Ang-2 to Tie-2; inhibits or does not inhibit Tie-2 phosphorylation mediated by Ang-2 and / or Ang-2; etc. Epitope Mapping and Related Technologies
[00072] To assess antibodies that bind to a particular epitope (for example, those that block IgE binding to its high affinity receptor), a routine cross-block assay such as the one described "Antibodies", Harlow and Lane ( Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scan mutants, peptide blots (Reineke (2004) Methods Mol. Biol.248: 443-63) or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496).
[00073] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. B-cell epitopes can be formed from either the contiguous amino acids or the non-contiguous amino acids juxtaposed by a protein's tertiary fold. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturation solvents, while tertiary fold epitopes are typically lost in treatment with denaturation solvents. An epitope typically includes at least 3, and, more commonly, at least 5 or 8-10 amino acids in a single spatial conformation.
[00074] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each category can reflect a unique epitope or distinctly different from or partially overlapping with an epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, so that characterization can be focused on genetically distinct antibodies. When applied to hybridoma evaluation, MAP can facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP can be used to classify the anti-Ang-2 antibodies of the invention into groups of antibodies binding to different epitopes.
[00075] Anti-Ang-2 antibodies can be attached to an epitope within the amino-terminal coiled helix domain or within the carboxy-terminal fibrinogen-like ("FD") domain. In preferred embodiments of the present invention, anti-Ang-2 antibodies and the antigen binding fragments thereof bind to an epitope within the FD.
[00076] The amino acids within the Ang-2 FD that interact with Tie-2 were determined from crystal structure analysis. See Barton et al., Nat. Struct. Mol. Biol. 13: 524-532 (May 2006). With respect to antibodies that block the binding of Ang-2 to Tie-2, but do not substantially block the binding of Ang-1 to Tie-2 (for example, H1H685P, see Examples 5 and 6 below), the epitope to which such antibodies they bind can include one or more Ang-2 amino acids that (a) interact with Tie-2 and (b) are non-identical to the corresponding Ang-1 amino acid. (See figure 1). In this way, the epitope to which such preferred Ang-2 antibodies bind can include one or more of the following hAng-2 amino acids (SEQ ID NO: 518); S-417; K- 432; I-434; N-467; F-469; Y-475 or S-480. For example, the present inventors have found that antibodies that interact with amino acids F-469, Y-475 and S-480 of Ang-2 (SEQ ID NO: 518) preferentially interact with Ang-2 over Ang-1, and this Preferential bonding can have therapeutic benefits. Thus, the present invention includes anti-Ang-2 antibodies that specifically bind to human angiopoietin-2 (hAng-2), but do not substantially bind to hAng-1, where the antibodies bind to an epitope in hAng- 2 (SEQ ID NO: 518) comprising amino acids F-469, Y-475 and S-480. Similarly, the present invention includes anti-Ang-2 antibodies that block the binding of hAng-2 to Tie-2, but do not substantially block the binding of hAng-1 to Tie-2, where the antibodies bind to an epitope in hAng-2 (SEQ ID NO: 518) comprising amino acids F-469, Y-475 and S-480.
[00077] The present invention includes anti-Ang-2 antibodies that bind to the same epitope as any of the specific exemplary antibodies described here (for example, H1H685, H1H690, H1H691, H1H696, H1H706, H1M724 and / or H2M744). Likewise, the present invention also includes anti-Ang-2 antibodies that compete for binding to Ang-2 with any of the exemplary antibodies described here (for example, H1H685, H1H690, H1H696, H1H706, H1M724 and / or H2M744).
[00078] A person can easily determine whether an antibody binds to the same epitope that, or competes to bind, a reference anti-Ang-2 antibody using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-Ang-2 antibody of the invention, the reference antibody is allowed to bind to an Ang-2 protein or peptide under saturation conditions. Then, the ability of a test antibody to bind to the Ang-2 molecule is assessed. If the test antibody was able to bind to Ang-2 following saturation binding with the reference anti-Ang-2 antibody, it can be concluded that the test antibody binds to an epitope other than the anti-Ang-2 antibody of reference. On the other hand, if the test antibody is unable to bind to the Ang-2 molecule following saturation binding with the reference anti-Ang-2 antibody, then the test antibody can bind to the same epitope as the bound epitope by the reference anti-Ang-2 antibody of the invention. Additional routine experimentation (for example, mutation and binding analyzes) can then be performed to confirm that the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the observed lack of connection. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antibodies bind to the same epitope (or overlapping) if, for example, an excess of 1, 5, 10, 20 or 100 times of one antibody inhibits binding of the other by at least 50% , but preferably 75%, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res., 1990: 50: 1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all of the amino acid mutations in the antigen that reduce or eliminate binding to one antibody reduce or eliminate binding to the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduces or eliminates binding of one antibody reduces or eliminates binding of the other.
[00079] To determine whether an antibody competes for binding with a reference anti-Ang-2 antibody, the binding methodology described above is carried out in two orientations: In a first orientation, the reference antibody is allowed to bind to a molecule of Ang-2 under saturation conditions followed by evaluation of binding of the test antibody to the Ang-2 molecule. In a second orientation, the test antibody is allowed to bind to an Ang-2 molecule under conditions of saturation followed by evaluation of binding of the reference antibody to the Ang-2 molecule. If, in both orientations, only the first antibody (saturation) is able to bind to the Ang-2 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to Ang-2. As will be understood by one of ordinary skill in the art, the antibody that competes for binding with a reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope. Species Selectivity and Species Cross Reactivity
[00080] According to certain embodiments of the invention, anti-Ang-2 antibodies bind to human Ang-2, but not to Ang-2 of other species. Alternatively, the anti-Ang-2 antibodies of the invention, in certain embodiments, bind to human Ang-2 and Ang-2 from one or more non-human species. For example, the Ang-2 antibodies of the invention may bind to human Ang-2 and may or may not bind, as the case may be, to one or more Ang-2 from mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cinomologist, marmot, rhesuse chimpanzee. Immunoconjugates
[00081] The invention comprises monoclonal anti-Ang-2 antibodies conjugated to a therapeutic moiety ("immunoconjugates"), such as a cytotoxin, chemotherapeutic drug, immunosuppressant or radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents and chemotherapeutic agents suitable for forming immunoconjugates are known in the art, see, for example, WO 05/10381). Multispecific Antibodies
[00082] The antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific mAbs can be specific for epitopes other than a target polypeptide or can contain specific antigen binding domains for more than one target polypeptide. See, for example, Tutt et al. (1991) J. Immunol.147: 60-69. The anti-Ang-2 antibodies of the present invention, or portions thereof, can be linked to or co-expressed with another functional molecule, for example, another peptide or protein, to form a multispecific molecule. For example, an antibody or fragment thereof can be functionally linked (for example, through chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecule entities, such as another antibody or fragment of antibody, to produce a bispecific or multispecific antibody with a second binding specificity.
[00083] An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin CH3 domain (lg) and a second lg CH2 domain, in which the first and second CH3 domains differ from one another. another in at least one amino acid, and in which at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody without the amino acid difference. In one embodiment, the first CH3 domain of lg binds to Protein A and the second CH3 domain of lg contains a mutation that reduces or abolishes binding to Protein A such as a modification of H95R (by exon numbering IMGT; H435R by numbering I). The second CH3 may further comprise a modification of Y96 (by IMGT; Y436F by the EU). Additional modifications that can be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by the EU) in the case of IgG antibodies; N44s, K52N and V821 (IMGT; N384S, N392N and V422I by the EU) in the case of lgG2 antibody; and Q15R, N44S, K52N, V57M, R69K, E79Q and V81I (by IMGT; Q355R, N384N, V397M, R409K, E419Q and V422I by the EU) in the case of lgG4 antibody. Variations of the bispecific antibody format described above are understood to be within the scope of the present invention. Therapeutic Formulation and Administration
[00084] The present invention provides therapeutic compositions comprising anti-Ang-2 antibodies or antigen binding fragments thereof of the present invention. The therapeutic compositions in the present invention can further comprise one or more carriers, excipients and other pharmaceutically acceptable agents which are incorporated into formulations to provide improved transfer, application, tolerance and the like (here collectively referred to as "pharmaceutically acceptable carriers or diluents") . A variety of appropriate formulations can be found in the form known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN®), DNA conjugates, anhydrous absorption pastes, oil-in-emulsions. water and water-in-oil, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. See also Powell and others "Compendium of excipients for parenteral formulations", PDA, 1998, J. Pharm. Know. Technol. 52: 238-311.
[00085] The dose of antibody may vary depending on the age and size of an individual to be administered, target disease, conditions, route of administration and the like. The preferred dose is typically calculated according to the body weight or body surface area. When an antibody of the present invention is used to treat a condition or disease associated with Ang-2 activity in an adult patient, it may be advantageous to administer the antibody of the present invention intravenously normally in a single dose of about 0.01 to about 20 mg / kg of body weight, more preferably about 0.02 to about 7, about 0.03 to about 5 or about 0.05 to about 3 mg / kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and programs for administering Ang-2 antibodies can be determined empirically, for example, patient progress can be monitored through periodic evaluation, and the dose adjusted accordingly. In addition, interspecies dosage increases can be performed using methods well known in the art (for example, Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).
[00086] Various application systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated encephalitis ( see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition can be administered via any convenient route, for example, by infusion or bolus injection, through absorption by epithelial or monocutaneous coatings (eg, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local.
[00087] A pharmaceutical composition of the present invention can be applied, for example, subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous application, a pen application device readily has applications in the application of a pharmaceutical composition of the present invention. Such a pen application device can be reusable or disposable. A reusable pen application device generally uses a replaceable cartridge that contains a pharmaceutical composition. Once the pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen application device can then be reused. In a disposable pen application device, there is no reusable cartridge. In contrast, the disposable pen application device comes pre-filled with the pharmaceutical composition kept in a reservoir within the device. Once the reservoir is without the pharmaceutical composition, the entire device is discarded.
[00088] Various pen application devices and reusable autoinjectors have applications in the subcutaneous application of a pharmaceutical composition of the present invention. Examples include, but are certainly not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 72 / 25® pen, HUMALOG® pen, pen HUMALIN 70 / 30® (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), to name a few. Examples of disposable pen application devices having subcutaneous application of a pharmaceutical composition of the present invention include, but are certainly not limited to, the SOLOSTAR® pen (Sanofi-aventis), FLEX-PEN® (Novo Nordisk) and KWIKPEN ® (Eli Lilly).
[00089] For the treatment of eye disorders, the antibodies and antigen binding fragments of the invention can be administered, for example, through eye drops, subconjunctival injection, subconjunctival implant, intravitreal injection, intravitreal implant, subcutaneous injection Teeth or sub-Tenon implant.
[00090] The pharmaceutical composition can also be applied to a vesicle, in particular a liposome (see Langer, 1990, Science, 249: 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez -Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., Pp. 317-327, see generally ibid.).
[00091] In certain situations, the pharmaceutical composition can be applied in a controlled release system. In one embodiment, a pump can be used (see Langer, supra, Sefton 1987, CRC, Crít. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled-release system can be placed in close proximity to the target of the composition, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applicatons of Controlled Release, supra, vol. 2, pp. 115-138).
[00092] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. Such injectable preparations can be prepared using publicly known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there is, for example, physiological saline, an isotonic solution containing glycosis and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (for example, ethanol), a polyalcohol (eg propylene glycol, polyethylene glycol), a nonionic surfactant [eg polysorbate 80, HCO-50 (polyoxyethylene adduct (50 moles) of hydrogenated castor oil)], etc. . As the oily medium is used, for example, sesame oil, soy oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection then prepared is preferably filled into an appropriate ampoule.
[00093] Advantageously, the pharmaceutical compositions for oral and parenteral uses described above are prepared in dosage forms in a unit dose suitable to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the injection form, it is preferred that the antibody mentioned above is contained in about 5 to about 100 mg and in about 10 to about 250 mg for other dosage forms. Therapeutic Uses of Antibodies
[00094] The antibodies of the invention are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with Ang-2 activity, including diseases or disorders associated with angiogenesis. The antibodies and antigen-binding fragments of the present invention can be used to treat, for example, primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract , muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, liver and urinary tract and special sensory organs such as the eye. In certain embodiments, the antibodies and antigen-binding fragments of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, breast cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer or melanoma.
[00095] The antibodies and antigen-binding fragments of the present invention may also be useful for the treatment of one or more disorders of the eye. Exemplary eye disorders that can be treated with the antibodies and antigen binding fragments of the invention include, for example, age-related macular degeneration, diabetic retinopathy and other eye disorders associated with choroidal neovascularization, vascular leakage, retinal edema and inflammation . In addition, the anti-Ang-2 antibodies of the invention can be administered as an adjunct to glaucoma surgery to prevent early hem- and lymphangiogenesis and recruitment of macrophages to the filter bubble after glaucoma surgery and improve the clinical outcome.
[00096] In other embodiments of the present invention, antibodies or antigen binding fragments are used to treat hypertension, diabetes (including non-insulin dependent diabetes mellitus), psoriasis, arthritis (including rheumatoid arthritis), asthma, sepsis, kidney disease and edema associated with injury, stroke or tumor.
[00097] Ang-2 expression has been shown to relate to the severity of various inflammatory and / or infectious diseases (see, for example, Siner et al., 2009, Shock, 31: 348-353; Yeo et al., 2008, Proc Natl. Acad. Sci. (USA): 105: 17097-17102). In this way, the anti-Ang-2 antibodies of the present invention can be used to treat, prevent or ameliorate one or more inflammatory or infectious diseases. Exemplary infectious diseases that can be treated, prevented or improved by administering the anti-Ang-2 antibodies of the invention include, but are not limited to: malaria (Plasmo-dium falciparum infection), viral hemorrhagic fevers (for example, fever dengue), rickettsial infection, toxic shock syndrome, sepsis, hepatitis C, infection by Bartonella bacilliformis, leishmaniasis, mycobacterial infection and infection by the Epstein-Barr virus. Combination Therapies
[00098] Combination therapies may include an anti-Ang-2 antibody of the invention and, for example, another Ang-2 antagonist (for example, an anti-Ang-2 antibody, peptibody or CovX-body such as CVX-060 (see US 7,521,425)). The anti-Ang-2 antibodies of the invention can also be administered together with another antiangiogenic agent such as, for example, a VEGF antagonist (for example, a VEGF-Trap, see, for example, US 7,087,411 (also referred to herein as "VEGF inhibition fusion protein"), anti-VEGF antibody (eg, bevacizumab), a small molecule kinase inhibitor of VEGF receptor (eg, sunitinib, sorafenib and pazopanib) and antibodies anti-DLL4 (for example, an anti-DLL4 antibody disclosed in US 2009/0142354 such as REG421), etc.) or with an epidermal growth factor receptor (EGFR) antagonist (for example, anti-EGFR antibody or inhibitor of small molecule of EGFR activity). Other agents that can be beneficially administered in combination with the anti-Ang-2 antibodies of the invention include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind cytokines such as IL-1, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18 or their respective receptors. The present invention also includes therapeutic combinations comprising any of the anti-Ang-2 antibodies mentioned herein and an inhibitor of one or more of VEGF, DLL4, EGFR or any of the cytokines mentioned above, wherein the inhibitor is an aptamer, a molecule antisense, a ribozyme, a siRNA, a peptibody, a nanobody or an antibody fragment (for example, Fab fragment; F (ab ') fragment 2j Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such such as corpses, tribodies, tetrabodies, minibodies and minimal reconnaissance units). The anti-Ang-2 antibodies of the invention can also be administered in combination with antivirals, antibiotics, analgesics, corticosteroids and / or NSAIDs. The anti-Ang-2 antibodies of the invention can also be administered as part of a treatment regimen that also includes treatment with radiation and / or conventional chemotherapy. When combined with one or more additional agents, the anti-Ang-2 antibodies of the invention can be administered prior to, simultaneously with (for example, in the same formulation or in separate formulations) or subsequent to the administration of the other (s) ) agent (s). Diagnostic Uses for Antibodies
[00099] The anti-Ang-2 antibodies of the present invention can also be used to detect and / or measure Ang-2 in a sample, for example, for diagnostic purposes. For example, an anti-Ang-2 antibody, or fragment thereof, can be used to diagnose a condition or disease characterized by the aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of Ang-2. Exemplary diagnostic assays for Ang-2 may include, for example, contacting a sample, obtained from a patient, with an anti-Ang-2 antibody of the invention, in which the anti-Ang-2 antibody is marked with a detectable marker or reporter molecule. Alternatively, an unlabeled anti-Ang-2 antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable marker or reporter molecule can be a radioisotope, such as 3H, 14C, 32P, 35S or 125l; a fluorescent or chemiluminescent portion such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase; β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure Ang-2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence-activated cell classification (FACS). EXAMPLES
[000100] The following examples are shown to provide those of ordinary skill in the art with full disclosure and description of how to make and use the methods and compositions of the 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 (eg quantities, temperature, etc.), but some errors and experimental deviations must be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade and pressure is at or near atmospheric. Example 1. Generation of Human Antibodies to Human Ang-2
[000101] Human Ang-2 antigen was administered directly, with an adjuvant to stimulate the immune response, to a VELOCIMMUNE® mouse comprising DNA encoding variable regions of human immunoglobulin heavy and light chain. The antibody immune response was monitored by an Ang-2 specific immunoassay. When a desired immune response was obtained, splenocytes were collected and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were evaluated and selected to identify cell lines that produce specific Ang-2 antibodies. Using this technique, several chimeric anti-Ang-2 antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this manner were called: H1M724, H1M727, H1M728, H2M730, H1M732, H1M737, H2M742, H2M743, H2M744, H1M749, H2M750 and H1M810.
[000102] Anti-Ang-2 antibodies were also isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. 2007 / 0280945A1. Using this method, several fully human anti-Ang-2 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained; exemplary antibodies generated in this way were called: H1H685, H1H690, H1H691, H1H693, H1H694, H1H695, H1H696, H1H704, H1H706 and H1H707.
[000103] The biological properties of the exemplary anti-Ang-2 antibodies generated according to the methods of this Example are described in detail in the Examples shown below. Example 2. Analysis of Variable Gene Use
[000104] To analyze the structure of antibodies produced, nucleic acids encoding variable regions of antibody were cloned and sequenced. From the nucleic acid sequence and the predicted amino acid sequence of the antibodies, gene use was identified for each heavy chain variable region (HCVR) and light chain variable region (LCVR) (Table 1). Table 1


[000105] Table 2 shows the selected light and heavy chain variable region amino acid sequence pairs of selected anti-Ang-2 antibodies and their corresponding antibody identifiers. The designations N, P and G refer to antibodies having heavy and light chains with identical CDR sequences, but with sequence variations in regions that are outside the CDR sequences (i.e., in the backbone regions). In this way, N, P and G variants of a particular antibody have identical CDR sequences within variable regions of heavy and light chains, but contain modifications within the main structure regions.

* The H1M737 LCVR amino acid sequence is not shown. Control Constructs Used in the Examples that Follow
[000106] Several control constructs (anti-Ang-2 antibodies and anti-Ang-2 peptibodies) have been included in the experiments that follow for comparative purposes. The control constructs are called: Control I: an anti-human Ang-2 antibody with variable domains of heavy and light chains having the amino acid sequences of the corresponding domains of "Ab536 (THW)", as shown in US 2006/0018909 (see also Oliner et al., 2004, Cancer Cell6: 507-516); Control II: a peptide that binds to human Ang-2 having the amino acid sequence of "2XCon4 (C)", as shown in US 7,205,275 (see also Oliner et al., 2004, Cancer Cell6: 507-516 ); Control III: a peptibody that binds to human Ang-2 having the "L1-7" amino acid sequence, as shown in U.S. 7,138,370; Control IV: a human anti-Ang-2 antibody with variable regions of heavy and light chains having the amino acid sequences of the corresponding domains of "3.19.3" as shown in U.S. 2006/0246071; and Control V: a human anti-Ang-2 antibody with variable regions of heavy and light chains having the amino acid sequences of the corresponding domains of "MEDI1 / 5" as shown in WO 2009/097325. (Not all control constructs were used in each Example). In the tables that follow, the notations "Ab" and "Pb" are included to identify antibody and peptibody controls, respectively (that is, Control 1 = Ab; Control II = Pb; Control III = Pb; Control IV = Ab; and Control V = Ab). Example 3. Determination of Antigen Binding Affinity
[000107] Balance dissociation constants (KD values) for the binding of selected purified Ang-2 antibodies to the dimeric fibrinogen-like domain of human Ang-2 (SEQ ID NO: 519), from mouse (Mus musculus', SEQ ID NO: 520) and monkey (Macca fascicula; SEQ ID NO: 521) (Ang-2FD) conjugated to human IgG1 (SEQ ID NO: 528) were determined by surface kinetics using a plasmon resonance assay real-time biosensor surface. Antibody was captured on a polyclonal anti-mouse IgG antibody surface, a goat anti-human K polyclonal antibody surface (Southern Biotech, Birmingham, AL) or a goat anti-human IgG polyclonal antibody surface (Jackson Immuno Research Lab, West Grove, PA) created by coupling direct amine to a chipBIACORE® CM5 sensor to form a captured antibody surface. Variable concentrations (ranging from 50 nM to 6.25 nM) of protein were injected at 100 pl / min into the surface of the captured antibody for 90 seconds. Antigen-antibody binding and dissociation were monitored in real time at room temperature. Kinetic analysis was performed to calculate the KDθ the dissociation half-life of the antigen / antibody complex. The results are summarized in Table 3 below.

[000108] The above experiment was repeated using selected purified anti-Ang-2 antibodies cloned into human IgG1. The results are summarized in Table 4 below.


[000109] Additional binding experiments were conducted using selected anti-Ang-2 antibodies at two different temperatures to further assess the affinity of cross species. Each selected antibody or control construct was captured at a flow rate of 40 pL / min for 1 minute on a goat anti-human kappa polyclonal antibody surface created by chemical coupling to a chipBIACORE® to form an antibody surface captured. Human, monkey and mouse Ang-2FC-Fc at a concentration of 25 nM or 0.78 nM was injected into the captured antibody surface at a flow rate of 60 pL / min for 3 minutes and the antigen-antibody dissociation was monitored in real time for 20 minutes at 25 ° C or 37 ° C.
[000110] The results are summarized in Tables 5 (25 ° C connection) and 6 (37 ° C connection) below.


[000111] In another experiment, KD values for selected purified antibodies that bind to a tetrameric construct "bow-Ang-2"("hBA2") were determined (using the methods described above). hBA2 consists of two dimers, each dimer containing two Ang-2 fibronectin-like domains connected to each other by a human Fc domain. The amino acid sequence of the constituents of the hBA2 dimer is represented by SEQ ID NO: 522. The results are summarized in Table 7 below. Table 7

[000112] In yet another experiment, KD values for selected purified antibodies that bind to human wild-type Ang-2 (hAng-2-WT; SEQ ID NO: 518) and the fibrinogen-like domain of human Ang2 (hAng-2FD ) have been determined (as described above). The results are summarized in Table 8 below. Table 8


[000113] Additional experiments were conducted to measure the binding properties of selected anti-Ang-2 antibodies to monomeric hAng-2Fd at 25 ° C and 37 ° C. Each selected antibody or control construct was captured at a flow rate of 40 pL / min for 1 minute on a goat anti-human IgG monoclonal antibody surface created by direct chemical coupling to a chipBIACORE® to form a captured antibody surface. Human Ang-2FD at a concentration of 500 nM or 7.8 nM was injected into the captured antibody surface at a flow rate of 60 pL / min for 3 minutes and antigen / antibody dissociation was monitored in real time for 20 minutes or at 25 ° C or 37 ° C.
[000114] The results are summarized in Tables 9 (25 ° C) and 10 (37 ° C) below. N / A = not determined.Table 9
Table 10


[000115] As shown in this Example, several of the anti-Ang-2 antibodies generated according to the methods of Example 1 bound to Ang-2 constructs with equivalent or greater affinities than the controls. For example, antibodies H1H685, H1H690, H1H724 and H1H744 bound to dimeric human Ang-2-FD with KD's of 71.4, 79, 115 and 114 pM, respectively, while Control I antibody bound to Ang-2-FD dimer with a Kode 339 pM (see Table 4). Similarly, H1H685, H1H690, H1H724 and H1H744 antibodies bind to human BA2 (a tetrameric Ang-2 fibrinogen-like domain construct) with KD'S of 11,9,17,9, 23,3 and 17,2 pM, respectively, while the Control antibody bound to hBA2 with a KD of 83 pM (see Table 7). In this way, as compared to control constructs, many of the antibodies of the invention exhibit increased binding to Ang-2. The H1H685P antibody showed particularly robust binding properties to Ang-2 compared to control constructs. Example 4. Preferential connection to Ang-2 over Ang-1
[000116] Binding experiments (plasmon resonance assays) were conducted to ascertain whether selected antibodies bound to both Ang-2 and Ang-1 or whether they preferentially bound to Ang-2 only. Each selected antibody or control construct was captured at a flow rate of 40 pL / min for 1 minute on a mouse anti-human IgG polyclonal antibody surface created by direct chemical coupling to a chipBIACORE® to form a captured antibody surface. . Full-length wild-type human Ang-1 or Ang-2 at a concentration of 25 nM or 0.78 nM was injected into the surface of antibody captured at a flow rate of 60 pL / min for 3 minutes and dissociation of an- antigen-antibody was monitored in real time for 20 minutes or at 25 ° C or 37 ° C.
[000117] The results of these experiments are summarized in Tables 11-14 below. N / D = not determined. "No binding" means that no detectable binding was observed under the particular experimental conditions used in these experiments.



[000118] These results show that H1H685P is unique among the antibodies tested in this experiment because it binds with high affinity to Ang-2, but does not bind to Ang-1. The only other construct that exhibits a link with Ang-2, but not Ang-1, is Control III. It should be emphasized, however, that Control III is a peptibody and that all other antibodies tested in this experiment were linked to both Ang-2 and Ang-1. Selectivity for binding to Ang-2 may confer therapeutic benefits to H1H685P that are not possessed by antibodies that bind to both Ang-2 and Ang-1. Example 5. Inhibition of binding of Ang-2 to Tie-2
[000119] Tie-2 is a natural receptor for Ang-2. Anti-Ang-2 antibodies were tested for their ability to block binding of Ang-2 to human Tie-2 (hTie-2). hTie-2-mFc (a chimeric construct consisting of human IgG-conjugated mouse IgG; SEQ ID NO: 525) was plated in 96-well plates at a concentration of 2 pg / ml and incubated overnight by washing four times in wash buffer (PBS with 0.05% Tween-20). The plate was then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 0.5% BSA (Sigma-Aldrich Corp., St. Louis, MO) for one hour at room temperature. On a separate plate, purified anti-Ang-2 antibodies, at a starting concentration of 50nM, were serially diluted by a factor of three across the entire plate. Human, mouse or monkey Ang-2FD protein conjugated to human IgG (Ang-2FD-hFc) was added to final concentrations of 2nM, 8nM or 2nM, respectively and incubated for one hour at room temperature. The antibody / Ang-2FD-Fc mixture was then added to the plate containing hTie-2-mFc and incubated for one hour at room temperature. Detection of Ang-2-FD-hFc linked to the hTie-2-mFc protein was determined with horseradish peroxidase (HPR) conjugated to anti-human IgG antibody (Jackson Immuno Research Lab, West Grove, PA) and developed using standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA). Absorbance was read at OD450 for 0.1 second. Percentage blockage of binding of Ang-2FD-hFC to hTIE-2-mFc by 16.67nM of selected anti-Ang-2 antibodies is shown in Table 15.Table 15

[000120] In a similar experiment, selected purified anti-Ang-2 antibodies cloned into human IgG1 were tested for their ability to block binding of Ang-2FD to hTie-2 (as described above). Percent blocking of Ang-2FD-hFc binding to hTie-2-mFc by 16.67 nM of selected anti-Ang-2 antibodies is shown in Table 16. NT: not tested. Table 16

[000121] In another experiment, selected purified anti-Ang-2 antibodies were tested for their ability to block binding of hBA2 to 20pM biotinylated hTie-2 (as described above). For this experiment, human Tie-2 conjugated to the histidine tag (hTie-2-His; SEQ ID NO: 526) was used in a similar manner to the hTie-2-mFc described above. Antibody concentrations of 5nM were serially diluted three times. An IC50 value (Inhibitory Concentration) was generated by calculating the amount of antibody required to block 50% of the biotin-hBA2 binding signal to Tie-2. An average IC50 value for each antibody was calculated based on two separate experiments. The results are summarized in Table 17. NB: no block observed at a concentration of 5nM. Table 17

[000122] In a similar experiment, selected purified anti-Ang-2 antibodies cloned into human IgG1 were tested for their ability to block binding of biotinylated hBA2 to hTie-2 (as described above). The results are shown in Table 18. NB: no block observed at a concentration of 5nM.

[000123] This Example illustrates that several of the anti-Ang-2 antibodies generated according to the methods of Example 1 blocked the interaction between the fibrinogen-like domain of Ang-2 and its receptor (TIE-2) to an equivalent or greater degree than the control antibody. For example, the antibodies H1H690, H1H691, H1H695, H1H696, H1H704, H1H706, H1H707, H1H724 and H1H744 each caused more than 95% blocking of human, mouse and monkey Ang-2FD constructs to the TIE receptor -2, similar to the results observed with the control constructs (see Table 16). Example 6: Inhibition of binding of full-length Ang-2 and Ang-1 to human Tie-2
[000124] Tie-2 is a receptor for Ang-1 as well as Ang-2. Thus, in the present Example, the ability of certain anti-Ang-2 antibodies to block binding of Ang-2 or Ang-1 to human Tie-2 was measured and compared.
[000125] The ELISA experiments shown in this Example were conducted in a similar manner to the experiments in Example 5. In short, hTie-2-mFc (a chimeric construct consisting of human Tie-2 conjugated to mouse IgG; SEQ ID NO: 525 ) was coated in 96 well plates at a concentration of 2 pg / ml and incubated overnight followed by washing four times in wash buffer (PBS with 0.05% Tween-20). The plate was then blocked with BPS (Irvine Scientific, Santa Ana, CA) containing 0.5% BSA (Sigma-AI-drich Corp., St. Louis, MO) for one hour at room temperature. On a separate plate, purified anti-Ang-2 antibodies and control constructs, at a starting concentration of 300 nM, were serially diluted by a factor of three across the plate. Full length human Ang-2 or Ang-1 protein conjugated to the 6X histidine tag (R&D Systems, Minneapolis, MN) was added to a final concentration of 0.6nM and incubated for one hour at room temperature. The antibody / antigen mixture was then added to the plate containing hTie-2-mFc and incubated for one hour at room temperature. Detection of Ang-2His and Ang-1 -His linked to the hTie-2-mFc protein was determined with Horseradish Peroxidase (HRP) conjugated to a Penta-His antibody (Qiagen, Valencia, CA) and developed using a standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA). Absorbance was read at OD450 for 0.1 second. An IC50 value (Inhibitory Concentration) was generated by calculating the amount of antibody required to block 50% of the signal from binding Ang-2 or human Ang-1 to Tie-2. The results, expressed in terms of IC50 are shown in Table 19, columns (1) and (2). The point up to which antibodies or control constructs block the interaction of hAng-2 / Tie-2 with respect to the interaction of hAng-1 / Tie-2 is reflected in the difference in times in IC 50 shown in column (3); that is, a larger number in column (3) indicates a greater ability to block the hAng-2 / Tie-2 interaction than the hAng-1 / Tie-2 interaction.
* Calculated by dividing the hAng-1 blocking IC50 (column 2) by the hAng-2 blocking IC50 (column 1)
[000126] In an effort to further evaluate the ability of selected anti-hAng-2 antibodies to block the binding of Ang-2 to Tie-2, a biosensor surface plasmon resonance experiment was conducted. In this experiment, a full-length extracellular domain construct of human Tie-2 (hTie-2-mFc-ecto) was coupled to the amide on a chipBIACORE® to create a receptor-coated surface. Selected anti-hAng-2 antibodies and control constructs, at 1 pm (100-fold excess with respect to the antigen), were pre-mixed with 10 nM hAng-1-WT, followed by 60 minutes of incubation at 25 ° C to allow antibody-antigen binding to achieve equilibrium to form balanced solutions. The balanced solutions were injected into the receiver surfaces at 5 pL / min for 5 minutes at 25 ° C. Changes in resonance units (RU) due to the binding of hAng-1-WT to hTie-2-mFc were determined. An irrelevant peptibody construct with no hAng-1 binding was included in this experiment to establish the 0% blocking baseline and a human Tie-2-mFc construct was used as a positive control for blocking. The amount of Ang-1 bound to Tie-2 following pre-incubation of the antibody, expressed as a percentage of the amount of Ang-1 bound to Tie-2 following pre-incubation of negative control, is shown in Table 20. A greater amount binding of Ang-1 to Tie-2 means a lesser degree of antibody blocking).


[000127] The above experiment was repeated using different amounts of Ang-2 blockers and controls. In particular, a tie-2 full-length extracellular domain construct (hTie-2-mFc-ecto) was coupled to the amine on a chipBIACORE® to create a receptor-coated surface. Selected anti-hAng-2 antibodies and control constructs (50 or 150 nM) were mixed with hAng-2-WT (25 nM) followed by 60 minutes of incubation at 25 ° C to allow antibody-antigen binding to reach equilibrium. The balanced solutions were injected into the recipient's surfaces at 10 pL / min for 5 minutes at 25 ° C. To assess the ability of selected anti-hAng-2 antibodies to block Ang-1-WT by binding to hTie-2, a similar procedure was followed except that the antibodies were tested in three concentrations (50, 100 or 1000 nM) and incubated with 10 nM hAng-1-WT. Changes in resonance units (RU) due to the binding of Ang-2-WT or hAng-1-WT to hTie-2-mFc were determined. An irrelevant antibody with no binding to any angiopoietin was included in these experiments to establish the 0% blocking baseline and a human Tie-2-mFc construct was used as a positive control for blocking. The results are summarized in Tables 21 (hAng-1 applied to an hTie-2 surface) and 22 (hAng-2 applied to an hTie-2 surface).


[000128] The results obtained from these experiments are in agreement with previous results that showed that H1H6685P preferably binds to Ang-2 with respect to Ang-1 (see example 4). In particular, the results of these Examples show that several anti-Ang-2 antibodies (for example, H1H685P and H1H706P) do not significantly block the binding of human Ang-1 to human Tie-2, however, in other experiments, it has been shown that these antibodies potently blocked the interaction between Ang-2 and Tie-2 (see Example 5, Table 16). Furthermore, in these experiments none of the control constructs, except the Control III peptibody, exhibited the same degree of preferential binding / blocking of Ang-2 with respect to Ang-1 as the exemplary anti-Ang-2 antibodies of the present invention, such as H1H685P. Example 7. Inhibition of Ang-2-Mediated Tie-2 Phosphorylation by Anti-Ang-2 Antibodies
[000129] The inventors of the present invention demonstrated that expression of Ang-2 can be induced in human umbilical vein endothelial cells (HUVECs) by the FOXO1 transcription factor (Daly et al., 2006. PNAS, 103: 15491). In addition, the inventors showed that infection of HUVECs with an adenovirus encoding FOXO1 results in expression and secretion of Ang-2, followed by activation of Tie-2 phosphorylation (Daly et al., 2006. PNAS, 103: 15491).
[000130] Anti-Ang-2 antibodies have been tested for their ability to inhibit Tie-2 phosphorylation. In short, 7x105 HUVECs (Vec Technologies, Rensselaer, NY) were plated on 6 cm cell culture plates in 3.5 ml MCDB131 Complete Medium (Vec Technologies, Rensselaer, NY). The next day, the cells were washed with Opti-MEM (Invitrogen Corp., Carlsbad, CA) and fed with 2 ml of Opti-MEM. Recombinant adenoviruses encoding either green fluorescent protein (GFP; control) or human FOXO1 (Daly et al., 2004, Genes Dev., 18: 1060) were added to the cells at a concentration of 10 pfu / cell and incubated for four hours. The cells were then washed with MCDB131 and fed with 2 ml of MCDB131 containing anti-Ang-2 antibodies in a concentration of 0.5 pg / ml. Twenty-four hours after infection, the cells were lysed and subjected to tie-2 immunoprecipitation as described by Daly et al., Proc. Natl. Acad. Know. USA 103: 15491-15496 (2006). Immunoglobulin was collected in protein A / G beads (Santa Cruz Biotechnology, Santa Cruz, SA) for one hour. The beads were washed with cold lysis buffer and resuspended in SDS sample buffer for analysis using wester blot with specific antibodies to phosphotyrosine (Millipore, Billerica, MA) or Tie-2. The signals were detected using secondary antibodies conjugated to HRP and ECL reagents (GE Healthcare, Piscataway, NJ). X-ray films were scanned and the signals of phospho-Tie-2 and Tie-2 were quantified using softwareImaged. The phospho-Tie-2 / Tie-2 ratios were used to determine the% inhibition for each anti-Ang-2 antibody (i.e., Percent inhibition = Reduction in phospho-Tie-2 / Tie-2 compared to control). For example, a reduction in Tie-2 phosphorylation to the level observed in the control sample is considered to be 100% inhibition. Relative inhibition (+, ++, +++) for each anti-Ang-2 antibody tested according to the observed percentage inhibition (25-50%, 50-75%, 75-100%, respectively) is shown in the Table 23.Table 23


[000131] As demonstrated in this Example, anti-Ang-2 antibodies generated according to the methods of Example 1 inhibited Tie-2 phosphorylation to a greater degree than the Control I antibody. Especially robust inhibition was observed with antibody H1H685, H1H690, H1H691, H1H693, H1H695, H1H696, H1H704, H1H706, H1H707, H1M724, H1M744 and H1M750 Example 8. Ang-1-Mediated Tie-2 Phosphorylation Inhibition
[000132] As shown in the previous example, Ang-2 can mediate Tie-2 phosphorylation. Ang-1 is also capable of promoting Tie-2 phosphorylation. In the present Example, the ability of selected anti-Ang-2 antibodies to block Ang-1-mediated Tie-2 phosphorylation was assessed.
[000133] EA.hy926 cells (Edgell et al., Proc. Natl. Acad. Sci. USA 80: 3734-3737 (1983)) were plated in 5x106 cells per 10 cm dish in 10 ml DMEM with 10% FBS, HAT , L-glutamine and penicillin / streptomycin. After 24 hours, the cells were deprived of serum for 1 hour in 10 ml DMEM + 1 mg / ml BSA. The cells were then stimulated for 10 minutes with 500 ng / ml of recombinant human Ang-1 (R&D Systems) in the presence of either an irrelevant isotype control antibody ("9E10") at 400 nM or the anti-Ang- 2 H1H685P, or control agents (Control I, Control II, Control IV or Control V) in concentrations ranging from 10 to 400 nM.
[000134] Following the incubation, the cells were lysed and Tie-2 was immunoprecipitated as described by Daly et al., Proc. Natl. Acad. Know. USA 103: 15491-15496 (2006). Immune complexes were collected by incubation with protein A / G beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 60 minutes. The beads were washed with cold lysis buffer and bound proteins were eluted by heating in SDS sample buffer. The samples were then subjected to Western blot analysis with monoclonal antibodies against Tie-2 or phosphorothirosine (clone 4G10, Millipore, Billerica, MA). The results are shown in figure 2.
[000135] The signals were detected using secondary antibodies conjugated to HRP and ECL reagents (GE Healthcare, Piscataway, NJ). X-ray films were scanned and the phospho-Tie-2 and Tie-2 signals were quantified using SoftwareImaged. The phospho-Tie-2 / Tie-2 ratios were used to determine the% inhibition for each antibody or peptidibody. Percent inhibition = reduction in phospho-Tie-2 / Tie-2 compared to the control sample (400 nM isotype control antibody).
[000136] In the presence of the control antibody 9E10, Ang-1 strongly activated Tie-2 phosphorylation (figure 2, panel A - compare lanes 2 and 3 vs. lane 1). All control agents that were tested significantly inhibited Tie-2 phosphorylation, with complete inhibition occurring at 50 nM for Control II (figure 2, panel B - range 17), 100 nM for Control IV (figure 2, panel A - range 11) and 200 nM for Control I (figure 2, panel B - range 24) and Control V (figure 2, panel C, range 9). In contrast, H1H685P had no significant inhibitory effects even at 400 nM (figure 2, panel A - ranges 4-8). These results provide further confirmation of the specificity of H1H685P for Ang-2 with respect to Ang-1. Example 9. Tumor Growth Inhibition by Anti-Ang-2 Antibodies
[000137] The effect of selected purified anti-Ang-2 antibodies on tumor growth was determined using two tumor cell lines.
[000138] PC3 (Human prostate cancer cell line). In short, 5x106 PC3 cells in 100 µl of growth factor-reduced Matrigel (BD Biosciences) were injected subcutaneously into the flanks of male NCr nude mice 6-8 weeks old (Taicon, Hudson, NY). After tumor volumes averaged about 200 mm3, the mice were randomized into groups for treatment. The mice in each treatment group were administered an anti-Ang-2 antibody, Fc protein, or controlled construct, at a concentration of 10 mg / kg by intraperitoneal injection twice a week for approximately three weeks (Table 24) or in concentrations of 2.5, 12.5 or 25 mg / kg through subcutaneous injection twice a week for approximately three weeks (Table 25). The tumor volumes were measured twice a week during the course of the experiment and the tumor weights were measured when the tumors were excised at the conclusion of the experiment. Means (mean +/- standard deviation) of weight and tumor growth were calculated for each treatment group. The percentage decrease in weight and tumor growth was calculated from the comparison for Fc protein measurements. The results are summarized in Tables 24 and 25.

[000139] As shown above, H1H744N and H1H685P antibodies demonstrated especially remarkable antitumor activity in the PC3 mouse tumor model compared to the control constructs.
[000140] The results of similar experiments using the PC3 mouse tumor model and different experimental antibodies (dosed at 2 mg / kg twice a week) are shown in Tables 26 and 27.

[000141] COLO 205 (Human colorectal adenocarcinoma cell line). In short, 2x106 COLO 205 cells in 100 µl serum-free medium were injected subcutaneously into the flank of male NCr nude mice 6-8 weeks old (Taconic, Hudson, NY). After the tumor volumes reached an average of about 150 mm3, the mice were randomized into groups for treatment with antibody or Fc protein. The mice in each treatment group were administered an anti-Ang-2 antibody or Fc protein at a concentration of 4 mg / kg by intraperitoneal injection twice a week for approximately two weeks. The tumor volumes were measured twice a week during the course of the experiment and the tumor weights were measured when the tumors were excised at the conclusion of the experiment. Means (mean +/- standard deviation) of weight and tumor growth were calculated for each treatment group. "Average Tumor Growth" represents the average growth at the time of treatment initiation (when tumors were approximately 150 mm3). The percentage decrease in weight and tumor growth is calculated from the comparison with Fc protein measurements. The results are summarized in Table 28.Table 28

[000142] A similar experiment was carried out to evaluate the effect of H1H685P, in particular, on COLO 205 tumor growth. In short, 2x106 COLO 205 cells in 100 pL of serum-free medium were implanted subcutaneously in the right rear flank of SCID mice CB17 males 9-11 weeks old. When the tumors reached ~ 125 mm3, the mice were randomized into 5 groups (n = 7-8 mice / group) and treated twice a week with Fc protein (15 mg / kg), H1H685P (5 or 25 mg / kg) or Control II (5 or 25 mg / kg) for a period of 19 days. Tumor volumes were measured twice a week during the course of the experiment and the tumor weights were measured when the tumors were excised at the end of the experiment. Mean weight and tumor growth at the start of treatment were calculated for each group. Percentage decrease in weight and tumor growth is calculated from the comparison with the control group Fc. The results are shown in Table 29.

[000143] As with the PC3 mouse tumor model, several of the antibodies of the invention, including H1H685P, exhibited substantial antitumor activities in the COLO 205 mouse model that were at least equivalent to the antitumor activities exhibited by the control molecules. Example 10. Growth Factor Inhibition and Tumor Perfusion by a Combination of an Anti-Ang-2 Antibody and a VEGF Inhibitor
[000144] To determine the effect of combining an anti-Ang-2 antibody with a VEGF inhibitor on the growth of COLO 205 xenografts, 2 x 106 cells were implanted subcutaneously in the right rear flank of female SCID mice 6-8 weeks old. When the tumors reached an average volume of ~ 350 mm3, the mice were randomized into 4 groups (n = 6 mice / group) and treated with: human Fc protein (7.5 mg / kg), H1H685P (5 mg / kg) , VEGF Trap (see US 7,087,411) (2.5 mg / kg) or the combination of H1H685P + VEGF Trap. The mice received a total of 3 doses during 10 days of treatment. Tumor volumes were measured twice a week during the course of the experiment. Mean tumor growth at baseline (mean +/- standard deviation) was calculated for each treatment group. Percentage decrease in tumor growth was calculated from the comparison of the control group Fc. The results are shown in Table 30. Note that in the VEGF Trap and H1H685P + VEGF Trap groups the mean tumor size was smaller at the end of treatment than at the beginning, that is, tumor regression was observed.


[000145] The results of this experiment demonstrate that the combination of H1H685P + VEGF Trap causes a decrease in tumor growth that is greater than the percentage decrease in tumor growth caused by any component alone.
[000146] To provide additional evidence of combination efficacy, the effect of the combination of H1H685P + VEGF TRAP on the growth of MMT tumors was evaluated. 0.5 x 106 MMT cells were implanted subcutaneously in the right rear flank of female SCID mice aged 6-8 weeks. When the tumors reached an average volume of ~ 400 mm3, the mice were randomized into 4 groups (n = 11 mice / group) and treated with: human Fc protein (17.5 mg / kg), H1H685P (12.5 mg / kg) kg), VEGF Trap (5 mg / kg) or the combination of H1H685P + VEGF Trap. The Fc and H1H685P groups received 3 doses for 9 days. The VEGF Trap and combination groups received 4 doses for 12 days. Tumor volumes were measured twice a week during the course of the experiment and tumor weights were measured when tumors were excised at the end of the experiment (due to their large size, tumors from the Fc and H1H685P groups were collected 3 days before the tumors of the VEGF Trap and combination groups). Means (mean +/- standard deviation) of tumor growth from the start of treatment and tumor weight were calculated for each group. The percentage decrease in weight and tumor growth is calculated from the comparison with the control group Fc. The results are shown in Table 31.

[000147] These results confirm the increased tumor inhibition effect of H1H685P + VEGF Trap with respect to single agent treatment.
[000148] To determine whether the combination of H1H685P + VEGF Trap has a greater effect on the tumor vessel function than the agents alone, a microultrasound (Vevo 770 imaging system from Visual-Sonics) was used to evaluate changes in perfusion tumor. COLO 205 tumors were grown to ~ 125 mm3 and the mice were then treated for 24 hours with H1H685P, VEG Trap or a combination of both agents. Following the treatment, tumor vessel perfusion was determined based on the acquisition of 2D image of enhanced contrast micro-ultrasound and analysis of a "wash-in" curve, which represents the amount of contrast agent that enters the tumor. Mean (mean +/- standard deviation) of tumor perfusion was calculated for each group. The percentage decrease was calculated from the comparison with the control group Fc. The results are shown in Table 32.


[000149] In line with the increased effect of the combination treatment on perfusion, anti-CD31 pretense of tumor sections demonstrated a more potent effect of the combination on tumor blood vessel density (data not shown). The increased effect of the H1H685P + VEGF Trap combination on tumor vasculature function provides a potential explanation for the increased effects of combination therapy on tumor growth. Example 11. Tumor Growth Inhibition by a Combination of an Anti-Ang-2 Antibody and a Chemotherapeutic Agent
[000150] To test the effect of H1H685P in combination with a chemotherapeutic agent on tumor growth, 2.5 x 106 COLO 205 tumor cells were implanted subcutaneously in the right rear flank of 8-9 week old male SCID mice. When the tumors reached an average volume of ~ 150 mm3 (day 17 after implantation), the mice were randomized into 4 groups (n = 5 mice / group) and treated as follows: the first group was treated sc with 15 mg / kg of hFc and intraperitoneally (ip) with 5-FU vehicle; the second group was treated sc with 15 mg / kg of H1H685P; the third group was treated ip with 75 mg / kg of 5-FU; the fourth group was treated with the combination of 15 mg / kg of H1H685P sc plus 75 mg / kg of 5-FU ip The mice received a total of three treatments, administered every 3-4 days. Tumor volumes were measured twice a week during the course of the experiment. The mean (mean +/- standard deviation) of tumor growth from the beginning of treatment until day 38 was calculated for each group. The percentage decrease in tumor growth was calculated from the comparison with the control group. The results are shown in Table 33.

[000151] The results of this experiment show that the combination of H1H685P and 5-FU caused a greater decrease in tumor growth than any agent administered separately. Example 12. Anti-Ang-2 Antibodies Mitigate Ocular Angiogenesis In Vivo
[000152] In this Example, the effects of selected anti-Ang-2 antibodies on retinal vascularization in a mouse model were evaluated.
[000153] In a set of wild-type mice experiments were used. In another set, mice expressing human Ang-2 in place of wild-type mouse Ang-2 (called "hu-Ang-2 mouse") were used. The two-day-old (P2) mice were injected subcutaneously or with Fc control or with anti-Ang-2 antibodies selected at a dose of 12.5 mg / kg. Three days later (in P5), the puppies were euthanized and the eyeballs were enucleated and fixed in 4% PFA for 30 minutes. The retinas were dissected, stained with Griffonia simplicifolia lectin-1 for 3 hours or overnight at 4 ° C to visualize the vasculature, and mounted smooth on microscope slides. The images were taken using a Nikon Eclipse 80i microscope camera and analyzed using Adobe Photoshop CS3, Fovea 4.0, and Scion 1.63 software.
[000154] Areas of the retina covered with superficial vasculature were measured and used as a reading of antibody activity. The reduction in the size of the vasculature areas in mice treated with antibody compared to controls treated with Fc is shown in Table 34. The percentage reduction in the vasculature area reflects the antiangiogenic potency of the antibody. (N / D = not determined).

[000155] As shown in this Example, the anti-Ang-2 antibodies selected from the present invention substantially inhibited ocular angiogenesis in vivo, thereby reflecting the likely antiangiogenic potential of these antibodies in other therapeutic settings. Example 13. Important Ang-2 Amino Acids for Antibody Binding
[000156] To further characterize the link between hAng2 and anti-hAng2 mAbs of the invention, seven hAng-2FD-mFc proteins were generated, each containing a single point mutation. The amino acids selected for mutation were based on the sequence difference between hAng-2 and hAng-1 in the region that interacts with hTie-2 (figure 1). In particular, amino acids within the fibrinogen-like (FD) domain of Ang-2 that are believed to interact with Tie-2 based on crystal structure analysis, but which differ from the corresponding Ang-1 amino acid, have been individually mutated for the residue corresponding hAng-1. The results of this example indicate the amino acid residues of hAng-2 with which the preferred binding antibodies to Ang-2 interact. That is, if a particular residue (or residues) of hAng-2 is / are changed to the corresponding hAng-1 residue, and the binding of a preferred Ang-2 binding antibody is substantially reduced, then it can be concluded that the antibody interacts with that particular hAng-2 residue (s).
[000157] In this experiment, each of the seven hAng-2-mFc mutant proteins was captured (-147-283 RU) on an anti-mouse-Fc surface created by direct chemical coupling to a BIACORE® chip. Then each Angi-2 antibody (or peptibody, as the case may be) at 100 nM was injected into the surface of mFc-labeled hAng-2FD protein at a flow rate of 50 pl / min for 180 seconds, and dissociation of hAng2- Variant FD-mFc and antibody was monitored in real time for 20 minutes at 25 ° C. The results are summarized in Tables 35a-35d and figure 3.

1] Amino acid numbering is based on the amino acid numbering of SEQ ID NO: 518, [2] WT = wild-type Ang-2FD-mFc construct N / B = no binding observed
[000158] For purposes of the present invention, an anti-Ang-2 antibody is considered to interact with a particular Ang-2 amino acid residue if, when the residue is mutated to the corresponding Ang-1 residue, the dissociation Ti / 2 it is at least 5 times less than the Ti / 2 dissociation observed for the wild type construct under the experimental conditions used in this Example. In view of this definition, the H1H685P antibody appears to be unique among the antibodies tested because it interacts with H469, Y475 and S480. Since H1H685P is also unique because of its strong preferential binding to Ang-2 over Ang-1, it can be concluded that F469, Y475 and S480 comprise an epitope that allows the immunological distinction of Ang-2 from Ang-1 . The other antibodies / peptidibodies tested in this experiment appear to interact with at most one or two of these residues; that is, H1H744 and Control I interact with Y475; Control III interacts with Y475 and S480; and Control V interacts with F469. Interestingly, Control II, which has been shown to block both Ang-1 and Ang-2 by binding to Tie-2 with equal potency, does not interact with any of the Ang-2-specific amino acids identified in this experiment.
[000159] The present invention should not be limited in scope by the specific modalities described here. In fact, various modifications of the invention in addition to those described here will become apparent to those skilled in the art from the above report. Such modifications are intended to fall within the scope of the appended claims.

权利要求:
Claims (10)
[0001]
1. Isolated antibody or antigen-binding fragment that specifically binds to human angiopoietin-2 (hAng-2), characterized by the fact that it comprises a heavy chain CDR-1 (HCDR-1) having the amino acid sequence of SEQ ID NO: 4, a CDR-2 HCDR-2 having the amino acid sequence of SEQ ID NO: 6, an HCDR-3 having the amino acid sequence of SEQ ID NO: 8, an LCDR-1 having the sequence of amino acids of SEQ ID NO: 12, LCDR-2 having the amino acid sequence of SEQ ID NO: 14, an LCDR-3 having the amino acid sequence of SEQ ID NO: 16.
[0002]
2. Isolated antibody or an antigen-binding fragment thereof that specifically binds to human angiopoietin-2 (hAng-2), characterized by the fact that it comprises a variable region of the heavy chain having the amino acid sequence of SEQ ID NO : 18 and a light chain variable region with the amino acid sequence of SEQ ID NO: 20.
[0003]
3. Pharmaceutical composition, characterized by the fact that it comprises the antibody or antigen binding fragment as defined in claim 1 and a pharmaceutically acceptable carrier or diluent.
[0004]
Pharmaceutical composition according to claim 3, characterized by the fact that it further comprises a vascular endothelial cell growth factor (VEGF) antagonist.
[0005]
Pharmaceutical composition according to claim 4, characterized in that the VEGF antagonist is selected from an anti-VEGF antibody, a VEGF receptor small molecule kinase inhibitor and a VEGF inhibition fusion protein.
[0006]
6. Pharmaceutical composition, characterized in that it comprises the antibody or antigen binding fragment as defined in claim 2 and a pharmaceutically acceptable carrier or diluent.
[0007]
Pharmaceutical composition according to claim 6, characterized by the fact that it further comprises a vascular endothelial cell growth factor (VEGF) antagonist.
[0008]
Pharmaceutical composition according to claim 7, characterized in that the VEGF antagonist is selected from the group consisting of an anti-VEGF antibody, a small molecule kinase inhibitor of the VEGF receptor and an inhibitory fusion protein of VEGF.
[0009]
Use of the isolated antibody or antigen-binding fragment according to either of claims 1 or 2, characterized in that it is for the manufacture of a medicament for the treatment of a patient having a tumor.
[0010]
10. Use of the isolated antibody or antigen-binding fragment according to either of claims 1 or 2, characterized by the fact that it is for the manufacture of a medicament for the treatment of an eye disease associated with angiogenesis.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-09-17| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-05-05| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 27/10/2020, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/07/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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